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Antigen delivery via respiratory mucosal surfaces is an interesting needle-free option for vaccination. Nonetheless, it demands for the design of especially tailored formulations. Here, lipid/poly(lactic-co-glycolic) acid (PLGA) hybrid nanoparticles (hNPs) for the combined delivery of an antigen, ovalbumin (Ova), and an adjuvant, synthetic unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG) motifs, is developed. A panel of Ova/CpG-loaded lipid@PLGA hNPs with tunable size and surface is attained by exploiting two lipid moieties, 1,2 distearoil-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) and monophosphoryl lipid A (MPLA), with or without polyethyleneimine (PEI). It is gained insights on the lipid@PLGA hNPs through a combination of techniques to analytically determine the specific moiety on the surface, the spatial distribution of the components and the internal structure of the nanoplatforms. The collected results suggest that PEI plays a role of paramount importance not only in promoting in vitro antigen escape from lysosomes and enhancing antigen cross-presentation, but also in determining the arrangement of the moieties in the final architecture of the hNPs. Though multicomponent PEI-engineered lipid@PLGA hNPs turn out as a viable strategy for delivery of antigens and adjuvant to the respiratory mucosa, tunable nanoparticle features are achievable only through the optimal selection of the components and their relative amounts., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Polymeric nanoparticles (NPs), specifically those comprised of biodegradable and biocompatible polyesters, have been heralded as a game-changing drug delivery platform. In fact, poly(α-hydroxy acids) such as polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and poly(ε-caprolactone) (PCL) have been heavily researched in the past three decades as the material basis of polymeric NPs for drug delivery applications. As materials, these polymers have found success in resorbable sutures, biodegradable implants, and even monolithic, biodegradable platforms for sustained release of therapeutics (e.g., proteins and small molecules) and diagnostics. Few fields have gained more attention in drug delivery through polymeric NPs than cancer therapy. However, the clinical translational of polymeric nanomedicines for treating solid tumors has not been congruent with the fervor or funding in this particular field of research. Here, we attempt to provide a comprehensive snapshot of polyester NPs in the context of chemotherapeutic delivery. This includes a preliminary exploration of the polymeric nanomedicine in the cancer research space. We examine the various processes for producing polyester NPs, including methods for surface-functionalization, and related challenges. After a detailed overview of the multiple factors involved with the delivery of NPs to solid tumors, the crosstalk between particle design and interactions with biological systems is discussed. Finally, we report state-of-the-art approaches toward effective delivery of NPs to tumors, aiming at identifying new research areas and re-evaluating the reasons why some research avenues have underdelivered. We hope our effort will contribute to a better understanding of the gap to fill and delineate the future research work needed to bring polyester-based NPs closer to clinical application. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies., (© 2024 The Author(s). WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC.)

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVβ3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVβ3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVβ3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVβ3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVβ3 and an αVβ3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVβ3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVβ3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVβ3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVβ3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

The strict dependence of the biological effects of nitric oxide (NO) on its concentration and generation site requires this inorganic free radical to be delivered with precise spatiotemporal control. Light-activation by suitable NO photoprecursors represents an ideal approach. Developing strategies to activate NO release using long-wavelength excitation light in the therapeutic window (650-1300 nm) is challenging. In this contribution, we demonstrate that NO release by a blue-light activatable NO photodonor (NOPD) with self-fluorescence reporting can be triggered catalytically by the much more biocompatible red light exploiting a supramolecular photosensitization process. Different red-light absorbing photosensitizers (PSs) are co-entrapped with the NOPD within different biocompatible nanocarriers such as Pluronic® micelles, microemulsions and branched cyclodextrin polymers. The intra-carrier photosensitized NO release, involving the lowest, long-lived triplet state of the PS as the key intermediate and its quenching by the NOPD, is competitive with that by molecular oxygen. This allows NO to be released with good efficacy, even under aerobic conditions. Therefore, the adopted general strategy provides a valuable tool for generating NO from an already available NOPD, otherwise activatable with the poorly biocompatible blue light, without requiring any chemical modification and using sophisticated and expensive irradiation sources.

Cannabidiol (CBD) has gained significant attention as a complementary and alternative medicine due to its promising therapeutic properties. However, CBD faces obstacles when administered orally due to its poor solubility in water, leading to limited absorption into the bloodstream and low and variable bioavailability. Therefore, the development of innovative delivery approaches that can enhance CBD's bioavailability, facilitate administration, and promote patient adherence is crucial. We propose a new approach for buccal delivery of CBD based on a self-assembling nanoemulsion (NE) made of a mixture of surfactants (Tween 80 and Labrasol) and medium chain triglycerides (MCTs). The NE formulation showed properties suitable for buccal administration, including appropriate size, CBD content, and surface properties, and, if compared to a CBD-MCT solution, it exhibited better control of administered doses, faster dissolution in buccal medium, and enhanced stability. The CBD-NE effectively released its active load within 5 h, remained stable even when diluted in simulated buccal fluids, and could be easily administered through a commercially available spray, providing consistent and reproducible doses of NE with optimized properties. In vitro permeation studies demonstrated that the CBD-NE facilitated swift and consistent permeation through the buccal mucosa, resulting in a higher concentration in the acceptor compartment compared to CBD-MCT. Furthermore, the in vivo study in mice showed that a single buccal administration of CBD-NE led to a quicker onset of action than a CBD solution in MCT, while maintaining the same plasma levels over time and leading to typically higher plasma concentrations compared to those usually achieved through oral administration. In conclusion, our CBD-NE represents a promising alternative formulation strategy for buccal CBD administration, overcoming the challenges associated with conventional formulations such as variable bioavailability and low control of administered doses., (© 2023. Controlled Release Society.)

The DisProt database is a resource containing manually curated data on experimentally validated intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) from the literature. Developed in 2005, its primary goal was to collect structural and functional information into proteins that lack a fixed three-dimensional structure. Today, DisProt has evolved into a major repository that not only collects experimental data but also contributes to our understanding of the IDPs/IDRs roles in various biological processes, such as autophagy or the life cycle mechanisms in viruses or their involvement in diseases (such as cancer and neurodevelopmental disorders). DisProt offers detailed information on the structural states of IDPs/IDRs, including state transitions, interactions and their functions, all provided as curated annotations. One of the central activities of DisProt is the meticulous curation of experimental data from the literature. For this reason, to ensure that every expert and volunteer curator possesses the requisite knowledge for data evaluation, collection and integration, training courses and curation materials are available. However, biocuration guidelines concur on the importance of developing robust guidelines that not only provide critical information about data consistency but also ensure data acquisition.This guideline aims to provide both biocurators and external users with best practices for manually curating IDPs and IDRs in DisProt. It describes every step of the literature curation process and provides use cases of IDP curation within DisProt. Database URL: https://disprot.org/., (© The Author(s) 2024. Published by Oxford University Press.)

Nowadays, the interest in research towards the local administration of drugs via the inhalation route is growing as it enables the direct targeting of the lung tissue, at the same time reducing systemic side effects. This is of great significance in the era of nucleic acid therapeutics and personalized medicine for the local treatment of severe lung diseases. However, the success of any inhalation therapy is driven by a delicate interplay of factors, such as the physiochemical profile of the payload, formulation, inhalation device, aerodynamic properties, and interaction with the lung fluids. The development of drug delivery systems tailored to the needs of this administration route is central to its success and to revolutionize the treatment of respiratory diseases. With this review, we aim to provide an up-to-date overview of advances in the development of nanoparticulate carriers for drug delivery to the lung tissue, with special regard concerning lipid and polymer-based nanocarriers (NCs). Starting from the biological barriers that the anatomical structure of the lung imposes, and that need to be overcome, the current strategies to achieve efficient lung delivery and the best support for the success of NCs for inhalation are highlighted.

Colorectal cancer (CRC) often involves wild-type p53 inactivation by MDM2 and MDM4 overexpression, promoting tumor progression and resistance to 5-fluoruracil (5-FU). Disrupting the MDM2/4 heterodimer can proficiently reactivate p53, sensitizing cancer cells to 5-FU. Herein, we developed 16 peptides based on Pep3 ( 1 ), the only known peptide acting through this mechanism. The new peptides, notably 3 and 9 , showed lower IC 50 values than 1 . When incorporated into tumor-targeted biodegradable nanoparticles, these exhibited cytotoxicity against three different CRC cell lines. Notably, NPs/ 9 caused a significant increase in p53 levels associated with a strong increment of its main downstream target p21 inducing apoptosis. Also, the combined treatment of 9 with 5-FU caused the activation of nucleolar stress and a synergic apoptotic effect. Hence, the co-delivery of MDM2/4 heterodimer disruptors with 5-FU through nanoparticles might be a promising strategy to overcome drug resistance in CRC.

DisProt (URL: https://disprot.org) is the gold standard database for intrinsically disordered proteins and regions, providing valuable information about their functions. The latest version of DisProt brings significant advancements, including a broader representation of functions and an enhanced curation process. These improvements aim to increase both the quality of annotations and their coverage at the sequence level. Higher coverage has been achieved by adopting additional evidence codes. Quality of annotations has been improved by systematically applying Minimum Information About Disorder Experiments (MIADE) principles and reporting all the details of the experimental setup that could potentially influence the structural state of a protein. The DisProt database now includes new thematic datasets and has expanded the adoption of Gene Ontology terms, resulting in an extensive functional repertoire which is automatically propagated to UniProtKB. Finally, we show that DisProt's curated annotations strongly correlate with disorder predictions inferred from AlphaFold2 pLDDT (predicted Local Distance Difference Test) confidence scores. This comparison highlights the utility of DisProt in explaining apparent uncertainty of certain well-defined predicted structures, which often correspond to folding-upon-binding fragments. Overall, DisProt serves as a comprehensive resource, combining experimental evidence of disorder information to enhance our understanding of intrinsically disordered proteins and their functional implications., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVβ3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVβ3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVβ3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVβ3 ligands. Moreover, we describe for the first time co-fractionation of αVβ3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer., Competing Interests: Declaration of Competing Interest FQ is an employee/contractor at Janssen Pharmaceutical. EC is a consultant of Dotquant and received funding under institutional SRA from Janssen Research, Bayer Pharmaceuticals, Forma Therapeutics, Foghorn, Kronos Bios, Genentech, Astra Zeneca, and MacroGenics. No potential conflict of interest was reported by the other authors., (Copyright © 2023. Published by Elsevier B.V.)

An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources., (© 2023. Springer Nature America, Inc.)

The role of nitric oxide (NO) as an "unconventional" therapeutic and the strict dependence of biological effects on its concentration require the generation of NO with precise spatiotemporal control. The development of precursors and strategies to activate NO release by excitation in the so-called "therapeutic window" with highly biocompatible and tissue-penetrating red light is desirable and challenging. Herein, we demonstrate that one-photon red-light excitation of Verteporfin, a clinically approved photosensitizer (PS) for photodynamic therapy, activates NO release, in a catalytic fashion, from an otherwise blue-light activatable NO photodonor (NOPD) with an improvement of about 300 nm toward longer and more biocompatible wavelengths. Steady-state and time-resolved spectroscopic and photochemical studies combined with theoretical calculations account for an NO photorelease photosensitized by the lowest triplet state of the PS. In view of biological applications, the water-insoluble PS and NOPD have been co-entrapped within water-dispersible, biodegradable polymeric nanoparticles (NPs) of mPEG-b-PCL (about 84 nm in diameter), where the red-light activation of NO release takes place even more effectively than in an organic solvent solution and almost independently by the presence of oxygen. Moreover, the ideal spectroscopic prerequisites and the restricted environment of the NPs permit the green-fluorescent co-product formed concomitantly to NO photorelease to communicate with the PS via Förster resonance energy transfer. This leads to an enhancement of the typical red emission of the PS offering the possibility of a double color optical reporter useful for the real-time monitoring of the NO release through fluorescence techniques. The suitability of this strategy applied to the polymeric NPs as potential nanotherapeutics was evaluated through biological tests performed by using HepG2 hepatocarcinoma and A375 melanoma cancer cell lines. Fluorescence investigation in cells and cell viability experiments demonstrates the occurrence of the NO release under one-photon red-light illumination also in the biological environment. This confirms that the adopted strategy provides a valuable tool for generating NO from an already available NOPD, otherwise activatable with the poorly biocompatible blue light, without requiring any chemical modification and the use of sophisticated irradiation sources.

Curcumin (CUR) is a naturally occurring pigment extensively studied due to its therapeutic activity and delivered by suitable nanocarriers to overcome poor solubility in aqueous media. The significant absorption of CUR in the visible blue region has prompted its use as a potential phototherapeutic agent in treating infectious and cancer diseases, although the mechanism underlying the phototoxic effects is still not fully understood. This contribution investigates the photobehaviour of CUR within polymeric micelles, microemulsions, and zein nanoparticles, chosen as biocompatible nanocarriers, and human serum albumin as a representative biomolecule. Spectroscopic studies indicate that in all host systems, the enolic tautomeric form of CUR is converted in a significant amount of the diketo form because of the perturbation of the intramolecular hydrogen bond. This leads to intermolecular H-abstraction from the host components by the lowest excited triplet state of CUR with the formation of the corresponding ketyl radical, detected by nanosecond laser flash photolysis. This radical is oxidized by molecular oxygen, likely generating peroxyl and hydroperoxyl radical species, unless in Zein, reasonably due to the poor availability of oxygen in the closely packed structure of this nanocarrier. In contrast, no detectable formation of singlet oxygen was revealed in all the systems. Overall these results highlight the key role of the H-abstraction process over singlet oxygen sensitization as a primary photochemical pathway strictly dictated by the specific features of the microenvironment, providing new insights into the photoreactivity of CUR in biocompatible hosts that can also be useful for a better understanding of its phototoxicity mechanism., Competing Interests: Declaration of Competing Interest No competing interest to declare., (Copyright © 2023. Published by Elsevier B.V.)

Food supplements (FS) containing red yeast rice (RYR) are largely employed to reduce lipid levels in the blood. The main ingredient responsible for biological activity is monacolin K (MoK), a natural compound with the same chemical structure as lovastatin. Concentrated sources of substances with a nutritional or physiological effect are marketed in "dose" form as food supplements (FS). The quality profile of the "dosage form" of FS is not defined in Europe, whereas some quality criteria are provided in the United States. Here, we evaluate the quality profile of FS containing RYR marketed in Italy as tablets or capsules running two tests reported in The European Pharmacopoeia 11 Ed. and very close to those reported in the USP. The results highlighted variations in dosage form uniformity (mass and MoK content) compliant with The European Pharmacopoeia 11 Ed. specifications, whereas the time needed for disintegrating tablets was longer for 44% of the tested samples. The bioaccessibility of MoK was also investigated to obtain valuable data on the biological behaviour of the tested FS. In addition, a method for citrinin (CIT) determination was optimized and applied to real samples. None of the analyzed samples demonstrated CIT contamination (LOQ set at 6.25 ng/mL). Considering the widespread use of FS, our data suggest that greater attention should be paid by fabricants and regulatory authorities to ensure the quality profile and the safe consumption of marketed products.

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Despite recent therapeutic advancements, resistance to 5-fluorouracil (5-FU) remains a major obstacle to the successful treatment of this disease. We have previously identified the ribosomal protein uL3 as a key player in the cell response to 5-FU, and loss of uL3 is associated with 5-FU chemoresistance. Natural products, like carotenoids, have shown the ability to enhance cancer cell response to drugs and may provide a safer choice to defeat chemoresistance in cancer. Transcriptome analysis of a cohort of 594 colorectal patients revealed a correlation between uL3 expression and both progression-free survival and response to treatment. RNA-Seq data from uL3-silenced CRC cells demonstrated that a low uL3 transcriptional state was associated with an increased expression of specific ATP-binding cassette ( ABC ) genes. Using two-dimensional (2D) and three-dimensional (3D) models of 5-FU-resistant CRC cells stably silenced for uL3, we investigated the effect of a novel therapeutic strategy by combining β-carotene and 5-FU using nanoparticles (NPs) as a drug delivery system. Our results indicated that the combined treatment might overcome 5-FU chemoresistance, inducing cell cycle arrest in the G2/M phase and apoptosis. Furthermore, the combined treatment significantly reduced the expression levels of analyzed ABC genes. In conclusion, our findings suggest that β-carotene combined with 5-FU may be a more effective therapeutic approach for treating CRC cells with low levels of uL3.

Surface functionalization of nanoparticles (NPs) with tumor-targeting peptides is an emerging approach with a huge potential to translate in the clinic and ameliorate the efficacy of nano-oncologicals. One major challenge is to find straightforward strategies for anchoring peptides on the surface of biodegradable NPs and ensuring their correct exposure and orientation to bind the target receptor. Here, we propose a non-covalent strategy to functionalize polyester aminic NPs based on the formation of either electrostatic or lipophilic interactions between NPs and the peptide modified with an anchoring moiety. We selected an iNGRt peptide containing a CendR motif (CRNGR) targeting neuropilin receptor 1 (NRP-1), which is upregulated in several cancers. iNGRt was linked with either a short poly(glutamic acid) chain (polyE) or a palmitoyl chain (Palm) and used to functionalize the surface of NPs made of a diamine poly(ε-caprolactone). iNGRt-PolyE was adsorbed on preformed cationic NPs through electrostatic interaction, whereas iNGRt-Palm was integrated into the forming NPs through interactions. In both cases, peptides were strongly associated with NPs of ∼100 nm, low polydispersity indexes, and positive zeta potential values. NPs entered MDA-MB231 breast cancer cells overexpressing NRP-1 via receptor-mediated endocytosis and showed a different cell localization depending on the mode of peptide anchoring. When loaded with the lipophilic anticancer drug docetaxel (DTX), NPs functionalized with the iNGRt-Palm variant exerted a time- and dose-dependent cytotoxicity similar to DTX in MDA-MB-231 cells but were less toxic than DTX toward control MRC-5 human fibroblasts, not expressing NRP-1. In a heterotopic mouse model of triple negative breast cancer, iNGRt-Palm NPs were tolerated better than free DTX and demonstrated superior anticancer activity and survival compared to both free DTX and NPs without peptide functionalization. We foresee that the functionalization strategy with palmitoylated peptides proposed here can be extended to other biodegradable NPs and peptide sequences designed for therapeutic or targeting purposes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5'-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Inokuchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Human Proteome Organization (HUPO) Proteomics Standards Initiative (PSI) has been successfully developing guidelines, data formats, and controlled vocabularies (CVs) for the proteomics community and other fields supported by mass spectrometry since its inception 20 years ago. Here we describe the general operation of the PSI, including its leadership, working groups, yearly workshops, and the document process by which proposals are thoroughly and publicly reviewed in order to be ratified as PSI standards. We briefly describe the current state of the many existing PSI standards, some of which remain the same as when originally developed, some of which have undergone subsequent revisions, and some of which have become obsolete. Then the set of proposals currently being developed are described, with an open call to the community for participation in the forging of the next generation of standards. Finally, we describe some synergies and collaborations with other organizations and look to the future in how the PSI will continue to promote the open sharing of data and thus accelerate the progress of the field of proteomics.

A novel approach for dynamic microwave modulation is proposed in the form of reconfigurable resonant circuits. This result is obtained through the monolithic integration of double split ring resonators (DSRRs) with microelectromechanical actuators (MEMS) for geometrical tuning. Two configurations were analyzed to achieve a controlled deformation of the DSRRs' metamaterial geometry by mutual rotation or extrusion along the azimuthal direction of the two constituent rings. Then, the transfer function was numerically simulated for a reconfigurable MEMS-DSRR hybrid architecture where the DSRR is embedded onto a realistic piezo actuator chip. In this case, a 370 MHz resonance frequency shift was obtained under of a 170 µm extrusion driven by a DC voltage. These characteristics in combination with a high Q factor and dimensions compatible with standard CMOS manufacturing techniques provide a step forward for the production of devices with applications in multiband telecommunications and wireless power transfer and in the IoT field.

Micro-electro-mechanical systems (MEMS) have enabled new techniques for the miniaturization of sensors suitable for Structural Health Monitoring (SHM) applications. In this study, MEMS-based sensors, specifically Piezoelectric Micromachined Ultrasonic Transducers (PMUT), are used to evaluate and monitor the pre-tensioning of a bolted joint structural system. For bolted joints to function properly, it is essential to maintain a suitable level of pre-tensioning. In this work, an array of PMUTs attached to the head and to the end of a bolt, serve as transmitter and receiver, respectively, in a pitch-catch Ultrasonic Testing (UT) scenario. The primary objective is to detect the Change in Time of Flight (CTOF) of the acoustic wave generated by the PMUT array and propagating along the bolt's axis between a non-loaded bolt and a bolt in service. To model the pre-tensioning of bolted joints and the transmission of the acoustic wave to and from a group of PMUTs through the bolt, a set of numerical models is created. The CTOF is found to be linearly related to the amount of pre-tensioning. The numerical model is validated through comparisons with the results of a preliminary experimental campaign.

The αVβ6 integrin, an epithelial-specific cell surface receptor absent in normal prostate and expressed during prostate cancer (PrCa) progression, is a therapeutic target in many cancers. Here, we report that transcript levels of ITGB6 (encoding the β6 integrin subunit) are significantly increased in metastatic castrate-resistant androgen receptor-negative prostate tumors compared to androgen receptor-positive prostate tumors. In addition, the αVβ6 integrin protein levels are significantly elevated in androgen receptor-negative PrCa patient derived xenografts (PDXs) compared to androgen receptor-positive PDXs. In vitro , the androgen receptor-negative PrCa cells express high levels of the αVβ6 integrin compared to androgen receptor-positive PrCa cells. Additionally, expression of androgen receptor (wild type or variant 7) in androgen receptor-negative PrCa cells downregulates the expression of the β6 but not αV subunit compared to control cells. We demonstrate an efficient strategy to therapeutically target the αVβ6 integrin during PrCa progression by using short interfering RNA (siRNA) loaded into PrCa cell-derived small extracellular vesicles (sEVs). We first demonstrate that fluorescently-labeled siRNAs can be efficiently loaded into PrCa cell-derived sEVs by electroporation. By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into PrCa cells. We show that sEV-mediated delivery of ITGB6 -targeting siRNAs into PC3 cells specifically downregulates expression of the β6 subunit. Furthermore, treatment with sEVs encapsulating ITGB6 siRNA significantly reduces cell adhesion and migration of PrCa cells on an αVβ6-specific substrate, LAP-TGFβ1. Our results demonstrate an approach for specific targeting of the αVβ6 integrin in PrCa cells using sEVs encapsulating ITGB6 -specific siRNAs.

Development of inhalable formulations for delivering peptides to the conductive airways and shielding their interactions with airway barriers, thus enhancing peptide/bacteria interactions, is an important part of peptide-based drug development for lung applications. Here, we report the construction of a biocompatible nanosystem where the antimicrobial peptide SET-M33 is encapsulated within polymeric nanoparticles of poly(lactide-co-glycolide) (PLGA) conjugated with polyethylene glycol (PEG). This system was conceived for better delivery of the peptide to the lungs by aerosol. The encapsulated peptide showed prolonged antibacterial activity, due to its controlled release, and much lower toxicity than the free molecule. The peptide-based nanosystem killed Pseudomonas aeruginosa in planktonic and sessile forms in a dose-dependent manner, remaining active up to 72 h after application. The encapsulated peptide showed no cytotoxicity when incubated with human bronchial epithelial cells from healthy individuals and from cystic fibrosis patients, unlike the free peptide, which showed an EC50 of about 22 µM. In vivo acute toxicity studies in experimental animals showed that the peptide nanosystem did not cause any appreciable side effects, and confirmed its ability to mitigate the toxic and lethal effects of free SET-M33.

The potential of intra-venous gallium nitrate (GaN) administration against Pseudomonas aeruginosa pneumonia was recently demonstrated in mice and in cystic fibrosis (CF) patients. Likewise, the added value of direct lung delivery of Ga(III) has been shown in rats. Therefore, the design of a drug delivery system specifically engineered for Ga(III) inhalation is imperative to improve its accumulation in lungs. To this purpose, Ga(III) was efficiently encapsulated into hyaluronic acid/chitosan nanoparticles (Ga_HA/CS NPs), whose features were tuned to facilitate access to the target by overcoming mucus and biofilm surrounding bacteria. Then, to improve in vivo lung deposition, Ga_HA/CS NPs were engineered into mannitol-based NEM (Ga_Man NEM). The powders showed optimal in vitro aerosol performance, and sustained release kinetics in lung lining fluids. Moreover, good tolerability and antimicrobial properties were shown in vitro. Intratracheal insufflation of Ga_Man NEM in rats resulted in a significant improvement of Ga(III) persistence in the lungs coupled to a lower Ga(III) concentration in plasma and urine, compared to GaN solution. Noteworthy, the developed formulation significantly modifies the unfavorable Ga(III) kinetic increasing the Ga(III) to the lung and preventing Ga(III) accumulation in the kidney, key responsible for adverse effects, conclusively demonstrating the benefit of Ga_Man NEM to exploit the therapeutic effect of Ga(III) via inhalation route., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Purpose: The aim of our study was to identify maternal characteristics of women who are responsive to the second application of vaginal dinoprostone in a cohort of patients with a low Bishop Score. Secondarily, we compared the outcome of the patients' response to a single application to that of the women's response to a double application. Materials and methods: This was a retrospective observational cohort study. Patients undergoing preinduction of labor with dinoprostone 10mg controlled-release vaginal device were included. Results: Among 216 included patients, 192 women (88.9%) achieved a cervical ripening after a single application of dinoprostone, while 24 (11.1%) required a second application. Patients notresponding to the first application of dinoprostone had a significantly higher body mass index (27.4 ± 6.7 kg/m 2 vs 24.9 ± 5.2 kg/m 2 ; p < 0.05) and a significant increase in gestational weight gain (14 ± 5.2 kg vs 11.6 ± 6.1; p < 0.005). Double application of dinoprostone resulted in spontaneousdelivery in 58.4% of cases, but it was related to poorer neonatal outcome, compared to a single application. Conclusions: Obese women, not responding to the first application of dinoprostone could respond to the second application of this vaginal prostaglandin. However, data related to the use of a double application are still very limited to recommend its use as a standardized procedurefor not responsive patients.

Androgen deprivation therapies aimed to target prostate cancer (PrCa) are only partially successful given the occurrence of neuroendocrine PrCa (NEPrCa), a highly aggressive and highly metastatic form of PrCa, for which there is no effective therapeutic approach. Our group has demonstrated that while absent in prostate adenocarcinoma, the αVβ3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVβ3 that promotes NE differentiation (NED). This novel pathway requires the expression of a GPI-linked surface molecule, NgR2, also known as Nogo-66 receptor homolog 1. We show here that NgR2 is upregulated by αVβ3, to which it associates; we also show that it promotes NED and anchorage-independent growth, as well as a motile phenotype of PrCa cells. Given our observations that high levels of αVβ3 and, as shown here, of NgR2 are detected in human and mouse NEPrCa, our findings appear to be highly relevant to this aggressive and metastatic subtype of PrCa. This study is novel because NgR2 role has only minimally been investigated in cancer and has instead predominantly been analyzed in neurons. These data thus pave new avenues toward a comprehensive mechanistic understanding of integrin-directed signaling during PrCa progression toward a NE phenotype., (© 2022. The Author(s).)

The design, synthesis, photochemical properties, and biological evaluation of a novel molecular dyad with double photodynamic action and its formulation within biodegradable polymeric nanoparticles (NPs) are reported. A BODIPY-based singlet oxygen ( 1 O 2 ) photosensitizer (PS) and a nitric oxide (NO) photodonor (NOPD) based on an amino-nitro-benzofurazan moiety have been covalently joined in a new molecular dyad, through a flexible alkyl spacer. Excitation of the dyad with visible light in the range 400-570 nm leads to the concomitant generation of the cytotoxic 1 O 2 and NO with effective quantum yields, being Φ Δ  = 0.49 ± 0.05 and Φ NO  = 0.18 ± 0.01, respectively. Besides, the non-fluorescent NOPD unit becomes highly fluorescent after the NO release, acting as an optical reporter for the NO photogenerated. The dyad is not soluble in water medium but can be effectively entrapped in water-dispersible, biodegradable polymeric NPs made of mPEG-PCL, ca. 66 nm in diameter. The polymeric nano-environment affects in an opposite way the photochemical performances of the dyad, reducing Φ Δ to 0.16 ± 0.02 and increasing Φ NO to 0.92 ± 0.03, respectively. The NPs effectively deliver the photoactive cargo into the cytoplasm of HepG2 hepatocellular carcinoma cells. A remarkable level of cell mortality is observed for the loaded NPs at very low concentrations of the dyad (1-5 µM) and very low light doses (≤0.8 J cm -2 ) more likely as the result of the combined photodynamic action of 1 O 2 and NO., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A biodegradable engineered nanoplatform combining anti-angiogenic activity and targeting of cancer cells to improve the anticancer activity of docetaxel (DTX) is here proposed. Indeed, we have developed biodegradable nanoparticles (NPs) of poly(ethylene glycol)-poly(ε-caprolactone), exposing on the surface both folate motifs (Fol) for recognition in cells overexpressing Folate receptor-α (FRα) and the anti-angiogenic hexapeptide aFLT1. NPs showed a size around 100 nm, the exposure of 60% of Fol moieties on the surface, and the ability to entrap DTX and sustain its release with time. NPs were stable in simulated biological fluids and slightly interacted with Fetal Bovine serum, especially in the formulation decorated with Fol and aFLT1. The presence of Fol on NPs did not impair the anti-angiogenic activity of aFLT1, as assessed by in vitro tube formation assay in HUVEC endothelial cells. In both 2D and 3D KB cell cultures in vitro, the cytotoxicity of DTX loaded in NPs was not significantly affected by Fol/aFLT1 double decoration compared to free DTX. Remarkably, NPs distributed differently in 3D multicellular spheroids of FRα-positive KB cancer cells depending on the type of ligand displayed on the surface. In particular, NPs unmodified on the surface were randomly distributed in the spheroid, whereas the presence of Fol promoted the accumulation in the outer rims of the spheroid. Finally, NPs with Fol and aFLT1 gave a uniform distribution throughout the spheroid structure. When tested in zebrafish embryos xenografted with KB cells, NPs displaying Fol/aFLT1 reduced DTX systemic toxicity and inhibited the growth of the tumor mass and associated vasculature synergistically. Overall, nanotechnology offers excellent ground for combining therapeutic concepts in cancer, paving the way to novel multifunctional nanopharmaceuticals decorated with bioactive elements that can significantly improve therapeutic outcomes., (© 2021. The Author(s).)

Six-Transmembrane Epithelial Antigen of the Prostate 1-4 (STEAP1-4) compose a family of metalloproteinases involved in iron and copper homeostasis and other cellular processes. Thus far, five homologs are known: STEAP1, STEAP1B, STEAP2, STEAP3, and STEAP4. In prostate cancer, STEAP1, STEAP2, and STEAP4 are overexpressed, while STEAP3 expression is downregulated. Although the metalloreductase activities of STEAP1-4 are well documented, their other biological functions are not. Furthermore, the properties and expression levels of STEAP heterotrimers, homotrimers, heterodimers, and homodimers are not well understood. Nevertheless, studies over the last few decades have provided sufficient impetus to investigate STEAP1-4 as potential biomarkers and therapeutic targets for prostate cancer. In particular, STEAP1 is the target of many emerging immunotherapies. Herein, we give an overview of the structure, physiology, and pathophysiology of STEAP1-4 to provide context for past and current efforts to translate STEAP1-4 into the clinic.

It has recently emerged that drugs such as the mTOR inhibitor rapamycin (Rapa) may play a key role in the treatment of airway inflammation associated with lung diseases, such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis. Nevertheless, Rapa clinical application is still prevented by its unfavorable chemical-physical properties, limited oral bioavailability, and adverse effects related to non-specific biodistribution. In this paper, the design and production of a novel formulation of Rapa based on nano into micro (NiM) particles are detailed. To achieve it, Rapa-loaded nanoparticles were produced by nanoprecipitation of an amphiphilic pegylated poly-ɛ-caprolactone/polyhydroxyethyl aspartamide graft copolymer. The obtained nanoparticles that showed a drug loading of 14.4 wt% (corresponding to an encapsulation efficiency of 82 wt%) did not interact with mucins and were able to release and protect Rapa from degradation in simulated lung and cell fluids. To allow their local administration to the lungs as a dry powder, particle engineering at micro-sized level was done by embedding nanoparticles into mannitol-based microparticles by spray drying. Obtained NiM particles had a mean diameter of about 2-µ, spherical shape and had good potential to be delivered to the lungs by a breath-activated dry powder inhalers. Rheological and turbidity experiments showed that these NiM particles can dissolve in lung simulated fluid and deliver the Rapa-loaded pegylated nanoparticles, which can diffuse through the mucus layer., (© 2022. The Author(s).)

The SARS-CoV-2 pandemic is maintained by the emergence of successive variants, highlighting the flexibility of the protein sequences of the virus. We show that experimentally determined intrinsically disordered regions (IDRs) are abundant in the SARS-CoV-2 viral proteins, making up to 28% of disorder content for the S1 subunit of spike and up to 51% for the nucleoprotein, with the vast majority of mutations occurring in the 13 major variants mapped to these IDRs. Strikingly, antigenic sites are enriched in IDRs, in the receptor-binding domain (RBD) and in the N-terminal domain (NTD), suggesting a key role of structural flexibility in the antigenicity of the SARS-CoV-2 protein surface. Mutations occurring in the S1 subunit and nucleoprotein (N) IDRs are critical for immune evasion and antibody escape, suggesting potential additional implications for vaccines and monoclonal therapeutic strategies. Overall, this suggests the presence of variable regions on S1 and N protein surfaces, which confer sequence and antigenic flexibility to the virus without altering its protein functions., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

DisProt is the major repository of manually curated data for intrinsically disordered proteins collected from the literature. Although lacking a stable three-dimensional structure under physiological conditions, intrinsically disordered proteins carry out a plethora of biological functions, some of them directly arising from their flexible nature. A growing number of scientific studies have been published during the last few decades to shed light on their unstructured state, their binding modes, and their functions. DisProt makes use of a team of expert biocurators to provide up-to-date annotations of intrinsically disordered proteins from the literature, making them available to the scientific community. Here we present a comprehensive description on how to use DisProt in different contexts and provide a detailed explanation of how to explore and interpret manually curated annotations of intrinsically disordered proteins. We describe how to search DisProt annotations, both using the web interface and the API for programmatic access. Finally, we explain how to visualize and interpret a DisProt entry, the SARS-CoV-2 Nucleoprotein, characterized by the presence of unstructured N-terminal and C-terminal regions and a flexible linker. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Performing a search in DisProt Support Protocol 1: Downloading options Support Protocol 2: Programmatic access with DisProt REST API Basic Protocol 2: Exploring the DisProt Ontology page Basic Protocol 3: Visualizing and interpreting DisProt entries-the SARS-CoV-2 Nucleoprotein use case., (© 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.)

This paper presents a numerical reduced-order modeling (ROM) approach for complex multi-layered arrays of piezoelectric micromachined ultrasonic transducers (PMUTs). The numerical modeling technique adopted to generate an array of PMUTs consisting of a considerable number of transducers allows for a large reduction in computational cost without reducing accuracy. The modeling idea is based on coupling shell elements applied to the PMUT structural layers with 3D-solid elements applied to the piezoelectric layer. A set of eigenfrequency and frequency domain analyses are presented considering a single ROM of a PMUT performing in different ambients and the performing central frequencies are obtained for every considered scenario. A unique arrangement of 228 PMUTs is presented and tested for its ability to transmit and receive acoustic waves. The operating frequency band of the array and the level of interference and cross-talk among different PMUTs in the near field are estimated. Finally, the results from a preliminary experimental test performed to analyze the acoustic abilities of an 8 × 8 array of PMUTs are presented. A corresponding numerical model is created and the obtained results matched the experimental data, leading to a validation of the modeling technique proposed in this work.

In this contribution, we report a strategy to enhance the therapeutic action of the chemotherapeutic Sorafenib (SRB) through its combination with a multifunctional β-cyclodextrin-based polymer able to deliver nitric oxide (NO) and emit green fluorescence upon visible light excitation (PolyCDNO). The basically water-insoluble SRB is effectively encapsulated in the polymeric host (1 mg mL -1 ) up to a concentration of 18 μg mL -1 . The resulting host-guest supramolecular complex is able to release SRB in sink conditions and to preserve very well the photophysical and photochemical properties of the free PolyCDNO, as demonstrated by the similar values of the NO release and fluorescence emission quantum efficiencies found. The complex PolyCDNO/SRB internalizes in HEP-G2 hepatocarcinoma, MCF-7 breast cancer and ACHN kidney adenocarcinoma cells, localizing in all cases mainly at the cytoplasmic level. Biological experiments have been performed at SRB concentrations below the IC 50 and with light doses producing NO at nontoxic concentrations. The results demonstrate exceptional mortality levels for PolyCDNO/SRB upon visible light irradiation in all the different cell lines tested, indicating a clear synergistic action between the chemotherapeutic drug and the NO. These findings can open up exciting avenues to potentiate the anticancer action of SRB and, in principle, to reduce its side effects through its use at low dosages when in combination with the photo-regulated release of NO.

The biological activity of a molecular hybrid ( DXNO-GR ) joining doxorubicin (DOX) and an N-nitroso moiety releasing nitric oxide (NO) under irradiation with the biocompatible green light has been investigated against DOX-sensitive (MCF7) and -resistant (MDA-MB-231) breast cancer cells in vitro . DXNO-GR shows significantly higher cellular internalization than DOX in both cell lines and, in contrast to DOX, does not experience cell efflux in MDR overexpressing MDA-MB-231 cells. The higher cellular internalization of the DXNO-GR hybrid seems to be mediated by bovine serum albumin (BSA) as a suitable carrier among serum proteins, according to the high binding constant measured for DXNO-GR , which is more than one order of magnitude larger than that reported for DOX. Despite the higher cellular accumulation, DXNO-GR is not toxic in the dark but induces remarkable cell death following photoactivation with green light. This lack of dark toxicity is strictly related to the different cellular compartmentalization of the molecular hybrid that, different from DOX, does not localize in the nucleus but is mainly confined in the Golgi apparatus and endoplasmic reticulum and therefore does not act as a DNA intercalator. The photochemical properties of the hybrid are not affected by binding to BSA as demonstrated by the direct detection of NO photorelease, suggesting that the reduction of cell viability observed under light irradiation is a combined effect of DOX phototoxicity and NO release which, ultimately, inhibits MDR1 efflux pump in DOX-resistant cells., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Functional impairment of the von Hippel-Lindau tumor suppressor (pVHL) is causative of a familiar increased risk of developing cancer. As an E3 substrate recognition particle, pVHL marks the hypoxia inducible factor 1α (HIF-1α) for degradation in normoxic conditions, thus acting as a key regulator of both acute and chronic cell adaptation to hypoxia. The male mice model carrying VHL gene conditional knockout presents significant abnormalities in testis development paired with defects in spermatogenesis and infertility, indicating that pVHL exerts testis-specific roles. Here we aimed to explore whether pVHL could have a similar role in humans by performing a testis-tissue library screening complemented with in-depth bioinformatics analysis. We identified 55 novel pVHL binding proteins directly involved in spermatogenesis, cell differentiation and reproductive metabolism. In addition, computational investigation of these new interactors identified multiple pVHL-specific binding motifs and demonstrated that somatic mutations described in human cancers reside in these binding regions. Collectively, these findings suggest that, in addition to its role in cancer formation, pVHL may also be pivotal in normal gonadal development in humans.

Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-κB (siNFκB) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl- sn -glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air-liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NFκB gene silencing was achieved in LPS-stimulated 16HBE14o- cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.

Intrinsically disordered regions (IDRs) lacking a fixed three-dimensional protein structure are widespread and play a central role in cell regulation. Only a small fraction of IDRs have been functionally characterized, with heterogeneous experimental evidence that is largely buried in the literature. Predictions of IDRs are still difficult to estimate and are poorly characterized. Here, an overview of the publicly available knowledge about IDRs is reported, including manually curated resources, deposition databases and prediction repositories. The types, scopes and availability of the various resources are analyzed, and their complementarity and overlap are highlighted. The volume of information included and the relevance to the field of structural biology are compared., (open access.)

Hand hygiene, social distancing, and face covering are considered the first protection against Coronavirus spreading. The high demand during the COVID-19 emergency has driven a frenetic production and marketing of hand sanitizer gels. Nevertheless, the effect of the gelling agent and its amount on the effectiveness of alcohol-based hand sanitizers (ABHSs) needs to be clarified. We presented a systematic study on the effect of the characteristics and concentration of the most employed excipients on the properties and antimicrobial activity of ABHSs. Three different gelling agents, carbopol, hydroxypropylmethylcellulose (HPMC), and hydroxyethylcellulose (HEC), at four different concentrations were used to prepare ABHSs. Viscosity, spreadability, delivery from commercial dispensers, evaporation rate, rubbing time, and hand distribution of the ABHSs were then explored. Biocidal activity of selected ABHSs was evaluated in vitro on ATCC and clinical strains. The studied ABHS can be considered bioactive and comfortable. Nevertheless, the cellulose polymers and ethanol interactions led to a slight but significant reduction in the biocidal activity compared with carbopol-based formulations. Our results underline the importance of the gelling agent properties and support the choice of carbopol as one of the best thickener agents in ABHS formulations.

3D-printed hydrogels are particularly advantageous as drug-delivery platforms but their loading with water-soluble active compounds remains a challenge requiring the development of innovative inks. Here, we propose a new 3D extrusion-based approach that, by exploiting the internal gelation of the alginate, avoids the post-printing crosslinking process and allows the loading of epirubicin-HCl (EPI). The critical combinations of alginate, calcium carbonate and d-glucono-δ-lactone (GDL) combined with the scaffold production parameters (extrusion time, temperature, and curing time) were evaluated and discussed. The internal gelation in tandem with 3D extrusion allowed the preparation of alginate hydrogels with a complex shape and good handling properties. The dispersion of epirubicin-HCl in the hydrogel matrix confirmed the potential of this self-crosslinking alginate-based ink for the preparation of 3D-printed drug-delivery platforms. Drug release from 3D-printed hydrogels was monitored, and the cytotoxic activity was tested against MCF-7 cells. Finally, the change in the expression pattern of anti-apoptotic, pro-apoptotic, and autophagy protein markers was monitored by liquid-chromatography tandem-mass-spectrometry after exposure of MCF-7 to the EPI-loaded hydrogels.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Evidence and Conclusion Ontology (ECO) is a community resource that provides an ontology of terms used to capture the type of evidence that supports biomedical annotations and assertions. Consistent capture of evidence information with ECO allows tracking of annotation provenance, establishment of quality control measures, and evidence-based data mining. ECO is in use by dozens of data repositories and resources with both specific and general areas of focus. ECO is continually being expanded and enhanced in response to user requests as well as our aim to adhere to community best-practices for ontology development. The ECO support team engages in multiple collaborations with other ontologies and annotating groups. Here we report on recent updates to the ECO ontology itself as well as associated resources that are available through this project. ECO project products are freely available for download from the project website (https://evidenceontology.org/) and GitHub (https://github.com/evidenceontology/evidenceontology). ECO is released into the public domain under a CC0 1.0 Universal license., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nowadays, the clinical administration of siRNA therapeutics is still challenging due to the need of safe and efficient delivery carriers. In this context, biodegradable and amphiphilic triblock copolymers (ABC) containing amine-based cationic segments could be a powerful tool for siRNA delivery. Herein, we propose a range of poly(ethylene glycol) (PEG)-poly(2-dimethyl(aminoethyl) methacrylate) (pDMAEMA)-polycaprolactone (PCL) copolymers with different lengths of the blocks and hydrophilic/lipophilic balance to deliver siRNA alone or in association with a conventional anticancer drug. mPEG-pDMAEMA-PCL copolymers were synthesized by a combination of techniques and characterized by NMR analysis, Fourier transform infrared (FTIR) spectroscopy, gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). Copolymers were then employed to prepare NPs through nanoprecipitation. NPs based on copolymers with long PCL chains (SSL-NPs and LLL-NPs) showed the best colloidal properties and a highly stable core-shell structure with a better orientation of the PEG fringe on the surface. Concerning siRNA delivery, SSL-NPs based on copolymers with short PEG and pDMAEMA chains showed optimized ability to complex and then deliver siRNA at the cell level. The strong interaction between the nucleic acid and the cationic pDMAEMA blocks of NPs was then confirmed by release studies that showed a sustained release of siRNA within 48 h. The transfection efficiency of NPs was assessed in human melanoma cells. NPs were complexed with a therapeutic siRNA against TUBB3 (TUB-siRNA). We observed the best results with SSL-NPs, probably due to the higher preserved buffer capacity of the pDMAEMA blocks. Finally, in order to give a proof of concept of a possible application in the combined chemo/gene-therapy of cancer, SSL-NPs complexed with TUB-siRNA were loaded with docetaxel (DTX) and then cytotoxicity was evaluated in the same cell line. The co-delivery of TUB-siRNA into NPs appeared to strongly potentiate the anti-proliferative activity of DTX, thus highlighting the combinatory activity of the NPs.

Bioresponsive nanoparticles (NPs) are of interest for anticancer nanomedicines, owing to the possibility to 'design in' selective modulation of drug release at target sites. Here we describe the double emulsion formulation of redox-responsive NPs based on modified polyethylene glycol (PEG)-co-poly(lactic-co-glycolic acid) (PLGA) block copolymers and oligo (β-aminoesters) (OBAE), both of which contained disulfide linkages, for the co-delivery of a cytotoxic small molecule drug and a nucleic acid. In particular, we focused our attention on docetaxel (DTX) and a siRNA against TUBB3, a gene that encodes for βIII-tubulin, in order to have a synergistic effect in the treatment of lung cancer. Spherical NPs of around 150 nm with negative zeta potential and high loading efficiencies of both drugs were obtained. Stability and release studies showed "on demand" drug release under reducing conditions. Unloaded NPs containing PEG-disulfide-PLGA and OBAE were well-tolerated by lung cancer cells, thus masking the intrinsic cytotoxicity of OBAE, while for intracellular siRNA delivery, redox responsive NPs demonstrated a higher cell internalization with a preferential cytoplasmic accumulation of siRNA, with a subsequent fast gene-silencing efficiency. The viability of cells treated with combined DTX/TUBB3-siRNA NPs significantly decreased as compared to NPs loaded only with DTX, thus showing an efficient combined anticancer effect, due to a substantial reduction of β-tubulin expression. Finally, in an in vivo feasibility study employing an orthotopic lung cancer model, NPs formulated with an anti-luciferase siRNA distributed throughout the lungs following oro-tracheal administration, and demonstrated effective gene knockdown and no apparent cytotoxicity. Taken together, these results show that the double emulsion formulated redox responsive PEG-PLGA and OBAE systems represent a promising new therapeutic approach for the local combined chemo- and gene-therapy of lung cancer.

The Protein Ensemble Database (PED; https://proteinensemble.org/) is the major repository of conformational ensembles of intrinsically disordered proteins (IDPs). Conformational ensembles of IDPs are primarily provided by their authors or occasionally collected from literature, and are subsequently deposited in PED along with the corresponding structured, manually curated metadata. The modeling of conformational ensembles usually relies on experimental data from small-angle X-ray scattering (SAXS), fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics (MD) simulations, or a combination of these techniques. The growing number of scientific studies based on these data, along with the astounding and swift progress in the field of protein intrinsic disorder, has required a significant update and upgrade of PED, first published in 2014. To this end, the database was entirely renewed in 2020 and now has a dedicated team of biocurators providing manually curated descriptions of the methods and conditions applied to generate the conformational ensembles and for checking consistency of the data. Here, we present a detailed description on how to explore PED with its protein pages and experimental pages, and how to interpret entries of conformational ensembles. We describe how to efficiently search conformational ensembles deposited in PED by means of its web interface and API. We demonstrate how to make sense of the PED protein page and its associated experimental entry pages with reference to the yeast Sic1 use case. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Performing a search in PED Support Protocol 1: Programmatic access with the PED API Basic Protocol 2: Interpreting the protein page and the experimental entry page-the Sic1 use case Support Protocol 2: Downloading options Support Protocol 3: Understanding the validation report-the Sic1 use case Basic Protocol 3: Submitting new conformational ensembles to PED Basic Protocol 4: Providing feedback in PED., (© 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Introduction : Years of tissue engineering research have clearly demonstrated the potential of integrating growth factors (GFs) into scaffolds for tissue regeneration, a concept that has recently been applied to wound dressings. The old concept of wound dressings that only take a passive role in wound healing has now been overtaken, and advanced dressings which can take an active part in wound healing, are of current research interest. Areas covered : In this review we will focus on the recent strategies for the delivery of GFs to wound sites with an emphasis on the different approaches used to achieve fine tuning of spatial and temporal concentrations to achieve therapeutic efficacy. Expert opinion : The use of GFs to accelerate wound healing and reduce scar formation is now considered a feasible therapeutic approach in patients with a high risk of infections and complications. The integration of micro - and nanotechnologies into wound dressings could be the key to overcome the inherent instability of GFs and offer adequate control over the release rate. Many investigations have led to encouraging outcomes in various in vitro and in vivo wound models, and it is expected that some of these technologies will satisfy clinical needs and will enter commercialization.

APICURON is an open and freely accessible resource that tracks and credits the work of biocurators across multiple participating knowledgebases. Biocuration is essential to extract knowledge from research data and make it available in a structured and standardized way to the scientific community. However, processing biological data-mainly from literature-requires a huge effort that is difficult to attribute and quantify. APICURON collects biocuration events from third-party resources and aggregates this information, spotlighting biocurator contributions. APICURON promotes biocurator engagement implementing gamification concepts like badges, medals and leaderboards and at the same time provides a monitoring service for registered resources and for biocurators themselves. APICURON adopts a data model that is flexible enough to represent and track the majority of biocuration activities. Biocurators are identified through their Open Researcher and Contributor ID. The definition of curation events, scoring systems and rules for assigning badges and medals are resource-specific and easily customizable. Registered resources can transfer curation activities on the fly through a secure and robust Application Programming Interface (API). Here, we show how simple and effective it is to connect a resource to APICURON, describing the DisProt database of intrinsically disordered proteins as a use case. We believe APICURON will provide biological knowledgebases with a service to recognize and credit the effort of their biocurators, monitor their activity and promote curator engagement. Database URL: https://apicuron.org., (© The Author(s) 2021. Published by Oxford University Press.)

The first wave of the COVID-19 pandemic brought about a broader use of masks by both professionals and the general population. This resulted in a severe worldwide shortage of devices and the need to increase import and activate production of safe and effective surgical masks at the national level. In order to support the demand for testing surgical masks in the Italian context, Universities provided their contribution by setting up laboratories for testing mask performance before releasing products into the national market. This paper reports the effort of seven Italian university laboratories who set up facilities for testing face masks during the emergency period of the COVID-19 pandemic. Measurement set-ups were built, adapting the methods specified in the EN 14683:2019+AC. Data on differential pressure (DP) and bacterial filtration efficiency (BFE) of 120 masks, including different materials and designs, were collected over three months. More than 60% of the masks satisfied requirements for DP and BFE set by the standard. Masks made of nonwoven polypropylene with at least three layers (spunbonded-meltblown-spunbonded) showed the best results, ensuring both good breathability and high filtration efficiency. The majority of the masks created with alternative materials and designs did not comply with both standard requirements, resulting in suitability only as community masks. The effective partnering between universities and industries to meet a public need in an emergency context represented a fruitful example of the so-called university "third-mission".