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In this study, the use of thermal desorption in on-line solid phase extraction coupled with reversed phase liquid chromatography (on-line SPE-LC) was for the first time proposed and demonstrated for the desorption of analytes strongly retained by multiple interaction polymeric sorbents. In detail, this analytical strategy was applied to the on-line SPE-LC targeted analysis of a model set of 34 human gut metabolites characterized by heterogeneous physicochemical properties (i.e., octanol-water partition coefficient in the range -0.3 - 3.4). The novel thermally assisted on-line SPE approach was investigated in comparison to conventional room temperature desorption strategies based on the use of (i) an optimized elution gradient or (ii) organic desorption followed by post-cartridge dilution. The thermally assisted desorption strategy has been shown to be better performing and suitable for the development of a reliable and sensitive method for the analysis of the model group of analytes in urine and serum. In more detail, under the optimized experimental conditions, the proposed method provided negligible matrix effects in both biofluids for almost all target analytes. Moreover, method quantification limits were in the ranges 0.026-7.2 μg L -1 and 0.033-23 μg L -1 for urine and serum, respectively, i.e., comparable to or lower than those reported in methods previously published., 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 early steps of viral infection involve protein complexes and structural lipid rearrangements which characterize the peculiar strategies of each virus to invade permissive host cells. Members of the human immune-related interferon-induced transmembrane (IFITM) protein family have been described as inhibitors of the entry of a broad range of viruses into the host cells. Recently, it has been shown that SARS-CoV-2 is able to hijack IFITM2 for efficient infection. Here, we report the characterization of a newly generated specific anti-IFITM2 mAb able to impair Spike-mediated internalization of SARS-CoV-2 in host cells and, consequently, to reduce the SARS-CoV-2 cytopathic effects and syncytia formation. Furthermore, the anti-IFITM2 mAb reduced HSVs- and RSV-dependent cytopathic effects, suggesting that the IFITM2-mediated mechanism of host cell invasion might be shared with other viruses besides SARS-CoV-2. These results show the specific role of IFITM2 in mediating viral entry into the host cell and its candidacy as a cell target for antiviral therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shareholders of the academic spin-off FIBROSYS s.r.l., which has filed a patent application relating to this work, include AR, MDM, MCT, VDL and LM. The other authors have nothing to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Quality by Design was adopted for developing an effective extraction procedure of (poly)phenolic compounds from bilberry (Vaccinium myrtillus L.) fruits, using a pooled sample of berries from different regions of Ukraine. Mechanical solvent extraction, microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) were investigated by screening matrices. Extraction time (Time, from 5 to 15 min), organic solvent type (OS type, methanol, ethanol and acetone), organic solvent percentage (OS%, from 50% to 90%), sample/extractant ratio (S/E ratio, from 0.025 to 0.1 g mL -1 ), and, only for MAE, extraction temperature (T, from 30 to 60°C), were selected as critical method parameters (CMPs). The spectrophotometric assays total soluble polyphenols (TSP), total monomeric anthocyanins (TMA), and radical scavenging activity (evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), the 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid), and the ferric reducing antioxidant power methods) were chosen as critical method attributes (CMAs). The screening procedure allowed for selecting UAE and methanol, while the other CMPs underwent further optimization through Response Surface Methodology. Target values for TSP, TMA and DPPH were selected and the method operable design region (MODR) was defined by means of Monte-Carlo simulations. The optimized conditions, with the corresponding MODR intervals in bracket, were the following: (i) Time, 17 min (15-23 min); OS%, 56% (44-59%); S/E ratio, 0.030 (0.022-0.034) g mL -1 . Under these experimental conditions, CMAs values of the pooled sample were the following (n = 3): TSP=4433±176 mg (+)-catechin eq/100 g dry weight (d.w.); TMA=3575±194 mg cyanidin-3-glucoside eq/100 g d.w.; DPPH=273±5 μg DPPH inhib./mg d.w. The optimized extraction method was tested for matrix effect (ME%) in the UHPLC-MS/MS analysis of 15 anthocyanins and 20 non-anthocyanins individual (poly)phenols commonly found in bilberries, as well as for luteolin, sinapic acid, and pelargonidin-3-glucoside, absent in this fruit and therefore added to the extracts as surrogate standards for evaluating apparent recovery (AR%). |ME%| was in any case ≤ 23% and AR% of the surrogate standards in the range 91-95%, confirming the very good performances of the optimized extraction method., 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.)

Background: BAG3 was identified as a co-chaperone of the heat shock protein (Hsp) 70, which helps, through the binding to the ATPase domain, the ADP release from the chaperone and the nucleotide cycling. By interacting with Hsp70, BAG3 modulates the activities of this chaperone, including the delivery of client proteins to proteasome. BAG3 can also carry out Hsp70- independent functions, through its interactions with other proteins involved in apoptosis, cytoskeleton dynamics and other pathways., Scope of Review: Here we provide a summary of the main mechanisms which encompass BAG3 as an intracellular factor involved in different pathways which regulate and modulate the physiological cell response. Furthermore, it has been shown that BAG3 can be secreted by some cell types and is able to activate the monocytes through the binding on a membrane cell receptor, indicating that the protein can act like an alarmin with different functions inside and outside the cell., Major Conclusions: Whereas intracellularly BAG3 sustains the levels of anti-apoptotic factors and other molecules, participates in protein quality control, drives the cytoskeleton dynamics, exerts structural and functional roles in myocytes, the discovery of a secreted BAG3 opened a new field of investigation in tumor development and progression, revealing its role in a new signaling pathway, mediated by the BAG3/BAG3R axis, which also includes monocytes and other stromal cells., General Significance: BAG3 is a multifunctional protein that is involved cell stress response through its participation in several regulatory pathways which control cell homeostatic response in physiological and pathological conditions., 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 © 2020 Elsevier B.V. All rights reserved.)

Trefoil Factor 1 belongs to a group of small secreted proteins (the Trefoil Factor Family proteins), that are localized within the mucous granules and are expressed and secreted by epithelial cells that line mucous membranes. Trefoil factors are mainly expressed in the gastrointestinal tract, where they normally contribute to maintain the integrity of the mucosa. We recently demonstrated a selective binding ability of Trefoil Factor 1 for copper ions, through its carboxy-terminal tail, and we also observed that copper levels influence the equilibrium between the monomeric and homodimeric forms of Trefoil Factor 1, thus modulating its biological activity. Here we report that transcriptional regulation of Trefoil Factor 1 is also affected by copper levels, through the modulated binding of the copper-sensing transcription factor Sp1 onto the responsive elements present in the regulatory region of the gene. In addition we demonstrate that copper overload causes an accumulation of the trefoil protein in the Trans-Golgi Network and that Trefoil Factor 1 levels can influence copper excretion and copper related toxicity. These findings suggest that the protein might play a role in the overall complex mechanisms of copper homeostasis in the gastrointestinal tissues., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-gamma and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT., (2009 Elsevier Ltd. All rights reserved.)

Stress-induced apoptosis regulates neoplasia pathogenesis and response to therapy. Indeed, cell transformation induces a stress response, that is overcome, in neoplastic cells, by alterations in apoptosis modulators; on the other hand, antineoplastic therapies largely trigger the apoptosis stress pathway, whose impairment results in resistance. Therefore, the study of the roles of apoptosis-modulating molecules in neoplasia development and response to therapy is of key relevance for our understanding of these processes. Among molecules that regulate apoptosis, a role is emerging for BAG3, a member of the BAG co-chaperone protein family. Proteins that share the BAG domain are characterized by their interaction with a variety of partners (heat shock proteins, steroid hormone receptors, Raf-1 and others), involved in regulating a number of cellular processes, including proliferation and apoptosis. BAG3, also known as CAIR-1 or Bis, forms a complex with the heat shock protein (Hsp) 70. This assists polypeptide folding, can mediate protein delivery to proteasome and is able to modulate apoptosis by interfering with cytochrome c release, apoptosome assembly and other events in the death process. It has been recently shown that, in human primary lymphoid and myeloblastic leukemias and other neoplastic cell types, BAG3 expression sustains cell survival and underlies resistance to therapy, through downmodulation of apoptosis. This review summarizes findings that assign an apoptotic role to BAG3 in some neoplastic cell types and identify the protein as a candidate target of therapy.

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes., (Copyright 1999 Academic Press.)

A new procedure was developed for enzyme immobilization by entrapment in copper alginate gel. The mechanical properties of the copper alginate gel were characterized and compared with those of the most widely used calcium alginate. The system was applied to the immobilization of a fungal phenol oxidase. Optimal conditions for enzyme immobilization were set up: the system immobilized 85% of the enzyme, and the remaining 15% was recovered in the aqueous immobilization medium. The stability and activity of the immobilized enzyme were studied. After immobilization, the enzyme was active in a wider pH range, the temperature of its optimal activity was shifted to lower values, and the possibility of storage at 4 degrees C was greatly improved. The immobilized enzyme generally increased the rate of oxidation of various substrates. The results indicate a potential use of this system for the construction of bioreactors to be used in the detoxification of polluted waste waters.