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Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type–containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

Silicon nanowires are held and manipulated in controlled optical traps based on counter-propagating beams focused by low numerical aperture lenses. The double-beam configuration compensates light scattering forces enabling an in-depth investigation of the rich dynamics of trapped nanowires that are prone to both optical and hydrodynamic interactions. Several polarization configurations are used, allowing the observation of optical binding with different stable structure as well as the transfer of spin and orbital momentum of light to the trapped silicon nanowires. Accurate modeling based on Brownian dynamics simulations with appropriate optical and hydrodynamic coupling confirms that this rich scenario is crucially dependent on the non-spherical shape of the nanowires. Such an increased level of optical control of multiparticle structure and dynamics open perspectives for nanofluidics and multi-component light-driven nanomachines.

The pandemic outbreak caused by SARS‐CoV‐2 coronavirus brought a crucial issue in public health causing up to now more than 600 million infected people and 6.5 million deaths. Conventional diagnostic methods are based on quantitative reverse transcription polymerase chain reaction (RT‐qPCR assay) and immuno‐detection (ELISA assay). However, despite these techniques have the advantages of being standardized and consolidated, they keep some main limitations in terms of accuracy (immunoassays), time/cost consumption of analysis, the need for qualified personnel, and lab constrain (molecular assays). There is crucial the need to develop new diagnostic approaches for accurate, fast and portable viral detection and quantification. Among these, PCR‐free biosensors represent the most appealing solution since they can allow molecular detection without the complexity of the PCR. This will enable the possibility to be integrated in portable and low‐cost systems for massive and decentralized screening of SARS‐CoV‐2 in a point‐of‐care (PoC) format, pointing to achieve a performant identification and control of infection. In this review, the most recent approaches for the SARS‐CoV‐2 PCR‐free detection are reported, describing both the instrumental and methodological features, and highlighting their suitability for a PoC application. PCR‐free biosensors for SARS‐CoV‐2 detection are characterized by a high degree of technological integration and miniaturization, proposing device on which the viral sample can be directly processed and detected in a simple and fast way. This allows the viral infections analysis to be decentralized, addressing the growing need of massive outdoor screening in a PoC format.

ABSTRACTPost-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24–39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.

Increasing attention is being given to lipid nanocarriers (LNs) as drug delivery systems, due to the advantages offered of a higher biocompatibility and lower toxicity compared with polymeric nanoparticles. Many administration routes are being investigated for LNs, including topical, oral and parenteral ones. LNs are also proposed for specific applications such as cancer treatment, gene therapy, diagnosis and medical devices production. However, the high number of published research articles does not match an equal amount of patents. A recent Review of ours, published in Pharmaceutical Patent Analyst, reported the patents proposing novel methods for the production of LNs. This review work discusses recent patents, filed in 2007–2013 and dealing with the industrial applications of lipid-based nanocarriers for the vectorization of therapeutically relevant molecules, as well as biotech products such as proteins, gene material and vaccines, in the pharmaceutical, diagnostic and biomedical areas.

Increasing attention is being given to lipid nanocarriers (LNs) as drug delivery systems, due to the advantages offered of a higher biocompatibility and lower toxicity compared with polymeric nanoparticles. Many administration routes are being investigated for LNs, including topical, oral and parenteral ones. LNs are also proposed for specific applications such as cancer treatment, gene therapy, diagnosis and medical devices production. However, the high number of published research articles does not match an equal amount of patents. A recent Review of ours, published in Pharmaceutical Patent Analyst, reported the patents proposing novel methods for the production of LNs. This review work discusses recent patents, filed in 2007–2013 and dealing with the industrial applications of lipid-based nanocarriers for the vectorization of therapeutically relevant molecules, as well as biotech products such as proteins, gene material and vaccines, in the pharmaceutical, diagnostic and biomedical areas.

Among the colloidal vectors proposed for the controlled delivery and targeting of drugs and other biologically active compounds, lipid-based nanocarriers are acquiring an increasing role due to a number of peculiar technological and physical features. Solid lipid nanoparticles, lipid nanocapsules, nanostructured lipid carriers, and drug–lipid conjugates are all examples of how it can be possible to combine the properties of the more acknowledged liposomal systems, such as biocompatibility and biodegradability, with the stability and compositional flexibility, distinctive of polymeric nanosystems. This article introduces recent patents, filed in years 2007–2013, that deal with novel or amended methods of production of the various types of lipid-based nanocarriers. Although a significant gap still remains between basic research and patenting activity in this field, many of the proposed methods can attain an industrial value. Furthermore, the critical analysis of these patents further supports the position that a general revision of patenting systems at an international level would be necessary for nanosized pharmaceutical systems.

Among the colloidal vectors proposed for the controlled delivery and targeting of drugs and other biologically active compounds, lipid-based nanocarriers are acquiring an increasing role due to a number of peculiar technological and physical features. Solid lipid nanoparticles, lipid nanocapsules, nanostructured lipid carriers, and drug–lipid conjugates are all examples of how it can be possible to combine the properties of the more acknowledged liposomal systems, such as biocompatibility and biodegradability, with the stability and compositional flexibility, distinctive of polymeric nanosystems. This article introduces recent patents, filed in years 2007–2013, that deal with novel or amended methods of production of the various types of lipid-based nanocarriers. Although a significant gap still remains between basic research and patenting activity in this field, many of the proposed methods can attain an industrial value. Furthermore, the critical analysis of these patents further supports the position that a general revision of patenting systems at an international level would be necessary for nanosized pharmaceutical systems.

The induction of senescence in tumor cells impairs transformation and promotes an anticancer immune response resulting from the production by senescent cells of cytokines and chemokines, an aspect known as “senescence-associated secretory phenotype” (SASP). Here we discuss recent findings regarding the role of NFκB in the modulation of the SASP and the consequent anticancer immune response.

CONTEXT: Micro-RNAs (miRNAs) have been recently involved in the modulation of several biological activities including cancer. Many human tumors show deregulated expression of miRNAs targeting oncogenes and/or tumor suppressors, thus identifying miRNAs as new molecular targets for cancer therapy. OBJECTIVES: Nuclear factor (NF)-B is strongly activated in human anaplastic thyroid carcinomas (ATCs). Because the regulation of miRNA expression is under control of RNA polymerase II-dependent transcription factors, we stably inactivated NF-B in the ATC-derived FRO cell line and analyzed its miRNA profile in comparison with the parental counterpart by using a miRNA chip microarray. RESULTS: The analysis revealed that a number of miRNAs were differentially expressed in the two cell lines. Among others, the miR-146a showed a strong down-regulation that was confirmed by quantitative real time RT-PCR. The expression of miR-146a was almost undetectable in mouse embryonic fibroblasts isolated from the RelA knockout mice and was restored after reexpression of RelA, thus indicating that miR-146a transcription was controlled by NF-B. The inhibition of miR-146a expression in FRO cells decreased their oncogenic potential and increased the susceptibility to chemotherapeutic drug-induced apoptosis. No difference was found in the growth rate between untransfected and miR-146a-null FRO cells. Importantly, the miR-146a resulted in overexpression of human ATC specimens compared with the normal thyroid tissue. CONCLUSIONS: Our results show that NF-B contributes to anaplastic thyroid cancer up-regulating the expression of miR-146a.

Activation of transcription factors such as NF-κB occurs through signaling pathways involving sequential phosphorylation of a variety of substrates by distinct kinases. Proper assemby and activation of these kinases require interaction with non-enzymatic and essential partners named scaffold proteins. Here, we describe how the NF-κB activating scaffold proteins involved in the signaling pathways triggered by the proinflammatory cytokines TNFα, IL-1β and by the CD40 ligand play such roles. We also illustrate the human genetic diseases that are linked to mutations affecting genes coding for these proteins. We suggest that these scaffold proteins may be specifically targeted by novel therapeutical agents for the treatment of inflammation or cancers.

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca2+ from the ER elicited by thapsigargin (a specific inhibitor of ER Ca2+-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca2+ strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.

Canonical activation of NF-κB is mediated via phosphorylation of the inhibitory IκB proteins by the IκB kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKKα and IKKβ subunits and a presumed regulatory protein termed NEMO (NF-κB essential modulator) or IKKγ. NEMO/IKKγ is indispensable for activation of the IKKs in response to many signals, but its mechanism of action remains unclear. Here we identify TANK (TRAF family member-associated NF-κB activator) as a NEMO/IKKγ-interacting protein via yeast two-hybrid analyses. This interaction is confirmed in mammalian cells, and the domains required are mapped. TANK was previously shown to assist NF-κB activation in a complex with TANK-binding kinase 1 (TBK1) or IKKε, two kinases distantly related to IKKα/β, but the underlying mechanisms remained unknown. Here we show that TBK1 and IKKε synergize with TANK to promote interaction with the IKKs. The TANK binding domain within NEMO/IKKγ is required for proper functioning of this IKK subunit. These results indicate that TANK can synergize with IKKε or TBK1 to link them to IKK complexes, where the two kinases may modulate aspects of NF-κB activation.

In the biomedical field, the demand for the development of broad-spectrum biomaterials able to inhibit bacterial growth is constantly increasing. Chronic infections represent the most serious and devastating complication related to the use of biomaterials. This is particularly relevant in the orthopaedic field, where infections can lead to implant loosening, arthrodesis, amputations and sometimes death. Antibiotics are the conventional approach for implanted-associated infections, but they have the limitation of increasing antibiotic resistance, a critical worldwide healthcare issue. In this context, the development of anti-infective biomaterials and infection-resistant surfaces can be considered the more effective strategy to prevent the implant colonisation and biofilm formation by bacteria, so reducing the occurrence of implant-associated infections. In the last years, inorganic nanostructures have become extremely appealing for chemical modifications or coatings of Ti surfaces, since they do not generate antibiotic resistance issues and are featured by superior stability, durability, and full compatibility with the sterilization process. In this work, we present a simple, rapid, and cheap chemical nanofunctionalization of titanium (Ti) scaffolds with colloidal ZnO and Mn-doped ZnO nanoparticles (NPs), prepared by a sol-gel method, exhibiting antibacterial activity. ZnO NPs and ZnxMn(1−x)O NPs formation with a size around 10–20nm and band gap values of 3.42 eV and 3.38 eV, respectively, have been displayed by characterization studies. UV–Vis, fluorescence, and Raman investigation suggested that Mn ions acting as dopants in the ZnO lattice. Ti scaffolds have been functionalized through dip coating, obtaining ZnO@Ti and ZnxMn(1−x)O@Ti biomaterials characterized by a continuous nanostructured film. ZnO@Ti and ZnxMn(1−x)O@Ti displayed an enhanced antibacterial activity against both Gram-positive Staphylococcus aureus(S. aureus) and Gram-negative Pseudomonas aeruginosa(P. aeruginosa) bacterial strains, compared to NPs in solution with better performance of ZnxMn(1−x)O@Ti respect to ZnO@Ti. Notably, it has been observed that ZnxMn(1−x)O@Ti scaffolds reach a complete eradication for S. aureusand 90 % of reduction for P. aeruginosa. This can be attributed to Zn2+and Mn2+metal ions release (as observed by ICP MS experiments) that is also maintained over time (72 h). To the best of our knowledge, this is the first study reported in the literature describing ZnO and Mn-doped ZnO NPs nanofunctionalized Ti scaffolds with improved antibacterial performance, paving the way for the realization of new hybrid implantable devices through a low-cost process, compatible with the biotechnological industrial chain method.

The conformational stability of TTF‐1HD has been determined by CD‐monitored thermal denaturation and isothermal urea unfolding studies. The Gibbs free energy of stabilization found are 1.44 and 1.26 kcal·mol−1, respectively. TTF‐1HD exhibits a Tmof 42°C and a δCpof 80 cal·mol−1·K−1indicating that TTF‐1HD, when free in solution, is a mobile flexible segment folded into loose helices. Such a flexibility would be relevant for the DNA‐binding function of this homeodomain. In fact, a small reduction of the α‐helical content of TTF‐1HD significally modifies its DNA‐binding activity.

The conformational stability of TTF-1HD has been determined by CD-monitored thermal denaturation and isothermal urea unfolding studies. The Gibbs free energy of stabilization found are 1.44 and 1.26 kcal·mol −1, respectively. TTF-1HD exhibits a Tmof 42°C and a δ Cpof 80 cal·mol −1·K −1indicating that TTF-1HD, when free in solution, is a mobile flexible segment folded into loose helices. Such a flexibility would be relevant for the DNA-binding function of this homeodomain. In fact, a small reduction of the α-helical content of TTF-1HD significally modifies its DNA-binding activity.

The 1H NMR solution structure of the rat thyroidtranscription factor 1 homeodomain (TTF-1 HD) showed that the molecule foldslike classical homeodomains. The C-terminal extension of helix III (fragment51–59) appeared to adopt a helical geometry, albeit not as rigid asthe preceding portion, but the hydrogen–deuterium exchange of backboneamides and the NOE data provided evidence of a discontinuity between the twomoieties of helix III at the highly conserved fragmentAsn51–His52–Arg53.Analysis of quantitative measurements of isotope exchange rates allows oneto recognize the general occurrence, in that region of HD motifs, ofopposite effects to helix III stability. Asparagine, histidine and arginineresidues occur most frequently at the beginning and end of protein helices.In TTF-1 HD a local fluctuation is observed in the fragment 51–53which either kinks or tightens the α-helix. A search through theprotein structure database reveals that the three most common variants of HDfragments 51–53 are often involved in helices and, frequently, inhelix initiation or termination. For homeodomains in general, the nature ofthe fragment 51–53 may be related to the conformational dynamics oftheir DNA-recognition helix (helix III). Besides the specific results onfragment 51–53, the complete isotope exchange analysis of TTF-1 HDdata shows that the partially solvent-exposed recognition helix isstabilized by hydrophobic interactions, like most of the structured regionsof the molecule. Hydrophobic stabilization of the contacting regions meetsthe requirements of a DNA-interaction mechanism which, as shown with otherDNA-protein complexes, should entail negative heat capacity variations dueto changes in solvent exposure of the nonpolar protein surface.

p52 is a subunit of nuclear factor (NF)-κB transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the IκB family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.

Tornatore: Kesios Therapeutics Ltd.: Consultancy, Equity Ownership, Honoraria, Patents & Royalties. Adams:Kesios Therapeutics Ltd.: Consultancy. Kelly:Kesios Therapeutics Ltd.: Consultancy. Ruvo:Kesios Therapeutics Ltd.: Equity Ownership, Patents & Royalties. Oakervee:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Schey:Celgene Corporation: Honoraria. Apperley:Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ARIAD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Franzoso:Kesios Therapeutics Ltd.: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

The NF-κB transcription factor pathway is aberrantly activated in multiple myeloma (MM) and many other cancers, where it promotes malignancy by upregulating survival genes, thus providing a compelling rationale for therapeutically targeting this pathway in MM. However, despite aggressive efforts to develop a specific NF-κB or IκB kinase (IKK)β inhibitor, no such inhibitor has been approved, due to the preclusive toxicities associated with the general suppression of NF-κB. As a key pathogenetic activity of NF-κB in MM is to block apoptosis through the induction of target genes, an attractive alternative to globally inhibiting NF-κB would be to therapeutically target the non-redundant, cancer-specific downstream effectors of the NF-κB survival pathway.

The thyroid transcription factor-1 homeodomain (TTF-1HD) shows a peculiar DNA binding specificity, preferentially recognizing sequences containing the 5′-CAAG-3′ core motif. Most other homeodomains instead recognize sites containing the 5-TAAT-3′ core motif. Here, we show that TTF-1HD efficiently recognizes another sequence, called D1, devoid of the 5′-CAAG-3′ core motif. Different experimental approaches indicate that TTF-1HD contacts the D1 sequence in a manner which is different to that used to interact with sequences containing the 5′-CAAG-3′ core motif. The binding activities that mutants of TTF-1HD display with the D1 sequence or with the sequence containing the 5′-CAAG-3′ core motif indicate that the role of several DNA-contacting amino acids is different. In particular, during recognition of the D1 sequence, backbone-interacting amino acids not relevant in binding to sequences containing the 5′-CAAG-3′ core motif play an important role. In the TTF-1HD, therefore, the contribution of several amino acids to DNA recognition depends on the bound sequence. These data indicate that although a common bonding network exists in all of the HD/DNA complexes, peculiarities important for DNA recognition may occur in single cases.