Recombinant humanized Fab fragments targeting the CFC domain of human Cripto-1.

In the era of immunotherapy, the targeting of disease-specific biomarkers goes hand in hand with the development of highly selective antibody-based reagents having optimal pharmacological/toxicological profiles. One interesting and debated biomaker for several types of cancers is the onco-fetal protein Cripto-1 that is selectively expressed in many solid tumours and has been actively investigated as potential theranostic target. Starting from previously described anti-CFC/Cripto-1 murine monoclonal antibodies, we have moved forward to prepare the humanized recombinant Fabs which have been engineered so as to bear an MTGase site useful for a one-step site-specific labelling. The purified and bioconjugated molecules have been extensively characterized and tested on Cripto-1-positive cancer cells through in vitro binding assays. These recombinant Fab fragments recognize the target antigen in its native form on intact cells suggesting that they can be further developed as reagents for detecting Cripto-1 in theranostic settings.

Glutamine Metabolism in Cancer Stem Cells: A Complex Liaison in the Tumor Microenvironment.

In this review we focus on the role of glutamine in control of cancer stem cell (CSC) fate. We first provide an overview of glutamine metabolism, and then summarize relevant studies investigating how glutamine metabolism modulates the CSC compartment, concentrating on solid tumors. We schematically describe how glutamine in CSC contributes to several metabolic pathways, such as redox metabolic pathways, ATP production, non-essential aminoacids and nucleotides biosynthesis, and ammonia production. Furthermore, we show that glutamine metabolism is a key regulator of epigenetic modifications in CSC. Finally, we briefly discuss how cancer-associated fibroblasts, adipocytes, and senescent cells in the tumor microenvironment may indirectly influence CSC fate by modulating glutamine availability. We aim to highlight the complexity of glutamine's role in CSC, which supports our knowledge about metabolic heterogeneity within the CSC population.

Alkoxysilane-Mediated Decoration of Si Nanowires Vertical Arrays with Au Nanoparticles as Improved SERS-Active Platforms.

The search for improved transducers to fabricate better-performing (bio)sensors is a challenging but rewarding endeavor aiming to better diagnose and treat diseases. In this paper, we report on the decoration of a dense vertical array of ultrathin silicon nanowires (Si NWs), produced by metal-assisted chemical etching, with 20 nm gold nanoparticles (Au NPs) for surface-enhanced Raman scattering (SERS) applications. To optimize the production of a uniform 3D SERS active platform, we tested different Si NW surface functionalizations with various alkoxysilanes before Au decoration. Scanning electron microscopy investigations confirm that Au NPs decorate both bare and (3-glycidiloxypropyl)trimethoxysilane (GPTMS)-modified Si NWs with a high surface coverage uniformity. The SERS response of the decorated NWs was probed using a model dye system (methylene blue; MB) at 633 and 785 nm excitation wavelengths. The GPTMS-modified NWs present the highest enhancements of 2.9 and 2.6 for the 450 cm-1 and 1625 cm-1 peaks under 785 nm excitation and of 10.8 and 5.3 for the 450 cm-1 and 1625 cm-1 peaks under 633 nm excitation. These results demonstrate the perspective role of Si NWs decorated with Au NPs as a low-cost 3D SERS platform.

Luminescent Silicon Nanowires as Novel Sensor for Environmental Air Quality Control.

Air quality monitoring is an increasingly debated topic nowadays. The increasing spillage of waste products released into the environment has contributed to the increase in air pollution. Consequently, the production of increasingly performing devices in air monitoring is increasingly in demand. In this scenario, the attention dedicated to workplace safety monitoring has led to the developing and improving of new sensors. Despite technological advancements, sensors based on nanostructured materials are difficult to introduce into the manufacturing flow due to the high costs of the processes and the approaches that are incompatible with the microelectronics industry. The synthesis of a low-cost ultra-thin silicon nanowires (Si NWs)-based sensor is here reported, which allows us the detection of various dangerous gases such as acetone, ethanol, and the ammonia test as a proof of concept in a nitrogen-based mixture. A modified metal-assisted chemical etching (MACE) approach enables to obtain ultra-thin Si NWs by a cost-effective, rapid and industrially compatible process that exhibit an intense light emission at room temperature. All these gases are common substances that we find not only in research or industrial laboratories, but also in our daily life and can pose a serious danger to health, even at small concentrations of a few ppm. The exploitation of the Si NWs optical and electrical properties for the detection of low concentrations of these gases through their photoluminescence and resistance changes will be shown in a nitrogen-based gas mixture. These sensing platforms give fast and reversible responses with both optical and electrical transductions. These high performances and the scalable synthesis of Si NWs could pave the way for market-competitive sensors for ambient air quality monitoring.

Hybrid Platforms of Silicon Nanowires and Carbon Nanotubes in an Ionic Liquid Bucky Gel.

Silicon nanowires (NWs) are appealing building blocks for low-cost novel concept devices with improved performances. In this research paper, we realized a hybrid platform combining an array of vertically oriented Si NWs with different types of bucky gels, obtained from carbon nanotubes (CNT) dispersed into an ionic liquid (IL) matrix. Three types of CNT bucky gels were obtained from imidazolium-based ionic liquids (BMIM-I, BIMI-BF4, and BMIM-Tf2N) and semiconductive CNTs, whose structural and optical responses to the hybrid platforms were analyzed and compared. We investigated the electrical response of the IL-CNT/NW hybrid junctions in dark and under illumination for each platform and its correlation to the ionic liquid characteristics and charge mobility. The reported results confirm the attractiveness of such IL-CNT/NW hybrid platforms as novel light-responsive materials for photovoltaic applications. In particular, our best performing cell reported a short-circuit current density of 5.6 mA/cm2 and an open-circuit voltage of 0.53 V.

Electrodeposition of Molybdenum Disulfide (MoS2) Nanoparticles on Monocrystalline Silicon.

Molybdenum disulfide (MoS2) has attracted great attention for its unique chemical and physical properties. The applications of this transition metal dichalcogenide (TMDC) range from supercapacitors to dye-sensitized solar cells, Li-ion batteries and catalysis. This work opens new routes toward the use of electrodeposition as an easy, scalable and cost-effective technique to perform the coupling of Si with molybdenum disulfide. MoS2 deposits were obtained on n-Si (100) electrodes by electrochemical deposition protocols working at room temperature and pressure, as opposed to the traditional vacuum-based techniques. The samples were characterized by X-ray Photoelectron Spectroscopy (XPS), Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Rutherford Back Scattering (RBS).

Estimating asymptomatic SARS-CoV-2 infections in a geographic area of low disease incidence.

BACKGROUND: The SARS-CoV-2 infection has emerged as a rapidly spreading infection. Today it is relatively easy to isolate Covid-19 symptomatic cases, while remains problematic to control the disease spread by infected but symptom-free individuals. The control of this possible path of contagion requires drastic measures of social distancing, which imply the suspension of most activities and generate economic and social issues. This study is aimed at estimating the percentage of asymptomatic SARS-CoV-2 infection in a geographic area with relatively low incidence of Covid-19. METHODS: Blood serum samples from 388 healthy volunteers were analyzed for the presence of anti-SARS-CoV-2 IgG by using an ELISA assay based on recombinant viral nucleocapsid protein. RESULTS: We found that 7 out of 388 healthy volunteers, who declared no symptoms of Covid-19, like fever, cough, fatigue etc., in the preceding 5 months, have bona fide serum anti-SARS-CoV-2 IgG, that is 1.8% of the asymptomatic population (95% confidence interval: 0.69-2.91%). CONCLUSIONS: The estimated range of asymptomatic individuals with anti-SARS-CoV-2 IgG should be between 26,565 and 112, 350. In the same geographic area, there are 4665 symptomatic diagnosed cases.

The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate.

SNAP-tag ® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag ® and its relative thermostable version (SsOGT-H5 ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

NGAL as a Potential Target in Tumor Microenvironment.

The signaling network between cancer and stromal cells plays a crucial role in tumor microenvironment. The fate of tumor progression mainly depends on the huge amount of information that these cell populations exchange from the onset of neoplastic transformation. Interfering with such signaling has been producing exciting results in cancer therapy: just think of anti-PD-1/anti-PD-L1/anti-CTLA-4 antibodies that, acting as immune checkpoint inhibitors, interrupt the inhibitory signaling exerted by cancer cells on immune cells or the CAR-T technology that fosters the reactivation of anti-tumoral immunity in a restricted group of leukemias and lymphomas. Nevertheless, many types of cancers, in particular solid tumors, are still refractory to these treatments, so the identification of novel molecular targets in tumor secretome would benefit from implementation of current anti-cancer therapeutical strategies. Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a secreted protein abundantly expressed in the secretome of various human tumors. It represents a promising target for the multiple roles that are played inside cancer and stromal cells, and also overall in their cross-talk. The review focuses on the different roles of NGAL in tumor microenvironment and in cancer senescence-associated secretory phenotype (SASP), highlighting the most crucial functions that could be eventually targetable in cancer therapy.

New Insights into Cancer Targeted Therapy: Nodal and Cripto-1 as Attractive Candidates.

The transforming growth factor beta (TGF-ß) signaling is fundamental for correct embryonic development. However, alterations of this pathway have been correlated with oncogenesis, tumor progression and sustaining of cancer stem cells (CSCs). Cripto-1 (CR-1) and Nodal are two embryonic proteins involved in TGF-ß signaling. Their expression is almost undetectable in terminally differentiated cells, but they are often re-expressed in tumor cells, especially in CSCs. Moreover, cancer cells that show high levels of CR-1 and/or Nodal display more aggressive phenotypes in vitro, while in vivo their expression correlates with a worse prognosis in several human cancers. The ability to target CSCs still represents an unmet medical need for the complete eradication of certain types of tumors. Given the prognostic role and the selective expression of CR-1 and Nodal on cancer cells, they represent archetypes for targeted therapy. The aim of this review is to clarify the role of CR-1 and Nodal in cancer stem populations and to summarize the current therapeutic strategy to target CSCs using monoclonal antibodies (mAbs) or other molecular tools to interfere with these two proteins.

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry.

BACKGROUND: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb's epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. METHODS: We generated a mAb against PRAME immunizing mice with PRAME fragment 161-415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. RESULTS: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202-212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. CONCLUSIONS: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody's epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

Optical Trapping, Optical Binding, and Rotational Dynamics of Silicon Nanowires in Counter-Propagating Beams.

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.

NF-κB Essential Modulator (NEMO) Is Critical for Thyroid Function.

The I-κB kinase (IKK) subunit NEMO/IKKγ (NEMO) is an adapter molecule that is critical for canonical activation of NF-κB, a pleiotropic transcription factor controlling immunity, differentiation, cell growth, tumorigenesis, and apoptosis. To explore the functional role of canonical NF-κB signaling in thyroid gland differentiation and function, we have generated a murine strain bearing a genetic deletion of the NEMO locus in thyroid. Here we show that thyrocyte-specific NEMO knock-out mice gradually develop hypothyroidism after birth, which leads to reduced body weight and shortened life span. Histological and molecular analysis indicate that absence of NEMO in thyrocytes results in a dramatic loss of the thyroid gland cellularity, associated with down-regulation of thyroid differentiation markers and ongoing apoptosis. Thus, NEMO-dependent signaling is essential for normal thyroid physiology.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

UNLABELLED: The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a ß-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE: Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a ß-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

Structural insights into the interaction of a monoclonal antibody and Nodal peptides by STD-NMR spectroscopy.

Nodal is a growth factor expressed during early embryonic development, but reactivated in several advanced-stage cancers. Targeting of Nodal signaling, which occurs via the binding to Cripto-1 co-receptor, results in inhibition of cell aggressiveness and reduced tumor growth. The Nodal binding region to Cripto-1 was identified and targeted with a high affinity monoclonal antibody (3D1). By STD-NMR technique, we investigated the interaction of Nodal fragments with 3D1 with the aim to elucidate at atomic level the interaction surface. Data indicate with high accuracy the antibody-antigen contact atoms and confirm the information previously obtained by immune-enzymatic methods. Main residues contacted by 3D1 are P46, V47, E49 and E50, which belong to the Nodal loop involved in the interaction with the co-receptor.

CIKS/DDX3X interaction controls the stability of the Zc3h12a mRNA induced by IL-17.

IL-17 is a proinflammatory cytokine that promotes the expression of different cytokines and chemokines via the induction of gene transcription and the posttranscriptional stabilization of mRNAs. In this study, we show that IL-17 increases the half-life of the Zc3h12a mRNA via interaction of the adaptor protein CIKS with the DEAD box protein DDX3X. IL-17 stimulation promotes the formation of a complex between CIKS and DDX3X, and this interaction requires the helicase domain of DDX3X but not its ATPase activity. DDX3X knockdown decreases the IL-17-induced stability of Zc3h12a without affecting the stability of other mRNAs. IKKε, TNFR-associated factor 2, and TNFR-associated factor 5 were also required to mediate the IL-17-induced Zc3h12a stabilization. DDX3X directly binds the Zc3h12a mRNA after IL-17 stimulation. Collectively, our findings define a novel, IL-17-dependent mechanism regulating the stabilization of a selected mRNA.

COMMD7 as a novel NEMO interacting protein involved in the termination of NF-κB signaling.

NEMO/IKKγ is the regulatory subunit of the IκB Kinase (IKK) complex, required for the activation of the NF-κB pathway, which is involved in a variety of key processes, including immunity, inflammation, differentiation, and cell survival. Termination of NF-κB activity on specific -κB responsive genes, which is crucial for the resolution of inflammatory responses, can be achieved by direct degradation of the chromatin-bound NF-κB subunit RelA/p65, a process mediated by a protein complex that contains Copper Metabolism Murr1 Domain 1 (COMMD1). In this study, we identify COMMD7, another member of the COMMDs protein family, as a novel NEMO-interacting protein. We show that COMMD7 exerts an inhibitory effect on NF-κB activation upon TNFα stimulation. COMMD7 interacts with COMMD1 and together they cooperate to down-regulate NF-κB activity. Accordingly, termination of TNFα-induced NF-κB activity on the -κB responsive gene, Icam1, is defective in cells silenced for COMMD7 expression. Furthermore, this impairment is not greatly increased when we silence the expression of both COMMD7 and COMMD1 indicating that the two proteins participate in the same pathway of termination of TNFα-induced NF-κB activity. Importantly, we have demonstrated that COMMD7's binding to NEMO does not interfere with the binding to the IKKs, and that the disruption of the IKK complex through the use of the NBP competitor impairs the termination of NF-κB activity. We propose that an intact IKK complex is required for the termination of NF-κB-dependent transcription and that COMMD7 acts as a scaffold in the IKK-mediated NF-κB termination.

Pregnancy-associated plasma protein-A promotes TF procoagulant activity in human endothelial cells by Akt-NF-κB axis.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase with a controversial role in pathophysiology of cardiovascular disease. It seems involved in progression of atherosclerosis and is widely represented in atherosclerotic plaque. PAPP-A plasma levels are elevated in patients with acute coronary syndromes (ACS), thus it has been suggested that it might be a prognostic marker for developing major cardiovascular events. However, the pathophysiological link(s) between PAPP-A and ACS are still unknown. Several studies have indicated that tissue factor (TF) plays a pivotal role in the pathophysiology of ACS by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates whether PAPP-A, at concentrations measurable in ACS patients, might induce TF expression in human endothelial cells in culture (HUVEC). In HUVEC, PAPP-A induced TF-mRNA transcription as demonstrated by real time PCR and expression of functionally active TF as demonstrated by FACS analysis and pro-coagulant activity assay. PAPP-A induced TF expression through the activation of Akt/NF-κB axis, as demonstrated by luciferase assay and by suppression of TF-mRNA transcription as well as of TF expression/activity by Akt and NF-κB inhibitors. These data indicate that PAPP-A promotes TF expression in human endothelial cells and support the hypothesis that this proteinase, besides being involved in progression of atherosclerosis, does not represent an independent risk factor for adverse cardiovascular events, but it rather might play an "active" role in the pathophysiology of ACS as an effector molecule able to induce a pro-thrombotic phenotype in endothelial cells.

Cytokine expression and ultrastructural alterations in fresh-frozen, freeze-dried and γ-irradiated human amniotic membranes.

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.

Fructose induces prothrombotic phenotype in human endothelial cells : A new role for "added sugar" in cardio-metabolic risk.

Intake of large amounts of added sweeteners has been associated with the pathogenesis of cardiometabolic risk. Several studies have shown that fructose increases the cardiovascular risk by modulating endothelial dysfunction and promoting atherosclerosis. Recently, a potential role for fructose in cardiovascular thrombosis has been suggested but with controversial results. Tissue factor (TF) plays a pivotal role in the pathophysiology of cardiovascular thrombosis by triggering the formation of intracoronary thrombi following endothelial injury. This study investigates the effects of fructose, in a concentration range usually observed in the plasma of patients with increased cardiovascular risk, on TF in human umbilical endothelial cells (HUVECs). Cells were stimulated with increasing concentrations of fructose (0.25, 1 and 2.5 mM) and then processed to evaluate TF-mRNA levels by real-time PCR as well as TF expression/activity by FACS analysis and procoagulant activity. Finally, a potential molecular pathway involved in modulating this phenomenon was investigated. We demonstrate that fructose induces transcription of mRNA for TF. In addition, we show that this monosaccharide promotes surface expression of TF that is functionally active. Fructose effects on TF appear modulated by the oxygen free radicals through activation of the transcription factor NF-κB since superoxide dismutase and NF-κB inhibitors suppressed TF expression. Data of the present study, although in vitro, indicate that fructose, besides promoting atherosclerosis, induces a prothrombotic phenotype in HUVECs, thus indicating one the mechanism(s) by which this sweetener might increase cardiometabolic risk.