Addressing adverse synergies between chemical and biological pollutants at schools-The 'SynAir-G' hypothesis.

While the number and types of indoor air pollutants is rising, much is suspected but little is known about the impact of their potentially synergistic interactions, upon human health. Gases, particulate matter, organic compounds but also allergens and viruses, fall within the 'pollutant' definition. Distinct populations, such as children and allergy and asthma sufferers are highly susceptible, while a low socioeconomic background is a further susceptibility factor; however, no specific guidance is available. We spend most of our time indoors; for children, the school environment is of paramount importance and potentially amenable to intervention. The interactions between some pollutant classes have been studied. However, a lot is missing with respect to understanding interactions between specific pollutants of different classes in terms of concentrations, timing and sequence, to improve targeting and upgrade standards. SynAir-G is a European Commission-funded project aiming to reveal and quantify synergistic interactions between different pollutants affecting health, from mechanisms to real life, focusing on the school setting. It will develop a comprehensive and responsive multipollutant monitoring system, advance environmentally friendly interventions, and disseminate the generated knowledge to relevant stakeholders in accessible and actionable formats. The aim of this article it to put forward the SynAir-G hypothesis, and describe its background and objectives.

A Förster Resonance Energy Transfer (FRET)-Based Immune Assay for the Detection of Microcystin-LR in Drinking Water.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

A Surface-Enhanced Raman Spectroscopy-Based Biosensor for the Detection of Biological Macromolecules: The Case of the Lipopolysaccharide Endotoxin Molecules.

The development of sensitive methods for the detection of endotoxin molecules, such as lipopolysaccharides (LPS), is essential for food safety and health control. Conventional analytical methods used for LPS detection are based on the pyrogen test, plating and culture-based methods, and the limulus amoebocyte lysate method (LAL). Alternatively, the development of reliable biosensors for LPS detection would be highly desirable to solve some critical issues, such as high cost and a long turnaround time. In this work, we present a label-free Surface-Enhanced Raman Spectroscopy (SERS)-based method for LPS detection in its free form. The proposed method combines the benefits of plasmonic enhancement with the selectivity provided by a specific anti-lipid A antibody (Ab). A high-enhancing nanostructured silver substrate was coated with Ab. The presence of LPS was quantitatively monitored by analyzing the changes in the Ab spectra obtained in the absence and presence of LPS. A limit of detection (LOD) and quantification (LOQ) of 12 ng/mL and 41 ng/mL were estimated, respectively. Importantly, the proposed technology could be easily expanded for the determination of other biological macromolecules.

Focusing the Clinical Supervision on the Therapist's Developmental Trauma: A Single Case Study.

The term developmental trauma (DT) refers to the impact of stressful events which occur cumulatively within the child's relevant relationships and contexts, and usually early in life. According to several authors, DT depends on the caregiver's inadequate intersubjective recognition of one or more aspects of the evolving individual's identity. In the clinical and empirical literature, the study of therapists' developmental trauma, and how it might constitute a relevant variable in the clinical exchange, seem to be underrepresented. In this paper, through the analysis of the supervision process of a clinical case, we show how the therapeutic relationship may implicitly take the form of a "dance" between the patient's and therapist's DT, that prevents the therapist from intersubjectively attuning with the patient; and how a supervision process peculiarly focused on the therapist's DT can effectively promote this attunement and a good clinical outcome.

Photonic Label-Free Biosensors for Fast and Multiplex Detection of Swine Viral Diseases.

In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody-antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.

SPR-Based Detection of ASF Virus in Cells.

African swine fever (ASF) is one of the most dangerous hemorrhagic infectious diseases that affect domestic and wild pigs. Currently, neither a vaccine nor effective treatments are available for this disease. As regards the degree of virulence, ASFV strains can be divided into high, moderate, or low virulence. The main detection methods are based on the use of the polymerase chain reaction (PCR). In order to prevent an uncontrolled spread of ASF, new on-site techniques that can enable the identification of an early-stage disease are needed. We have developed a specific immunological SPR-based assay for ASFV antigen detection directly in liquid samples. The developed assay allows us to detect the presence of ASFV at the dose of 103 HAD50/mL.

The Porcine Odorant-Binding Protein as a Probe for an Impedenziometric-Based Detection of Benzene in the Environment.

Odorant-binding proteins (OBPs) are a group of small and soluble proteins present in both vertebrates and insects. They have a high level of structural stability and bind to a large spectrum of odorant molecules. In the environmental field, benzene is the most dangerous compound among the class of pollutants named BTEX (benzene, toluene, ethylbenzene, and xylene). It has several effects on human health and, consequently, it appears to be important to monitor its presence in the environment. Commonly, its detection requires the use of very sophisticated and time-consuming analytical techniques (GC-MS, etc.) as well as the presence of specialized personnel. Here, we present the application of an odorant-binding protein (pOBP) isolated from pigs as a molecular recognition element (MRE) for a low-energy impedenziometric biosensor for outdoor and real-time benzene detection. The obtained results show that the biosensor can detect the presence of 64 pM (5 µg/m3) benzene, the limit value of exposure for human health set by the European Directive 2008/50/EC.

Emergent Biosensing Technologies Based on Fluorescence Spectroscopy and Surface Plasmon Resonance.

The purpose of this work is to provide an exhaustive overview of the emerging biosensor technologies for the detection of analytes of interest for food, environment, security, and health. Over the years, biosensors have acquired increasing importance in a wide range of applications due to synergistic studies of various scientific disciplines, determining their great commercial potential and revealing how nanotechnology and biotechnology can be strictly connected. In the present scenario, biosensors have increased their detection limit and sensitivity unthinkable until a few years ago. The most widely used biosensors are optical-based devices such as surface plasmon resonance (SPR)-based biosensors and fluorescence-based biosensors. Here, we will review them by highlighting how the progress in their design and development could impact our daily life.

Spectroscopic Properties of Two 5'-(4-Dimethylamino)Azobenzene Conjugated G-Quadruplex Forming Oligonucleotides.

The synthesis of two 5'-end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K+-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5'-(4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5'-end of TBA and T30695 showed that, unlike the free dye, in K+-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer.

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate.

Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed of a subunit of 8 kDa and another of 50 kDa. The two protein subunits are noncovalently associated. The cloning and expression of the two protein subunits in Escherichia coli and their subsequent purification to homogeneity under native conditions result in the production of an active HPSE enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the nonreducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on HPSE, as the HPSE cleavage of fluorogenic tag would result in a measurable response.

Osmolyte-Like Stabilizing Effects of Low GdnHCl Concentrations on d-Glucose/d-Galactose-Binding Protein.

The ability of d-glucose/d-galactose-binding protein (GGBP) to reversibly interact with its ligands, glucose and galactose, makes this protein an attractive candidate for sensing elements of glucose biosensors. This potential is largely responsible for attracting researchers to study the conformational properties of this protein. Previously, we showed that an increase in the fluorescence intensity of the fluorescent dye 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN) is linked to the holo-form of the GGBP/H152C mutant in solutions containing sub-denaturing concentrations of guanidine hydrochloride (GdnHCl). It was hypothesized that low GdnHCl concentrations might lead to compaction of the protein, thereby facilitating ligand binding. In this work, we utilize BADAN fluorescence spectroscopy, intrinsic protein UV fluorescence spectroscopy, and isothermal titration calorimetry (ITC) to show that the sub-denaturing GdnHCl concentrations possess osmolyte-like stabilizing effects on the structural dynamics, conformational stability, and functional activity of GGBP/H152C and the wild type of this protein (wtGGBP). Our data are consistent with the model where low GdnHCl concentrations promote a shift in the dynamic distribution of the protein molecules toward a conformational ensemble enriched in molecules with a tighter structure and a more closed conformation. This promotes the increase in the configurational complementarity between the protein and glucose molecules that leads to the increase in glucose affinity in both GGBP/H152C and wtGGBP.

On the possibility of ephedrine detection: time-resolved fluorescence resonance energy transfer (FRET)-based approach.

Ephedrine is one of the main precursor compounds used in the illegal production of amphetamines and related drugs. Actually, conventional analytical methods such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography-mass spectrometry (GC-MS) are used for the detection of ephedrine; sadly, these methods require qualified personnel and are time-consuming and expensive. In order to overcome these problems, in recent years, different methods have been developed based on the surface plasmon resonance (SPR) and electrochemical method. In this work, we present a simple, rapid, and effective method to detect the presence of ephedrine in solution, based on competitive fluorescence resonance energy transfer (FRET) assay. The antibody anti-ephedrine and ephedrine derivative were produced and labeled respectively, with two different fluorescent probes (donor and acceptor). The change in FRET signal intensity between donor and acceptor ephedrine compounds gives the possibility of detecting ephedrine traces of at least 0.81 ± 0.04 ppm (LOD). Graphical abstract A new Time-resolved Fluorescence Resonance Energy Transfer (FRET) assay for ephedrine detection.

A near-infrared fluorescence assay method to detect patulin in food.

Patulin (PAT) is a toxic secondary metabolite (mycotoxin) of different fungal species belonging to the genera Penicillium, Aspergillus, and Byssochlamys. They can grow on a large variety of food, including fruits, grains, and cheese. The amount of PAT in apple derivative products is a crucial issue because it is the measure of the quality of both the used raw products and the performed production process. Actually, all current methodologies used for the quantification of PAT are time-consuming and require skilled personnel beyond the sample pretreatment methods (e.g., high-performance liquid chromatography, mass spectrometry, and electrophoresis techniques). In this work, we present a novel fluorescence polarization approach based on the use of emergent near-infrared (NIR) fluorescence probes. The use of these fluorophores coupled to anti-PAT antibodies makes possible the detection of PAT directly in apple juice without any sample pretreatment. This methodology is based on the increase of fluorescence polarization emission of a fluorescence-labeled PAT derivative on binding to specific antibodies. A competition between PAT and the fluorescence-labeled PAT derivative allowed detecting PAT. The limit of detection of the method is 0.06 µg/L, a value that is lower than maximum residue limit of PAT fixed at 50 µg/L from European Union regulation.

Tryptophan residue of the D-galactose/D-glucose-binding protein from E. Coli localized in its active center does not contribute to the change in intrinsic fluorescence upon glucose binding.

Changes of the characteristics of intrinsic tryptophan fluorescence of the wild type of D-galactose/D-glucose-binding protein from Escherichia coli (GGBPwt) induced by D-glucose binding were examined by the intrinsic UV-fluorescence of proteins, circular dyhroism in the near-UV region, and acrylamide-induced fluorescence quenching. The analysis of the different characteristics of GGBPwt and its mutant form GGBP-W183A together with the analysis of the microenvironment of tryptophan residues of GGBPwt revealed that Trp 183, which is directly involved in sugar binding, has the least influence on the provoked by D-glucose blue shift and increase in the intensity of protein intrinsic fluorescence in comparison with other tryptophan residues of GGBP.

What makes us more susceptible to false memories in the era of COVID-19? A focus on vaccines and Green Pass.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic was accompanied by an overabundance of fake news increasing the risk of developing false memories (FMs). Previous studies have shown that the relationship between fake news and FMs could be mediated by some individual variables, including attitudinal biases. We explored the role of these variables in true memories (TMs) and FMs formation, with special emphasis on vaccine- and Green Pass (GP)-related topics. METHOD: We set up a large online survey exploring several constructs including media usage, attitude toward vaccines and GP, perceived (PK) and objective knowledge (OK) about COVID-19-related information, fear of the disease, depression and anxiety symptoms, coping mechanisms, and reasoning skills. Then, we asked participants whether they remembered certain news (true or fake), providing confidence ratings. RESULTS: Data from 289 respondents (198 females) from the general population were analyzed. Participants with positive attitude reported a greater fear that their loved ones contracted the COVID-19, a more frequent use of traditional media, and a higher PK when compared with respondents with negative attitude. On the whole sample, participants reported higher confidence levels when required to judge their memory of true than fake news; however, participants with positive attitude reported a higher confidence for both true and fake news. The relationship between attitude and TM confidence was mediated by the PK, whereas the relationship between attitude and FM confidence was probably affected by OK. CONCLUSION: Attitude can modulate individual behaviors in the context of health issues. The PK and OK may interact with attitude in the memory formation.

An Impedance-Based Immunosensor for the Detection of Ovalbumin in White Wine.

Food allergies are an exceptional response of the immune system caused by the ingestion of specific foods. The main foods responsible for allergic reactions are milk, eggs, seafood, soy, peanuts, tree nuts, wheat, and their derived products. Chicken egg ovalbumin (OVA), a common allergen molecule, is often used for the clarification process of wine. Traces of OVA remain in the wine during the fining process, and they can cause significant allergic reactions in sensitive consumers. Consequently, the European Food Safety Authority (EFSA) and the American Food and Drug Administration (FDA) have shown the risks for allergic people to assume allergenic foods and food ingredients, including eggs. Commonly, OVA detection requires sophisticated and time-consuming analytical techniques. Intending to develop a faster assay, we designed a proof-of-concept non-Faradaic impedimetric immunosensor for monitoring the presence of OVA in wine. Polyclonal antibodies anti-OVA were covalently immobilised onto an 11-mercaptoundecanoic-acid (11-MUA)-modified gold surface. The developed immunosensor was able to detect OVA in diluted white wine without the need for an external probe or any pre-treatment step with a sensitivity of 0.20 µg/mL, complying with the limit established by the resolution OIV/COMEX 502-2012 for the quantification of allergens in wine.

An Impedimetric Biosensor for Detection of Volatile Organic Compounds in Food.

The demand for a wide choice of food that is safe and palatable increases every day. Consumers do not accept off-flavors that have atypical odors resulting from internal deterioration or contamination by substances alien to the food. Odor response depends on the volatile organic compounds (VOCs), and their detection can provide information about food quality. Gas chromatography/mass spectrometry is the most powerful method available for the detection of VOC. However, it is laborious, costly, and requires the presence of a trained operator. To develop a faster analytic tool, we designed a non-Faradaic impedimetric biosensor for monitoring the presence of VOCs involved in food spoilage. The biosensor is based on the use of the pig odorant-binding protein (pOBP) as the molecular recognition element. We evaluated the affinity of pOBP for three different volatile organic compounds (1-octen-3-ol, trans-2-hexen-1-ol, and hexanal) related to food spoilage. We developed an electrochemical biosensor conducting impedimetric measurements in liquid and air samples. The impedance changes allowed us to detect each VOC sample at a minimum concentration of 0.1 µM.

The Endoscopic Retrograde Cholangiopancreatography and Endoscopic Ultrasound Connection: Unity Is Strength, or the Endoscopic Ultrasonography Retrograde Cholangiopancreatography Concept.

Endoscopic ultrasound (EUS) and endoscopic retrograde cholangiopancreatography (ERCP) are both crucial for the endoscopic management of biliopancreatic diseases: the combination of their diagnostic and therapeutic potential is useful in many clinical scenarios, such as indeterminate biliary stenosis, biliary stones, chronic pancreatitis and biliary and pancreatic malignancies. This natural and evident convergence between EUS and ERCP, which by 2006 we were calling the "Endoscopic ultrasonography retrograde colangiopancreatography (EURCP) concept", has become a hot topic in the last years, together with the implementation of the therapeutic possibilities of EUS (from EUS-guided necrosectomy to gastro-entero anastomoses) and with the return of ERCP to its original diagnostic purpose thanks to ancillary techniques (extraductal ultrasound (EDUS), intraductal ultrasound (IDUS), cholangiopancreatoscopy with biopsies and probe-based confocal laser endomicroscopy (pCLE)). In this literary review, we retraced the recent history of EUS and ERCP, reported examples of the clinical applicability of the EURCP concept and explored the option of performing the two procedures in only one endoscopic session, with its positive implications for the patient, the endoscopist and the health care system. In the last few years, we also evaluated the possibility of combining EUS and ERCP into a single endoscopic instrument in a single step, but certain obstacles surrounding this approach remain.

Engineering resonance energy transfer for advanced immunoassays: the case of celiac disease.

Celiac disease (CD) is an immune-mediated disorder affecting genetically predisposed subjects. It is caused by the ingestion of wheat gluten and related prolamins. A final diagnosis for this disease can be obtained by examination of jejunal biopsies. Nevertheless, different analytical approaches have been established to detect the presence of anti-tissue transglutaminase antibodies that represent a serological hallmark of the disease. In this work, we explored a new method for the diagnosis of CD based on the detection of serum anti-transglutaminase antibodies by resonance energy transfer (RET) between donor molecules and acceptor molecules. In particular, we labeled the liver transglutaminase (tTG) enzyme from guinea pig and the rabbit anti-tTG antibodies with a couple of fluorescence probes that are able to make RET if they are located within with Förster distance. We labeled tTG with the fluorescence probe DyLight 594 as donor and the anti-tTG antibodies with the fluorescence probe DyLight 649 as acceptor. However, due to the large size of the formed complex (tTG/anti-tTG), and consequently to the low efficiency energy transfer process between the donor-acceptor molecules, we explored a new experimental approach that allows us to extend the utilizable range of RET between donor:acceptor pairs by using one single molecule as donor and multiple molecules as energy acceptors, instead of using a single acceptor molecule as usually occurs in RET experiments. The obtained results clearly show that the use of one donor and multiacceptor strategy enables for a simple and rapid detection of serum anti-transglutaminase antibodies. In addition, our results point out that it is possible to consider this approach as a new method for a wide variety of analytical assays.