Unveiling the folding mechanism of the bromodomains

Bromodomains (BRDs) are small protein domains often present in large multidomain proteins involved in transcriptional regulation in eukaryotic cells. They currently represent valuable targets for the development of inhibitors of aberrant transcriptional processes in a variety of human diseases. Here we report urea-induced equilibrium unfolding experiments monitored by circular dichroism (CD) and fluorescence on two structurally similar BRDs: BRD2(2) and BRD4(1), showing that BRD4(1) is more stable than BRD2(2). Moreover, we report a description of their kinetic folding mechanism, as obtained by careful analysis of stopped-flow and temperature-jump data. The presence of a high energy intermediate for both proteins, suggested by the non-linear dependence of the folding rate on denaturant concentration in the millisec time regime, has been experimentally observed by temperature-jump experiments. Quantitative global analysis of all the rate constants obtained over a wide range of urea concentrations, allowed us to propose a common, three-state, folding mechanism for these two BRDs. Interestingly, the intermediate of BRD4(1) appears to be more stable and structurally native-like than that populated by BRD2(2). Our results underscore the role played by structural topology and sequence in determining and tuning the folding mechanism

Innovative method for recovery and valorization of hydroxytyrosol from olive mill wastewaters

The nutritional properties of olive oil can be attributed to its oleic acid and phenolic compounds content, acting as natural oxidants to prevent human diseases. In particular, hydroxytyrosol has an anti-inflammatory action similar to omega 3 fatty acids from fish oil. The olive oil production was conducted by two extraction procedures: first, a two-phase extraction giving extra-virgin olive oil and humid pomace, second, a three-phase working process of humid pomace, obtaining another minimum quantity of extra-virgin olive oil, 'dry' pomace devoid of polyphenols, and mill wastewaters rich in anti-oxidant compounds. The aim of this processing was to employ water to extract the highest concentration of polyphenols from humid pomace and convey them in oil mill wastewaters for extraction. Processed olives were 37,200 kg, pomace deprived of polyphenols was equal to 20,400 kg and processing was performed with 500 kg of olives per hour. This method offers advantages of using cheap equipment and technical simplicity

Structural Effects of Metal Single-Atom Catalysts for Enhanced Photocatalytic Degradation of Gemfibrozil

The development of efficient catalysts is a highly necessary but challenging task within the field of environmental water remediation. Single-atom catalysts are promising nanomaterials within this respect, but in-depth studies encompassing this class of catalysts remain elusive. In this work, we systematically study the degradation of gemfibrozil, a persistent pollutant, on a series of carbon nitride photocatalysts, investigating both the effect of (i) catalyst textural properties and (ii) metal single atoms on the contaminant degradation. Tests in the absence of the catalyst result in negligible degradation rates, confirming the stability of the contaminant when dispersed in water. Then, photocatalytic tests at optimal pH, solvent, and wavelength reveal a correlation between the support surface area and the degradation. This points to the role of carbon nitride surface nanostructure on gemfibrozil degradation. In particular, the use of silver on mesoporous carbon nitride single-atom catalyst (Ag@mpgC(3)N(4)) leads to an unprecedented degradation of gemfibrozil (>90% within 60 min). The possible degradation intermediates and products were identified by mass spectrometry and were inert by cytotoxicity evaluation. We anticipate that, with further refinement and customization, the carbon nitride catalysts reported herein may find broad applications for light-driven degradation of other contaminants of emerging concern

Development of a Desmocollin-3 Active Mouse Model Recapitulating Human Atypical Pemphigus

Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. It is often associated with autoantibodies to the desmosomal adhesion proteins Desmoglein 3 (DSG3) and Desmoglein 1 (DSG1). Recently, auto-antigens, such as desmocollins and others have been described in PV and in atypical pemphigus forms such as Pemphigus Herpetiformis (PH), Pemphigus Vegetans (PVeg), and Paraneoplastic Pemphigus (PP). Desmocollins belong to a cadherin subfamily that provides structure to the desmosomes and play an important role in cell-to-cell adhesion. In order to verify the pathogenic activity of anti-Desmocollin 3 (DSC3) antibodies, we developed an active disease model of pemphigus expressing anti-DSC3 autoantibodies or antiDSC3 and anti-DSG3 antibodies. This approach included the adoptive transfer of DSC3 and/or DSG3 lymphocytes to Rag2(-/-) immunodeficient mice that express DSC3 and DSG3. Our results show that the presence of anti-DSC3 auto-antibodies is sufficient to determine the appearance of a pathological phenotype relatable to pemphigus, but with features not completely super-imposable to those observed in the DSG3 active model, suggesting that the DSC3 active model might mimic the atypical pemphigus. Moreover, the presence of both anti-DSC3 and anti-DSG3 antibodies determines a more severe phenotype and a slower response to prednisolone. In conclusion, we have developed an adult DSC3 pemphigus mouse model that differs from the DSG3 model and supports the concept that antigens other than desmogleins may be responsible for different phenotypes in human pemphigus

Poorly differentiated clusters (PDC) in colorectal cancer: Does their localization in tumor matter?

Poorly differentiated clusters (PDC) are aggregates of at least five neoplastic cells lacking evidence of glandular differentiation. By definition, they can be present at the invasive front (peripheral PDC or pPDC) and within the tumor stroma (central PDC or cPDC). In colorectal cancer (CRC), PDC are considered adverse prognosticators and seem to reflect epithelial mesenchymal transition (EMT). In this study, we have investigated the immuno-expression of two EMT-related proteins, E-cadherin and \u3b2-catenin, in PDC of primary CRCs and matched liver metastases. pPDC always showed nuclear \u3b2-catenin staining and diffusely reduced/absence of E-cadherin expression as opposed cPDC which showed nuclear \u3b2-catenin immunoreactivity and E-cadherin expression in about 50% of cases. In addition, the pattern of \u3b2-catenin and E-cadherin expression differed between PDC and the main tumor, and between primary CRC and liver metastasis (LM), in a percentage of cases. A discordant pattern of \u3b2-catenin and E-cadherin expression between pPDC and cPDC, between main tumor and cPDC, and between primary CRC and LM, confirms that EMT is a dynamic and reversible process in CRC. On the overall, this suggests that pPDC and cPDC are biologically different. We may advocate that PDC develop at the tumor center (cPDC) and then some of them migrate towards the tumor periphery while progressively completing EMT process (pPDC). Based on these results, PDC presence and counting may have different prognostic relevance if the assessment is done at the invasive front of the tumor or in the intratumor stroma

Photoacoustic spectroscopy for estimating nutritional indices in Lepidopteran defoliators

Lymantria dispar L. and Malacosoma neustrium (L.) are the most serious defoliators of cork oak in the Mediterranean region. For this reason, information on their feeding behaviour are important in pest management. A non-destructive approach by using photoacoustic spectroscopy (PAS) combined with a partial least squares regression analyses (PLS), has been used to provide a rapid and cost-effective analysis to assess foliage chemistry and to estimate some nutritional indices of these insects

Development of a screening tool to assess dehydration in hospitalized older population: a diagnostic, observational study

INTRODUCTION: dehydration is a frequent condition in older people and is associated with an increased risk of negative health outcomes. In order to adopt strategies to prevent complications, an early recognition of this status is of primary importance. For this reason, a comprehensive assessment tool to monitor hydration status in older people could be useful.AIM: to develop a screening tool to detect dehydration in older people in hospital settings.METHODS: this is a diagnostic, observational study. The new tool is a modified version of the Geriatric Dehydration Screening Tool (GDST), integrated with seven questions and two clinical signs based on updated literature. We tested the new tool with people aged 65 or over. We used as reference standard serum osmolarity. Cronbach's alpha was used to measure the tool's reliability and subscales. We calculated the Area Under ROC Curve (AUC) to choose the cut-off that gave the best balance between sensibility and specificity.RESULTS: 127 patients participated in the study. The reliability of the new GDST was acceptable (Cronbach's alpha 0.63). The diagnostic accuracy, measured with AUC analysis, was 0.83 ± 0.04, p<0.0001 95% CI 0.72-0.87. The best cut-off value was 6 and showed a sensibility of 78%, specificity of 70%. Tongue dryness proved to be the most significant clinical sign associated with poor hydration status (AUC 0.78; p<0.0001, 95% CI 0.69-0.86).CONCLUSION: The new GDST presented an acceptable reliability and diagnostic accuracy that increased with the assessment of some items, such as tongue dryness. This is the first screening tool that presents a promising cut-off value.KEYWORDS: dehydration, aged, screening, inpatients, sensibility, specificity.Sviluppo di uno strumento di screening per valutare la disidratazione nella popolazione anziana ospedalizzata: uno studio diagnostico, osservazionaleRIASSUNTOINTRODUZIONE: La disidratazione è una condizione comune nella persona anziana ed è associata a numerosi rischi per la salute e ad esiti negativi. È importante il precoce riconoscimento di questa condizione, al fine di adottare strategie per prevenirne le complicanze. Per questa ragione è necessario sviluppare strumenti validati per valutare il rischio di disidratazione nelle persone anzianeOBIETTIVO: sviluppare uno strumento di screening per individuare la disidratazione nelle persone anziane ospedalizzate.METODO: il disegno di studio adottato è di tipo diagnostico, osservazionale. Lo strumento creato è basato sul "Geriatric Dehydration Screening Tool" (GDST), che è stato modificato aggiungendo sette domande e due segni clinici, basati sulla letteratura recente. Lo strumento è stato testato in persone con un'etí  maggiore o uguale a 65 anni ospedalizzate. Come reference standard è stata usata l'osmolarití  sierica. È stato calcolato l'alfa di Cronbach per testare l'affidabilití  della consistenza interna dello strumento e delle sue sotto scale. È stata calcolata l'area sotto la curva di ROC (AUC) per individuare il cut-off che dava il miglior bilanciamento tra sensibilití  e specificití .RISULTATI: 127 pazienti hanno partecipato allo studio. L'affidabilití  dello strumento è risultata discreta (Alfa di Cronbach=0.63). L'accuratezza diagnostica, misurata con l'AUC era 0.83±0.04, p<0.0001, 95% IC 0.72-0.87. Il cut-off migliore è risultato essere il valore 6, con una sensibilití  del 78% e specificití  70%. La secchezza della lingua è risultato essere il segno clinico più associato con uno stato di disidratazione (AUC 0.78, p<0.0001 95%CI 0.69-0.86).CONCLUSIONI: il nuovo GDST ha dimostrato un'accettabile affidabilití  e accuratezza diagnostica che aumenta con la valutazione di alcuni items, come la secchezza della lingua. Questo è il primo strumento che presenta un valore di cut-off promettente.PAROLE CHIAVE: disidratazione, anziano, screening, pazienti ospedalizzati, sensibilití , specificití 

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes

Background: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. Methods: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. Results: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. Conclusion: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development