Hermetia illucens and Poultry by-Product Meals as Alternatives to Plant Protein Sources in Gilthead Seabream (Sparus aurata) Diet: A Multidisciplinary Study on Fish Gut Status

The attempt to replace marine-derived ingredients for aquafeed formulation with plant-derived ones has met some limitations due to their negative side effects on many fish species. In this context, finding new, sustainable ingredients able to promote fish welfare is currently under exploration. In the present study, poultry by-products and Hermetia illucens meal were used to replace the vegetable protein fraction in diets totally deprived of fish meal intended for gilthead seabream (Sparus aurata). After a 12-week feeding trial, a multidisciplinary approach including histological, molecular, and spectroscopic techniques was adopted to investigate intestine and liver responses to the different dietary formulations. Regardless of the alternative ingredient used, the reduction in dietary vegetable proteins resulted in a lower incidence of intestine histological alterations and inflammatory responses. In addition, the dietary inclusion of insect meal positively affected the reduction in the molecular inflammatory markers analyzed. Spectroscopic analyses showed that poultry by-product meal improved lipid absorption in the intestine, while insect meal induced increased liver lipid deposition in fish. The results obtained demonstrated that both poultry by-products and H. illucens meal can successfully be used to replace plant-derived ingredients in diets for gilthead seabream, promoting healthy aquaculture

Cytotoxic Effects of 5-Azacytidine on Primary Tumour Cells and Cancer Stem Cells from Oral Squamous Cell Carcinoma: An In Vitro FTIRM Analysis

In the present study, the cytotoxic effects of 5-azacytidine on primary Oral Squamous Cell Carcinoma cells (OSCCs) from human biopsies, and on Cancer Stem Cells (CSCs) from the same samples, were investigated by an in vitro Fourier Transform InfraRed Microscospectroscopy (FTIRM) approach coupled with multivariate analysis. OSCC is an aggressive tumoral lesion of the epithelium, accounting for ~90% of all oral cancers. It is usually diagnosed in advanced stages, and this causes a poor prognosis with low success rates of surgical, as well as radiation and chemotherapy treatments. OSCC is frequently characterised by recurrence after chemotherapy and by the development of a refractoriness to some employed drugs, which is probably ascribable to the presence of CSCs niches, responsible for cancer growth, chemoresistance and metastasis. The spectral information from FTIRM was correlated with the outcomes of cytotoxicity tests and image-based cytometry, and specific spectral signatures attributable to 5-azacytidine treatment were identified, allowing us to hypothesise the demethylation of DNA and, hence, an increase in the transcriptional activity, together with a conformational transition of DNA, and a triggering of cell death by an apoptosis mechanism. Moreover, a different mechanism of action between OSSC and CSC cells was highlighted, probably due to possible differences between OSCCs and CSCs response

Towards the Identification of a Suitable Commercial Diet for Carpione (Salmo carpio, Linnaeus 1758): A Multidisciplinary Study on Fish Performances, Animal Welfare and Quality Traits

SIMPLE SUMMARY: Carpione (Salmo carpio, Linnaeus 1758) is an endangered precious endemism of Lake Garda (Northern Italy), the largest Italian lake. To date, several bottlenecks about its culture remain unsolved, including the identification of a proper growth-out diet. In the present study, four different grossly isolipidic, isoproteic, and isoenergetic diets containing ingredients from different origins were used for S. carpio culture. Specifically, a diet largely based on marine ingredients, and currently used for carpione farming, was used as control. Three other diets were formulated in order to include relevant percentages of vegetable ingredients or processed animal proteins (at two different inclusion levels). After a three-month feeding trial, fish zootechnical performances, welfare, and flesh quality were evaluated through a multidisciplinary approach, including histology, gene expression, chemical analysis, and Fourier transform infrared spectroscopy (FTIR). This study provided the first insights on carpione physiological responses to different commercial dietary formulations. ABSTRACT: Carpione (Salmo carpio, Linnaeus 1758) is an endangered precious endemism of Lake Garda (Northern Italy), the largest Italian lake. To date, several bottlenecks about its culture remain unsolved, including the identification of a proper growth-out diet. The aim of the present study was to test four different grossly isolipidic, isoproteic, and isoenergetic diets in which the main ingredients had a different origin. Specifically, a diet currently used by local farmers for carpione culture, largely based on marine ingredients, was used as control (CTRL), while the other three diets were formulated by partially replacing marine ingredients with plant ones (VEG) or with different percentages of processed animal proteins (PAP1 and PAP2). The feeding trial was run in triplicate, over a three-month period. No significant differences in growth performance among the experimental groups were observed. However, remarkable histological alterations and inflammatory markers upregulation were observed in VEG group, while PAP inclusion played a role in attenuating inflammation and improving nutrient uptake. Fillet analyses highlighted significant differences in marketable traits and flesh fatty acid composition among the experimental groups, including the reduction of polyunsaturated fatty acids related to PAPs inclusion. In conclusion, PAPs used in the present study promoted S. carpio gut health and absorption capacity, while further studies are required to maintain proper quality traits of the final product

DECLINE OF PREVALENCE OF RESISTANCE ASSOCIATED SUBSTITUTIONS TO NS3 AND NS5A INHIBITORS AT DAA- FAILURE IN HEPATITIS C VIRUS IN ITALY OVER THE YEARS 2015 TO 2018

Background: A minority of patients fails to eliminate HCV and resistance-associated substitutions (RASs) are commonly detected at failure of interferon-free DAA regimens . Methods: Within the Italian network VIRONET-C, the prevalence of NS3/NS5A/NS5B RASs was retrospectively evaluated in patients who failed an EASL recommended DAA-regimen in 2015-2018 . The geno2pheno system and Sorbo MC et al. Drug Resistance Updates 2018 were used to infer HCV- genotype/subtype and predict drug resistance . The changes in prevalence of RASs over time were evaluated by chi-square test for trend, predictors of RASs at failure were analysed by logistic regression . Results: We included 386 HCV infected patients: 75% males, median age was 56 years (IQR 52-61), metavir fibrosis stage F4 in 76%; 106 (28%) were treatment- experienced: 91 (86%) with IFN-based treatments, 26 (25%) with DAAs. Patients with HIV and HBV coinfection were 10% (33/317) and 8% (6/72), respectively. HCV genotype was 1b in 122 pts (32%), 3 in 109 (28%), 1a in 97 (25%), 4 in 37 (10%), 2 in 21 (5%). DAA regimens were: LDV/SOF in 115 (30%), DCV/SOF in 103 (27%), 3D in 83 (21%), EBR/GRZ in 32 (8%), VEL/SOF in 29 (7%), GLE/PIB in 18 (5%) and 2D in 6 (2%); ribavirin was administered in 123 (32%) . The NS5A fasta-sequence was available for all patients, NS5B for 361 (94%), NS3 for 365 (95%) . According to the DAA failed the prevalence of any RASs was 90%, namely 80/135 (59%) in NS3, 313/359 (87%) in NS5A, 114/286 (40%) in NS5B . The prevalence of any RASs significantly declined from 2015 to 2018 (93% vs 70%, p=0.004): NS5A RASs from 90% to 72% (p=0 .29), NS3 RASs from 74% to 18% (p<0 .001), while NS5B RASs remained stable . Independent predictors of any RASs included advanced fibrosis (AOR 6.1, CI 95% 1.8-20.3, p=0 .004) and genotype (G2 vs G1a AOR 0 .03, CI 95% 0 .002- 0 .31, p=0 .004; G3 vs G1a AOR 0 .08, CI 95% 0 .01-0 .62, p=0 .02; G4 vs G1a AOR 0 .05, CI 95% 0 .006-0 .46, p=0 .008), after adjusting for age, previous HCV treatment and year of genotype . Notably, full activity was predicted for GLE/PIB in 75% of cases and for at least two components of VEL/SOF/VOX in 53% of cases, no case with full-resistance to either regimen was found . Conclusion: Despite decreasing prevalence over the years, RASs remain common at virological failure of DAA treatment, particularly in patients with the highest grade of liver fibrosis. The identification of RASs after failure could play a crucial role in optimizing retreatment strategies

Multivariate curve Resolution-Alternating least squares coupled with Raman microspectroscopy: new insights into the kinetic response of primary oral squamous carcinoma cells to cisplatin

Raman MicroSpectroscopy (RMS) is a powerful label-free tool to probe the effects of drugs at a cellular/subcellular level. It is important, however, to be able to extract relevant biochemical and kinetic spectroscopic signatures of the specific cellular responses. In the present study, a combination of Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Principal Component Analysis (PCA) is used to analyse the RMS data for the example of exposure of primary Oral Squamous Carcinoma Cells (OSCC) to the chemotherapeutic agent cisplatin. Dosing regimens were established by cytotoxicity assays, and the effects of the drug on cellular spectral profiles were monitored from 16 to 72 hours post-exposure using an apoptosis assay, to establish the relative populations of viable (V), early (EA) and late apoptotic/dead (LA/D) cells after the drug treatment. Based on a kinetic model of the progression from V > EA > D, MCR-ALS regression analysis of the RMS responses was able to extract spectral profiles associated with each stage of the cellular responses, enabling a quantitative comparison of the response rates for the respective drug treatments. Moreover, PCA was used to compare the spectral profiles of the viable cells exposed to the drug. Spectral differences were highlighted in the early stages (16 hours exposure), indicative of the initial cellular response to the drug treatment, and also in the late stages (48-72 hours exposure), representing the cell death pathway. The study demonstrates that RMS coupled with multivariate analysis can be used to quantitatively monitor the progression of cellular responses to different drugs, towards future applications for label-free, in vitro, pre-clinical screening

Vibrational Imaging Techniques for the Characterization of Hard Dental Tissues: From Bench-Top to Chair-Side

Currently, various analytical techniques, including scanning electron microscopy, X-Ray diffraction, microcomputed tomography, and energy dispersive X-ray spectroscopy, are available to study the structural or elemental features of hard dental tissues. In contrast to these approaches, Raman Microspectroscopy (RMS) has the great advantage of simultaneously providing, at the same time and on the same sample, a morpho-chemical correlation between the microscopic information from the visual analysis of the sample and its chemical and macromolecular composition. Hence, RMS represents an innovative and non-invasive technique to study both inorganic and organic teeth components in vitro. The aim of this narrative review is to shed new light on the applicative potential of Raman Microspectroscopy in the dental field. Specific Raman markers representative of sound and pathological hard dental tissues will be discussed, and the future diagnostic application of this technique will be outlined. The objective and detailed information provided by this technique in terms of the structure and chemical/macromolecular components of sound and pathological hard dental tissues could be useful for improving knowledge of several dental pathologies. Scientific articles regarding RMS studies of human hard dental tissues were retrieved from the principal databases by following specific inclusion and exclusion criteria

New insights on collagen structural organization and spatial distribution around dental implants: a comparison between machined and laser-treated surfaces

Abstract Background One of the main factors for the osseointegration of dental implants is the development of an adequate soft tissue barrier, mainly composed by collagen, which protects the implant from bacterial development. The structural features of the peri-implant collagen are influenced by the implant components and, in particular, by the type of the surface. In the clinical practice, healing abutments are characterized by smooth surfaces, named machined. Recently, a new laser technique, Synthegra, has been developed to obtain a topography-controlled surface with micrometric regular pores that seems reducing the risk of peri-implantitis. Based on this background, this study aims investigating the structural organization and spatial distribution of collagen surrounding healing abutments characterized by laser-treated and machined surfaces. Methods Gingiva portions surrounding custom-made healing abutments (HA), characterized by alternated laser-treated and machined surfaces, were collected and analyzed by combining Fourier Transform InfraRed Imaging (FTIRI) spectroscopy, a non-invasive and high-resolution bidimensional analytical technique, with histological and multivariate analyses. Results Masson’s trichrome staining, specific for collagen, highlighted a massive presence of collagen in all the analyzed samples, evidencing a surface-related spatial distribution. The nature of collagen, investigated by the FTIRI spectroscopy, appeared more abundant close to the laser-treated surface, with a perpendicular disposition of the bundles respect to the HA; conversely, a parallel distribution was observed around the machined surface. A different secondary structure was also found, with a higher amount of triple helices and a lower quantity of random coils in collagen close to the laser treated surfaces. Conclusions FTIRI spectroscopy demonstrates that the use of a laser treated transmucosal surface can improve the morphological organization of the peri-implant collagen, which presents a distribution more similar to that of natural teeth. Trial registration: This trial is registered with ClinicalTrials.gov Identifier: (Registration Number: NCT05754970). Registered 06/03/2023, retrospectively registered, https://clinicaltrials.gov/show/NCT05754970

Hyperspectral characterization of the MSTO-211H cell spheroid model: A FPA–FTIR imaging approach

none9noneValentina Notarstefano, Simona Sabbatini, Maurizio Sabbatini, Aldo Arrais, Alessia Belloni, Chiara Pro, Lisa Vaccari, Domenico Osella, Elisabetta GiorginiNotarstefano, Valentina; Sabbatini, Simona; Sabbatini, Maurizio; Arrais, Aldo; Belloni, Alessia; Pro, Chiara; Vaccari, Lisa; Osella, Domenico; Giorgini, Elisabett