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The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons.

Neonatal calf diarrhoea (NCD) poses a significant health challenge in cattle herds, resulting in considerable economic losses and antimicrobial use. In response to the escalating threat of antimicrobial resistance, viable alternatives are imperative, aligning with European policies. This study evaluated the in-milk supplementation of the chestnut and quebracho tannin extract in preweaning calves on performance, diarrhoea occurrence, Cryptosporidium spp. shedding, protein digestibility, and intestinal health. Twenty newborn calves were divided, after colostrum administration, into two experimental groups for 30 days as follows: the control (CTRL) was fed with whole milk and solid feed, and tannins (TAN) were fed whole milk supplemented with 6/g day of tannin extract and solid feed. Faecal samples were collected on days 0, 3, 7, 14, and 30 for the evaluation of Cryptosporidium oocyst shedding and protein digestibility. Faecal consistency was evaluated during the sampling using the faecal score scale (0–3 scale, considering diarrhoea > 1). The results showed a significant reduction in diarrhoea frequency in the TAN compared to the CTRL group (p < 0.05) over 30 days of the trial. The prevalence of Cryptosporidium spp. was generally low (12%), considering all analysed samples. Protein digestibility revealed comparable values for the TAN and CTRL groups, suggesting that tannins did not negatively affect milk protein availability. In conclusion, the in-milk supplementation of 6/g day of the chestnut and quebracho tannin extract could be considered a valuable functional feed additive to decrease NCD occurrence, thus supporting animal health and decreasing antibiotic use in livestock.

In this work, we present a multiphysics modeling approach capable of simulating electrochemical impedance spectroscopy (EIS) responses of screen-printed electrodes (SPEs) modified with self-assembled monolayers of 11-Mercaptoundecanoic acid (MUA). Commercially available gold SPEs are electrochemically characterized through experimental cyclic voltammetry and EIS measurements with 10 mM [Fe(CN)6]3−/4− redox couple in phosphate buffered saline before and after the surface immobilization of MUA at different concentrations. We design the multiphysics model through COMSOL Multiphysics® based on the 3D geometry of the devices under test. The model includes four different physics considering the metal/solution interface electrochemical phenomena, the ion and electron potentials and currents, and the measurement set-up. The model is calibrated through a set of experimental measurements, allowing the tuning of the parameters used by the model. We use the calibrated model to simulate the EIS response of MUA-modified SPEs, comparing the results with experimental data. The simulations fit the experimental curves well, following the variation of MUA concentration on the surface from 1 µM to 100 µM. The EIS parameters, retrieved through a CPE-modified Randles’ circuit, confirm the consistency with the experimental data. Notably, the simulated surface coverage estimates and the variation of charge transfer resistance due to MUA-immobilization are well matched with their experimental counterparts, reporting only a 2% difference and being consistent with the experimental electrochemical behavior of the SPEs.

Meat analogues play an increasing role in meeting global nutritional needs. However, while it is well known that meat possesses inherent characteristics that create favourable conditions for the growth of various pathogenic bacteria, much less is known about meat analogues. This study aimed to compare the growth and survival of Escherichia coli HEHA16, Listeria monocytogenes, Salmonella enterica Typhi, Cronobacter sakazakii, and a cocktail of these bacteria in sterile juices from minced chicken, pig, and beef, as well as pea-based and soy-based minced meat. Traditional microbiology and next-generation sequencing of those metagenomes were employed to analyse the pathogen variability, abundance, and survival after an incubation period. Our findings show that all the meat juices provided favourable conditions for the growth and proliferation of the studied bacteria, with the exception of E. coli HEHA16, which showed lower survival rates in the chicken matrix. Meat analogue juice mainly supported L. monocytogenes survival, with C. sakazakii survival supported to a lesser extent. A correlation was observed between the traditional culturing and metagenomic analysis results, suggesting that further work is needed to compare these technologies in foodborne setups. Our results indicate that plant-based meats could serve as vectors for the transmission of certain, but likely not all, foodborne pathogens, using two accurate detection methods. This warrants the need for additional research to better understand and characterise their safety implications, including their potential association with additional pathogens.

The present study was designed to evaluate the effects of dietary levels of bioactive peptides (BPs) derived from salmon processing by-products on the presence and distribution of peptic cells (oxyntopeptic cells, OPs) and enteric endocrine cells (EECs) that contain GHR, NPY and SOM in the gastric mucosa of European seabass and gilthead seabream. In this study, 27 seabass and 27 seabreams were divided into three experimental groups: a control group (CTR) fed a control diet and two groups fed different levels of BP to replace fishmeal: 5% BP (BP5%) and 10% BP (BP10%). The stomach of each fish was sampled and processed for immunohistochemistry. Some SOM, NPY and GHR-IR cells exhibited alternating “open type” and “closed type” EECs morphologies. The BP10% group (16.8 ± 7.5) showed an increase in the number of NPY-IR cells compared to CTR (CTR 8.5 ± 4.8) and BP5% (BP10% vs. CTR p ≤ 0.01; BP10% vs. BP5% p ≤ 0.05) in the seabream gastric mucosa. In addition, in seabream gastric tissue, SOM-IR cells in the BP 10% diet (16.8 ± 3.5) were different from those in CTR (12.5 ± 5) (CTR vs. BP 10% p ≤ 0.05) and BP 5% (12.9 ± 2.5) (BP 5% vs. BP 10% p ≤ 0.01). EEC SOM-IR cells increased at 10% BP (5.3 ± 0.7) compared to 5% BP (4.4 ± 0.8) (5% BP vs. 10% BP p ≤ 0.05) in seabass. The results obtained may provide a good basis for a better understanding of the potential of salmon BPs as feed ingredients for seabass and seabream.

The main objective of this study was to investigate the dynamic of SARS-CoV-2 viral excretion in rectal swab (RS), saliva, and nasopharyngeal swab (NS) samples from symptomatic patients and asymptomatic contacts. In addition, in order to evaluate the replication potential of SARS-CoV-2 in the gastrointestinal (GI) tract and the excretion of infectious SARS-CoV-2 from feces, we investigated the presence of subgenomic nucleoprotein gene (N) mRNA (sgN) in RS samples and cytopathic effects in Vero cell culture. A prospective cohort study was performed to collect samples from symptomatic patients and contacts in Rio de Janeiro, Brazil, from May to October 2020. One hundred and seventy-six patients had samples collected at home visits and/or during the follow up, resulting in a total of 1633 RS, saliva, or NS samples. SARS-CoV-2 RNA was detected in 130 (73.9%) patients who had at least one sample that tested positive for SARS-CoV-2. The presence of replicating SARS-CoV-2 in RS samples, measured by the detection of sgN mRNA, was successfully achieved in 19.4% (6/31) of samples, whilst infectious SARS-CoV-2, measured by the generation of cytopathic effects in cell culture, was identified in only one RS sample. Although rare, our results demonstrated the replication capacity of SARS-CoV-2 in the GI tract, and infectious viruses in one RS sample. There is still a gap in the knowledge regarding SARS-CoV-2 fecal–oral transmission. Additional studies are warranted to investigate fecal or wastewater exposure as a risk factor for transmission in human populations.

Surface plasmon resonance (SPR) is a common and useful measurement technique to perform fast and sensitive optical detection. SPR instrumentations usually comprise optical systems of mirrors and lenses which are quite expensive and impractical for point-of-care applications. In this work, we presented a novel and compact SPR device called SPECTRA, designed as a spectrophotometer add-on with a grating coupling configuration. The device is conceived as a marketable solution to perform quick SPR measurements in grating configuration without the requirement of complex instrumentation. The device can be customized either in a vertical structure to reach lower incident light angles, or in a horizontal configuration, which is suitable for SPR analysis using liquid solutions. The SPECTRA performance was evaluated through SPR measurements in typical applications. The vertical SPECTRA system was employed to detect different functionalization molecules on gold 720 nm-period grating devices. Meanwhile, the horizontal SPECTRA configuration was exploited to carry out fluid-dynamic measurements using a microfluidic cell with glycerol solutions at increasing concentrations to account for different refractive indexes. The experimental tests confirmed that the SPECTRA design is suitable for SPR measurements, demonstrating its capability to detect the presence of analytes and changes in surface properties both in static and dynamic set-ups.

Satellite interferometry has recently developed as a powerful tool for monitoring displacements on structures for structural health monitoring (SHM), as it allows obtaining information on past deformation and performing back analysis on structural behavior. Despite the increasing literature on this subject, the lack of protocols for applying and interpreting interferometric data for structural assessment prevents these techniques from being employed alongside conventional SHM. This paper proposes a methodology for exploiting satellite interferometric data aiming at remotely detecting displacements and buildings’ criticalities at different levels of analysis, i.e., urban scale and single-building scale. Moreover, this research exploits the capability of satellite monitoring for damage diagnosis, comparing the millimeter scale displacements to information derived from on-site inspections. Different data-driven algorithms were applied to detect seasonal and irreversible components of displacements, such as statistical models for damage identification derived from traditional on-site monitoring. Thus, the proposed methodology was applied to a XVI-century case study located in the city center of Rome (Italy), Palazzo Primoli, and two stocks of COSMO-SkyMed (CSK) images processed through the Small BAseline Subset Differential Interferometry (SBAS-DInSAR) technique were used to assess displacements for an eight-year-long (between 2011 and 2019) monitoring period.

In Brazil, a yellow fever (YF) outbreak was reported in areas considered YF-free for decades. The low vaccination coverage and the increasing forest fragmentation, with the wide distribution of vector mosquitoes, have been related to yellow fever virus (YFV) transmission beyond endemic areas since 2016. Aiming to elucidate the molecular and phylogenetic aspects of YFV spread on a local scale, we generated 43 new YFV genomes sampled from humans, non-human primates (NHP), and primarily, mosquitoes from highly heterogenic areas in 15 localities from Rio de Janeiro (RJ) state during the YFV 2016–2019 outbreak in southeast Brazil. Our analysis revealed that the genetic diversity and spatial distribution of the sylvatic transmission of YFV in RJ originated from at least two introductions and followed two chains of dissemination, here named the YFV RJ-I and YFV RJ-II clades. They moved with similar dispersal speeds from the north to the south of the RJ state in parallel directions, separated by the Serra do Mar Mountain chain, with YFV RJ-I invading the north coast of São Paulo state. The YFV RJ-I clade showed a more significant heterogeneity across the entire polyprotein. The YFV RJ-II clade, with only two amino acid polymorphisms, mapped at NS1 (I1086V), present only in mosquitoes at the same locality and NS4A (I2176V), shared by all YFV clade RJ-II, suggests a recent clustering of YFV isolates collected from different hosts. Our analyses strengthen the role of surveillance, genomic analyses of YVF isolated from other hosts, and environmental studies into the strategies to forecast, control, and prevent yellow fever outbreaks.

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016–2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.

The blood ammonia (NH3) level is one of the most important hepatic biomarkers for the diagnosis and monitoring of liver pathologies and infections. In this work, we developed an optimized optical biosensing method to extract and quantify the ammonia contained in complex-matrix samples emulating the blood serum. First, the approach was tested with solutions of phosphate-buffered saline (PBS) and ammonia chloride. Then, further trials were carried out with solutions of fetal bovine serum (FBS). The ammonia was extracted from the tested samples through a customized cell, and it was optically quantified by exploiting the indophenol reaction. The extraction cell included a cation-exchange membrane in Nafion, which was chemically pre-treated through cleaning procedures of sulfuric acid and hydrogen peroxide to keep a basic pH in the ammonia solution and to avoid contaminants in the membrane. From the NH3 solution, the indophenol reaction produced light-reactive indophenol dye molecules, which were used as colorimetric indicators. Through absorbance measurements of the indophenol dye solution at 670 nm wavelength, we were able to detect and quantify the ammonia level in the samples both with a spectrophotometer and a customized miniaturized read-out system, obtaining a detection limit of 0.029 µmol/mL.

Yellow fever virus (YFV) caused an outbreak in the Brazilian Southeast from 2016 to 2019, of the most significant magnitude since the 1900s. An investigation of the circulating virus revealed that most of the genomes detected in this period carried nine unique amino acid polymorphisms, with eight located in the non-structural proteins NS3 and NS5, which are pivotal for viral replication. To elucidate the effect of these amino acid changes on viral infection, we constructed viruses carrying amino acid alterations in NS3 and NS5, performed infection in different cells, and assessed their neurovirulence in BALB/c mice and infected AG129 mice. We observed that the residues that compose the YFV 2016–2019 molecular signature in the NS5 protein might have been related to an attenuated phenotype, and that the alterations in the NS3 protein only slightly affected viral infection in AG129 mice, increasing to a low extent the mortality rate of these animals. These results contributed to unveiling the role of specific naturally occurring amino acid changes in the circulating strain of YFV in Brazil.

The possibility of a Zika virus epidemic resurgence requires studies to understand its mechanisms of pathogenicity. Here, we describe the isolation of the Zika virus from breast milk (Rio-BM1) and compare its genetic and virological properties with two other isolates (Rio-U1 and Rio-S1) obtained during the same epidemic period. Complete genomic analysis of these three viral isolates showed that they carry characteristics of the American isolates and belong to the Asian genotype. Furthermore, we detected eight non-synonymous single nucleotide variants and multiple nucleotide polymorphisms that reflect phenotypic changes. The new isolate, Rio-BM1, showed the lowest replication rates in mammalian cells, induced lower cell death rates, was more susceptible to treatment with type I IFN, and was less pathogenic than Rio-U1 in a murine model. In conclusion, the present study shows evidence that the isolate Rio-BM1 is more attenuated than Rio-U1, probably due to the impact of genetic alterations in the modulation of virulence. The results obtained in our in vitro model were consistent with the pathogenicity observed in the animal model, indicating that this method can be used to assess the virulence level of other isolates or to predict the pathogenicity of reverse genetic constructs containing other polymorphisms.

The current work was designed to assess the effect of feed supplemented with essential oils (EOs) on the histological features in sea bass’s gastric mucosa. Fish were fed three diets: control diet (CTR), HERBAL MIX® made with natural EOs (N-EOs), or HERBAL MIX® made with artificial EOs obtained by synthesis (S-EOs) during a 117-day feeding trial. Thereafter, the oxyntopeptic cells (OPs) and the ghrelin (GHR) and somatostatin (SOM) enteroendocrine cells (EECs) in the gastric mucosa were evaluated. The Na+K+-ATPase antibody was used to label OPs, while, for the EECs, anti-SOM and anti-GHR antibody were used. The highest density of OP immunoreactive (IR) area was in the CTR group (0.66 mm2 ± 0.1). The OP-IR area was reduced in the N-EO diet group (0.22 mm2 ± 1; CTR vs. N-EOs, p < 0.005), while in the S-EO diet group (0.39 mm2 ± 1) a trend was observed. We observed an increase of the number of SOM-IR cells in the N-EO diet (15.6 ± 4.2) compared to that in the CTR (11.8 ± 3.7) (N-EOs vs. CTR; p < 0.05), but not in the S-EOs diet. These observations will provide a basis to advance current knowledge on the anatomy and digestive physiology of this species in relation to pro-heath feeds.

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

Several classes of flavonoids, such as anthocyanins, flavonols, flavanols, and flavones, undergo a slow H/D exchange on aromatic ring A, leading to full deuteration at positions C(6) and C(8). Within the flavanol class, H-C(6) and H-C(8) of catechin and epicatechin are slowly exchanged in D2O to the corresponding deuterated analogues. Even quercetin, a relevant flavonol representative, shows the same behaviour in a D2O/DMSOd6 1:1 solution. Detailed kinetic measurements of these H/D exchange processes are here reported by exploiting the time-dependent changes of their peak areas in the 1H-NMR spectra taken at different temperatures. A unifying reaction mechanism is also proposed based on our detailed kinetic observations, even taking into account pH and solvent effects. Molecular modelling and QM calculations were also carried out to shed more light on several molecular details of the proposed mechanism.

Obesity is a complex disease resulting in several metabolic co-morbidities and is increasing at epidemic rates. The marine environment is an interesting resource of novel compounds and in particular cyanobacteria are well known for their capacity to produce novel secondary metabolites. In this work, we explored the potential of cyanobacteria for the production of compounds with relevant activities towards metabolic diseases using a blend of target-based, phenotypic and zebrafish assays as whole small animal models. A total of 46 cyanobacterial strains were grown and biomass fractionated, yielding in total 263 fractions. Bioactivities related to metabolic function were tested in different in vitro and in vivo models. Studying adipogenic and thermogenic gene expression in brown adipocytes, lipid metabolism and glucose uptake in hepatocytes, as well as lipid metabolism in zebrafish larvae, we identified 66 (25%) active fractions. This together with metabolite profiling and the evaluation of toxicity allowed the identification of 18 (7%) fractions with promising bioactivity towards different aspects of metabolic disease. Among those, we identified several known compounds, such as eryloside T, leptosin F, pheophorbide A, phaeophytin A, chlorophyll A, present as minor peaks. Those compounds were previously not described to have bioactivities in metabolic regulation, and both known or unknown compounds could be responsible for such effects. In summary, we find that cyanobacteria hold a huge repertoire of molecules with specific bioactivities towards metabolic diseases, which needs to be explored in the future.

Skeletal muscles are the agent of motion and one of the most important tissues responsible for the control of metabolism. The maintenance of muscle homeostasis is finely regulated by the balance between catabolic and anabolic process. Macroautophagy (or autophagy) is a catabolic process that provides the degradation of protein aggregation and damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagy flux is fundamental for the homeostasis of skeletal muscles during physiological situations and in response to stress. Defective as well as excessive autophagy is harmful for muscle health and has a pathogenic role in several forms of muscle diseases. This review will focus on the role of autophagy in muscle homeostasis and diseases.