Genetic Complexity of CC5 Staphylococcus aureus Isolates Associated with Sternal Bursitis in Chickens: Antimicrobial Resistance, Virulence, Plasmids, and Biofilm Formation.

Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of Staphylococcus aureus isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 S. aureus isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems.

Chalcone derivatives as promising antifoulants: Molecular optimization, bioactivity evaluation and performance in coatings.

Marine biofouling remains a huge concern for maritime industries and for environmental health. Although the current biocide-based antifouling coatings can prevent marine biofouling, their use has been associated with toxicity for the marine environment, being urgent to find sustainable alternatives. Previously, our research group has identified a prenylated chalcone (1) with promising antifouling activity against the settlement of larvae of the macrofouling species Mytilus galloprovincialis (EC50 = 16.48 µM and LC50 > 200 µM) and lower ecotoxicity when compared to Econea®, a commercial antifouling agent in use. Herein, a series of chalcone 1 analogues were designed and synthesized in order to obtain optimized antifouling compounds with improved potency while maintaining low ecotoxicity. Compounds 8, 15, 24, and 27 showed promising antifouling activity against the settlement of M. galloprovincialis larvae, being dihydrochalcone 27 the most potent. The effect of compound 24 was associated with the inhibition of acetylcholinesterase activity. Among the synthesized compounds, compound 24 also showed potent complementary activity against Navicula sp. (EC50 = 4.86 µM), similarly to the lead chalcone 1 (EC50 = 6.75 µM). Regarding the structure-activity relationship, the overall results demonstrate that the substitution of the chalcone of the lead compound 1 by a dihydrochalcone scaffold resulted in an optimized potency against the settlement of mussel larvae. Marine polyurethane (PU)-based coatings containing the best performed compound concerning anti-settlement activity (dihydrochalcone 27) were prepared, and mussel larvae adherence was reduced compared to control PU coatings.

Correction: Palmeira et al. Preliminary Virtual Screening Studies to Identify GRP78 Inhibitors Which May Interfere with SARS-CoV-2 Infection. Pharmaceuticals 2020, 13, 132.

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Meat juice as a feasible alternative sample for tuberculosis surveillance in large game.

In hunted animals, quality of blood samples may often be compromised. Alternative samples, such as meat juice, may offer an advantage to perform serological tests. This study evaluates if meat juice is a feasible alternative sample to perform the Tuberculosis ELISA test in hunted large game. Between 2017 and 2022, 175 samples were collected from 97 animals (14 red deer + 83 wild boar) in Portugal and Spain. Cohen's kappa coefficient was calculated at 0.71, pointing out a good agreement using 156 paired samples. The sensitivity of the ELISA test with serum was 37.6%, considering Tuberculosis-like lesions (TBL) detected during the initial examination (26 TBL+/ELISA+ in a total of 78 serum samples). Using meat juice as matrix, the sensitivity increased to 37.5% (33 TBL+/ELISA+ in 97 meat juice samples). According to the agreement score and sensitivity being so close between the two matrices tested, meat juice could be a feasible alternative matrix.

Seroprevalence study of brucellosis in wild boar hunted for private consumption in northeast Portugal.

Brucellosis is an important infectious disease caused by bacteria of the genus Brucella. In the northeast region of Portugal, infection with Brucella melitensis is endemic in small ruminants, and there are also humans' cases. However, the epidemiological role of the wild boar in the dynamics of this disease in this region is unknown. In this study, a total of 332 blood samples were collected from wild boar hunted in thirty-six hunting areas during the 2022/2023 hunting season. All were taken by the hunters for private consumption, with no evisceration or examination in the field. Serum samples were tested by indirect ELISA (i-ELISA). It was observed that 88 wild boars were exposed to Brucella spp., pointing to a seroprevalence of 26.5% (95% CI: 21.8 - 31.3%). This high prevalence underlines the importance that wild boar may have in the dynamics of this disease in the region and its potential transmission to other animals, and to humans (for example, during the handling of carcasses). Increased awareness and knowledge of brucellosis in wild boar is essential for the implementation of effective practices and habits and, consequently, for the control and prevention of this important zoonosis.

Antibacterial Potential of Symmetrical Twin-Drug 3,6-Diaminoxanthones.

Global health faces a significant issue with the rise of infectious diseases caused by bacteria, fungi, viruses, and parasites. The increasing number of multi-drug resistant microbial pathogens severely threatens public health worldwide. Antibiotic-resistant pathogenic bacteria, in particular, present a significant challenge. Therefore, there is an urgent need to identify new potential antimicrobial targets and discover new chemical entities that can potentially reverse bacterial resistance. The main goal of this research work was to create and develop a library of 3,6-disubstituted xanthones based on twin drugs and molecular extension approaches to inhibit the activity of efflux pumps. The process involved synthesizing 3,6-diaminoxanthones through the reaction of 9-oxo-9H-xanthene-3,6-diyl bis(trifluoromethanesulfonate) with various primary and secondary amines. The resulting 3,6-disubstituted xanthone derivatives were then tested for their in vitro antimicrobial properties against a range of pathogenic strains and their efficacy in inhibiting the activity of efflux pumps, biofilm formation, and quorum-sensing. Several compounds have exhibited effective antibacterial properties against the Gram-positive bacterial species tested. Xanthone 16, in particular, has demonstrated exceptional efficacy with a remarkable MIC of 11 µM (4 µg/mL) against reference strains Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212, and 25 µM (9 µg/mL) against methicillin-resistant S. aureus 272123. Furthermore, some derivatives have shown potential as antibiofilm agents in a crystal violet assay. The ethidium bromide accumulation assay pinpointed certain compounds inhibiting bacterial efflux pumps. The cytotoxic effect of the most promising compounds was examined in mouse fibroblast cell line NIH/3T3, and two monoamine substituted xanthone derivatives with a hydroxyl substituent did not exhibit any cytotoxicity. Overall, the nature of the substituent was critical in determining the antimicrobial spectra of aminated xanthones.

The Role of Natural and Synthetic Flavonoids in the Prevention of Marine Biofouling.

Marine biofouling is a major concern for the maritime industry, environment, and human health. Biocides which are currently used in marine coatings to prevent this phenomenon are toxic to the marine environment, and therefore a search for antifoulants with environmentally safe properties is needed. A large number of scientific papers have been published showing natural and synthetic compounds with potential to prevent the attachment of macro- and microfouling marine organisms on submerged surfaces. Flavonoids are a class of compounds which are highly present in nature, including in marine organisms, and have been found in a wide range of biological activities. Some natural and synthetic flavonoids have been evaluated over the last few years for their potential to prevent the settlement and/or the growth of marine organisms on submerged structures, thereby preventing marine biofouling. This review compiles, for the first-time, natural flavonoids as well as their synthetic analogues with attributed antifouling activity against macrofouling and microfouling marine organisms.

Evaluation of Antitumor Activity of Xanthones Conjugated with Amino Acids.

Cancer is a complex disease characterized by several alterations, which confer, to the cells, the capacity to proliferate uncontrollably and to resist cellular death. Multiresistance to conventional chemotherapy drugs is often the cause of treatment failure; thus, the search for natural products or their derivatives with therapeutic action is essential. Chiral derivatives of xanthones (CDXs) have shown potential inhibitory activity against the growth of some human tumor cell lines. This work reports the screening of a library of CDXs, through viability assays, in different cancer cell lines: A375-C5, MCF-7, NCI-H460, and HCT-15. CDXs' effect was analyzed based on several parameters of cancer cells, and it was also verified if these compounds were substrates of glycoprotein-P (Pgp), one of the main mechanisms of resistance in cancer therapy. Pgp expression was evaluated in all cell lines, but no expression was observed, except for HCT-15. Also, when a humanized yeast expressing the human gene MDR1 was used, no conclusions could be drawn about CDXs as Pgp substrates. The selected CDXs did not induce significant differences in the metabolic parameters analyzed. These results show that some CDXs present promising antitumor activity, but other mechanisms should be triggered by these compounds.

BP-M345 as a Basis for the Discovery of New Diarylpentanoids with Promising Antimitotic Activity.

Recently, the diarylpentanoid BP-M345 (5) has been identified as a potent in vitro growth inhibitor of cancer cells, with a GI50 value between 0.17 and 0.45 µM, showing low toxicity in non-tumor cells. BP-M345 (5) promotes mitotic arrest by interfering with mitotic spindle assembly, leading to apoptotic cell death. Following on from our previous work, we designed and synthesized a library of BP-M345 (5) analogs and evaluated the cell growth inhibitory activity of three human cancer cell lines within this library in order to perform structure-activity relationship (SAR) studies and to obtain compounds with improved antimitotic effects. Four compounds (7, 9, 13, and 16) were active, and the growth inhibition effects of compounds 7, 13, and 16 were associated with a pronounced arrest in mitosis. These compounds exhibited a similar or even higher mitotic index than BP-M345 (5), with compound 13 displaying the highest antimitotic activity, associated with the interference with mitotic spindle dynamics, inducing spindle collapse and, consequently, prolonged mitotic arrest, culminating in massive cancer cell death by apoptosis.

Polymersomes for Sustained Delivery of a Chalcone Derivative Targeting Glioblastoma Cells.

Glioblastoma (GBM) is a primary malignant tumor of the central nervous system responsible for the most deaths among patients with primary brain tumors. Current therapies for GBM are not effective, with the average survival of GBM patients after diagnosis being limited to a few months. Chemotherapy is difficult in this case due to the heterogeneity of GBM and the high efficacy of the blood-brain barrier, which makes drug absorption into the brain extremely difficult. In a previous study, 3',4',3,4,5-trimethoxychalcone (MB) showed antiproliferative and anti-invasion activities toward GBM cells. Polymersomes (PMs) are an attractive, new type of nanoparticle for drug administration, due to their high stability, enhanced circulation time, biodegradability, and sustained drug release. In the present study, different MB formulations, PEG2000-PCL and PEG5000-PCL, were synthesized, characterized, and compared in terms of 14-day stability and in vitro cytotoxicity (hCMEC/D3 and U-373 MG).

The Synthesis and Characterization of a Delivery System Based on Polymersomes and a Xanthone with Inhibitory Activity in Glioblastoma.

Glioblastoma (GBM) is the most common and deadly primary malignant brain tumor. Current therapies are insufficient, and survival for individuals diagnosed with GBM is limited to a few months. New GBM treatments are urgent. Polymeric nanoparticles (PNs) can increase the circulation time of a drug in the brain capillaries. Polymersomes (PMs) are PNs that have been described as having attractive characteristics, mainly due to their stability, prolonged circulation period, biodegradability, their ability to sustain the release of drugs, and the possibility of surface functionalization. In this work, a poly(ethylene glycol)-ε-caprolactone (PEG-PCL) copolymer was synthesized and PMs were prepared and loaded with an hydrolytic instable compound, previously synthesized by our research team, the 3,6-bis(2,3,4,6-tetra-O-acetyl-ß-glucopyranosyl)xanthone (XGAc), with promising cytotoxicity on glioblastoma cells (U-373 MG) but also on healthy cerebral endothelial cells (hCMEC/D3). The prepared PMs were spherical particles with uniform morphology and similar sizes (mean diameter of 200 nm) and were stable in aqueous suspension. The encapsulation of XGAc in PMs (80% encapsulation efficacy) protected the healthy endothelial cells from the cytotoxic effects of this compound, while maintaining cytotoxicity for the glioblastoma cell line U-373 MG. Our studies also showed that the prepared PMs can efficiently release XGAc at intratumoral pHs.

The regulatory landscape of 5' UTRs in translational control during zebrafish embryogenesis.

The 5' UTRs of mRNAs are critical for translation regulation, but their in vivo regulatory features are poorly characterized. Here, we report the regulatory landscape of 5' UTRs during early zebrafish embryogenesis using a massively parallel reporter assay of 18,154 sequences coupled to polysome profiling. We found that the 5' UTR is sufficient to confer temporal dynamics to translation initiation, and identified 86 motifs enriched in 5' UTRs with distinct ribosome recruitment capabilities. A quantitative deep learning model, DaniO5P, revealed a combined role for 5' UTR length, translation initiation site context, upstream AUGs and sequence motifs on in vivo ribosome recruitment. DaniO5P predicts the activities of 5' UTR isoforms and indicates that modulating 5' UTR length and motif grammar contributes to translation initiation dynamics. This study provides a first quantitative model of 5' UTR-based translation regulation in early vertebrate development and lays the foundation for identifying the underlying molecular effectors.

A Preliminary Investigation of Salmonella Populations in Indigenous Portuguese Layer Hen Breeds.

The sustainability of agroecological systems, biodiversity protection, animal welfare, and consumer demand for higher quality products from alternative and extensive farming methods have reinforced interest in local breeds that are well adapted to low-input environments. However, food safety needs to be safeguarded to reinforce consumer confidence. The aim of this study was to conduct a preliminary investigation on the occurrence of Salmonella spp. in eggshells, hen's cloaca, and litter materials from autochthonous Portuguese laying hens raised in a semi-extensive system for small-scale production. A total of 279 samples from 31 flocks belonging to 12 farms were obtained, with 63 samples from the "Preta Lusitânica" breed, and 72 samples each from the remaining autochthonous breeds, namely, "Branca", "Amarela", and "Pedrês Portuguesa". None (0%) of the samples analyzed were positive for Salmonella spp. To the best of our knowledge, these are the first results of Salmonella evaluation from hen's cloaca, eggshells, and litter materials in autochthonous Portuguese chickens, suggesting that a semi-extensive production system can contribute to better food security and a lower risk to public health and the environment.

Structure-Activity Relationship Studies of Chalcones and Diarylpentanoids with Antitumor Activity: Potency and Selectivity Optimization.

We previously reported that chalcone CM-M345 (1) and diarylpentanoid BP-C4 (2) induced p53-dependent growth inhibitory activity in human cancer cells. Herein, CM-M345 (1) and BP-C4 (2) analogues were designed and synthesized in order to obtain more potent and selective compounds. Compounds 16, 17, 19, 20, and 22-24 caused pronounced in vitro growth inhibitory activity in HCT116 cells (0.09 < GI50 < 3.10 µM). Chemical optimization of CM-M345 (1) led to the identification of compound 36 with increased selectivity for HCT116 cells expressing wild-type p53 compared to its p53-null isogenic derivative and low toxicity to non-tumor HFF-1 cells. The molecular modification of BP-C4 (2) resulted in the discovery of compound 16 with more pronounced antiproliferative activity and being selective for HCT116 cells with p53, as well as 17 with enhanced antiproliferative activity against HCT116 cells and low toxicity to non-tumor cells. Compound 16 behaved as an inhibitor of p53-MDM2 interaction, and compound 17 was shown to induce apoptosis, associated with an increase in cleaved PARP and decreased levels of the anti-apoptotic protein Bcl-2. In silico studies allowed us to predict the druglikeness and ADMET properties for 16 and 17. Docking and molecular dynamics studies predicted that 16 could bind stably to the MDM2 binding pocket.

First Serologic Survey of Erysipelothrix rhusiopathiae in Wild Boars Hunted for Private Consumption in Portugal.

Erysipelothrix rhusiopathiae is a relevant zoonotic infectious agent causing swine erysipelas (SE) in wild boar. In Portugal, there is no information on its occurrence. For this reason, this study aims to perform a first serosurvey of SE in hunted wild boars in Portugal. During the 2019/2020 hunting season, 111 sera from hunted wild boar were collected and analysed serologically in the laboratory with a commercial ELISA kit. No animals were eviscerated and examined after the hunt. The hunters took it all for private consumption. The results identified 18 animals that were exposed to SE, corresponding to a seroprevalence of 16.2% (95% CI: 19.9-24.4%). No statistical significance was observed on the effect of gender and age on seropositivity. However, wild boar hunted in Pinhel County, had five times more likely to be seropositivity (p-value < 0.05; OD = 5.4). Apart from its potential debilitating capacity and chronicity in the wild boar population, SE is also a very serious occupational zoonosis. Thus, the result of this first serosurvey in Portugal should raise awareness and alert competent national veterinary authorities and those involved in the hunting sector, especially hunters who directly handle these carcasses. Further studies should be conducted to better understand the role of wild boar as a reservoir and spillover of this disease to other animals and humans.

Hepatitis E Virus in the Wild Boar Population: What Is the Real Zoonotic Risk in Portugal?

Hepatitis E virus (HEV) is an important zoonosis in wild boar. Reported zoonotic cases are mainly associated with the consumption of raw/undercooked meat and/or liver. This study aims to determine the occurrence of HEV in the Portuguese wild boar population. During the hunting season 2021/2022, 123-matched samples (liver, faeces, and blood) were collected from hunted wild boars throughout Portugal. An RT-PCR assay tested liver and faeces samples to detect HEV-RNA. From blood samples, an ELISA test was performed. Only one liver sample was positive for HEV (0,8%) and one other from faeces. A total of 34 sera were seropositive (26.7%). At the same time, in a survey of 106 hunters, 21 consumed/ate the liver of wild boars (19.8%). Only three recognised the possibility of consuming it undercooked. Contrary to previous studies in Portugal, the prevalence of HEV in liver and faeces is low, but the seropositivity is higher. But, when analyzing in detail, it could be observed that an HEV hotspot exists in the southeast of central Portugal and that it is a zoonotic risk for hunters of this region. The data of this study reinforce the importance of including HEV in surveillance programs for wildlife diseases to expand the potential zoonotic information.

Salmonella spp., Escherichia coli and Enterobacteriaceae Control at a Pig Abattoir: Are We Missing Lairage Time Effect, Pig Skin, and Internal Carcass Surface Contamination?

To provide meat safety and consumer protection, appropriate hygiene control measures at an abattoir are required. This study aimed to evaluate the influence of visual fecal contamination level (VFCL) and lairage time (LT) on pig skin (PS) and external (ECS) and internal (ICS) carcass surfaces. The presence of Enterobacteriaceae, Escherichia coli (E. coli) and Salmonella in PS, ECS, and ICS were evaluated. A total of 300 paired samples were collected from 100 pigs. Results underlined the importance of the skin (Enterobacteriaceae: 3.27 ± 0.68 log CFU/cm2; E. coli: 3.15 ± 0.63 log CFU/cm2; Salmonella: 21% of samples) as a direct or indirect source of carcass contamination. Although VFCL revealed no significant effect (p > 0.05), the increase of LT had a significant impact (p < 0.001) on Enterobacteriaceae and E. coli levels across all analysed surfaces, and Salmonella presence on ICS (p < 0.01), demanding attention to LT. Also, the ICS showed a higher level of these bacteria compared to ECS. These results highlight the need of food business operators to consider ICS as an alternative area to sample for Salmonella, as a criterion for process hygiene based on EC Regulation No. 2073/2005, and as a potential contamination source to be integrated in the hazard analysis critical control point (HACCP) plans.

15 years overview of European zoonotic surveys in wild boar and red deer: A systematic review.

Wild boar (Sus scrofa) and red deer (Cervus elaphus) are the main large game species hunted in Europe. They can also be a source of zoonotic infections for cohabiting humans. The purpose of this systematic review was to examine the spatiotemporal tendencies and sanitary profiles of surveys on zoonotic diseases of wild boars and red deer in Europe in 15 years (2006-2020). Through the search strategy "((sus scrofa OR wild boar OR cervus elaphus OR red deer) AND (zoonosis OR zoonot* OR infectious disease))" in Pubmed and ScienceDirect databases, 1419 articles were assessed in February of 2021. Pursuing the inclusion criteria: species of interest - wild boar and red deer, established zoonosis and presence of natural infection and the exclusion filters: European study, specified a timeline (2006-2020), printed in English and with open-access. To conduct this systematic review, 194 European surveys issued in indexed journals were included after revising all abstracts and eliminating 323 unrepeated articles. Geographically, dissimilarity in the pattern of distribution of surveys was uttered. In the short term, the pattern of the number of publications about zoonotic diseases in wild boars and red deer oscillates, but with an increasing tendency over 15 years under study. When examining the sanitary profile of the eligible surveys, the focus is mostly on zoonoses such as Hepatitis E virus, Toxoplasmosis, Trichinellosis, Salmonellosis and Tuberculosis. With the high growth in the population of these large game species in Europe and the previous gaps in their sanitary profile, the number of surveys has been endorsed in the defined 15 years period. Based on the One Health concept and prioritizing the issue of the occurrence of zoonoses as a matter of Public Health, there must be increasing apprehension about that and enhanced knowledge about their potential risk for veterinarians, hunters and other agents involved in the hunting sector.

Discovery of a New Chalcone-Trimethoxycinnamide Hybrid with Antimitotic Effect: Design, Synthesis, and Structure-Activity Relationship Studies.

In this work, the design and synthesis of a new chalcone-trimethoxycinnamide hybrid (7) based on the combination of subunits of two promising antiproliferative compounds (CM-M345 (1) and BP-M345 (2)), previously obtained by our research group, are reported. In order to expand the structure-activity relationship (SAR) knowledge, a new series of 7-analogues was also designed and synthetized. All the compounds were evaluated for their antitumor activity against melanoma (A375-C5), breast adenocarcinoma (MCF-7), and colorectal carcinoma (HCT116) cell lines, as well as non-tumor HPAEpiC cells. Three of the newly synthesized compounds (6, 7, and 13) exhibited potent antiproliferative activity, mainly on colorectal tumor cells (GI50 = 2.66-3.26 µM), showing hybrid 7 selectivity for tumor cells. We performed molecular mechanism studies to evaluate the potential interference of compounds with the p53 pathway, namely, p53-MDM2 interaction and mitosis in HCT116 cells. The antiproliferative activities of compounds were shown to be p53-independent. Compound 7 emerged as an antimitotic agent by inducing the mitotic arrest of colorectal tumor cells, and subsequently, cell death.

Synthesis and Anti-Inflammatory Evaluation of a Library of Chiral Derivatives of Xanthones Conjugated with Proteinogenic Amino Acids.

In recent decades, the relationship between drug chirality and biological activity has been assuming enormous importance in medicinal chemistry. Particularly, chiral derivatives of xanthones (CDXs) have interesting biological activities, including enantioselective anti-inflammatory activity. Herein, the synthesis of a library of CDXs is described, by coupling a carboxyxanthone (1) with both enantiomers of proteinogenic amino esters as chiral building blocks (2-31), following the chiral pool strategy. The coupling reactions were performed at room temperature with good yields (from 44 to 99.9%) and very high enantiomeric purity, with most of them presenting an enantiomeric ratio close to 100%. To afford the respective amino acid derivatives (32-61), the ester group of the CDXs was hydrolyzed in mild alkaline conditions. Consequently, in this work, sixty new derivatives of CDXs were synthetized. The cytocompatibility and anti-inflammatory activity in the presence of M1 macrophages were studied for forty-four of the new synthesized CDXs. A significant decrease in the levels of a proinflammatory cytokine targeted in the treatment of several inflammatory diseases, namely interleukin 6 (IL-6), was achieved in the presence of many CDXs. The amino ester of L-tyrosine (X1AELT) was the most effective in reducing IL-6 production (52.2 ± 13.2%) by LPS-stimulated macrophages. Moreover, it was ≈1.2 times better than the D-enantiomer. Indeed, enantioselectivity was observed for the majority of the tested compounds. Thus, their evaluation as promising anti-inflammatory drugs should be considered.