First Insights on Resistance and Virulence Potential of Escherichia coli from Captive Birds of Prey in Portugal.

Captive birds of prey are often used for pest control in urban areas, while also participating in falconry exhibitions. Traveling across the country, these birds may represent a public health concern as they can host pathogenic and zoonotic agents and share the same environment as humans and synanthropic species. In this work, Escherichia coli from the cloacal samples of 27 captive birds of prey were characterized to determine their pathogenic potential. Isolates were clustered through ERIC-PCR fingerprinting, and the phylogenetic groups were assessed using a quadruplex PCR method. Their virulence and resistance profile against nine antibiotics were determined, as well as the isolates' ability to produce extended-spectrum ß-lactamases (ESBLs). The 84 original isolates were grouped into 33 clonal types, and it was observed that more than half of the studied isolates belonged to groups D and B2. Most isolates presented gelatinase activity (88%), almost half were able to produce biofilm (45%), and some were able to produce α-hemolysin (18%). The isolates presented high resistance rates towards piperacillin (42%), tetracycline (33%), and doxycycline (30%), and 6% of the isolates were able to produce ESBLs. The results confirm the importance of these birds as reservoirs of virulence and resistance determinants that can be disseminated between wildlife and humans, stressing the need for more studies focusing on these animals.

Impact of infrasound exposure and streptozotocin-induced glucose intolerance on bone composition in Wistar rats.

The elemental composition of chemical elements can vary between healthy and diseased tissues, providing essential insights into metabolic processes in physiological and diseased states. This study aimed to evaluate the calcium (Ca) and phosphorus (P) levels in the bones of rats with/without streptozotocin-induced diabetes and/or exposure to infrasound. X-ray fluorescence spectroscopy was used to determine the concentrations of Ca and P in Wistar rat tibiae samples.The results showed a significant decrease in bone P concentration in streptozotocin-induced diabetic rats compared to untreated animals. Similarly, the Ca/P ratio was higher in the streptozotocin-induced diabetic group. No significant differences were observed in bone Ca concentration between the studied groups or between animals exposed and not exposed to infrasound.Moreover, streptozotocin-induced diabetic rats had lower bone P concentration but unaltered bone Ca concentration compared to untreated rats. Infrasound exposure did not impact bone Ca or P levels. The reduced bone P concentration may be associated with an increased risk of bone fractures in diabetes.

When the solution becomes the problem: a review on antimicrobial resistance in dairy cattle.

Antibiotics' action, once a 'magic bullet', is now hindered by widespread microbial resistance, creating a global antimicrobial resistance (AMR) crisis. A primary driver of AMR is the selective pressure from antimicrobial use. Between 2000 and 2015, antibiotic consumption increased by 65%, reaching 34.8 billion tons, 73% of which was used in animals. In the dairy cattle sector, antibiotics are crucial for treating diseases like mastitis, posing risks to humans, animals and potentially leading to environmental contamination. To address AMR, strategies like selective dry cow therapy, alternative treatments (nanoparticles, phages) and waste management innovations are emerging. However, most solutions are in development, emphasizing the urgent need for further research to tackle AMR in dairy farms.

Antibiotics are becoming less effective at fighting infections because of antimicrobial resistance (AMR). This phenomenon is mainly caused by the abuse and misuse of antibiotics in both human and veterinary medicine. In the dairy cow industry, the use of antibiotics to treat diseases is a big concern. Ways to tackle this include the promotion of the responsible use of antibiotics, the development of alternative treatments and the discovery of better methods to deal with animal waste. However, much of these are still in development.

Design of a multi-epitope-based vaccine candidate against Bovine Genital Campylobacteriosis using a reverse vaccinology approach.

BACKGROUND: Bovine Genital Campylobacteriosis (BGC), a worldwide distributed venereal disease caused by Campylobacter fetus subsp. venerealis (Cfv), has a relevant negative economic impact in cattle herds. The control of BGC is hampered by the inexistence of globally available effective vaccines. The present in silico study aimed to develop a multi-epitope vaccine candidate against Cfv through reverse vaccinology. RESULTS: The analysis of Cfv strain NCTC 10354 proteome allowed the identification of 9 proteins suitable for vaccine development. From these, an outer membrane protein, OmpA, and a flagellar protein, FliK, were selected for prediction of B-cell and T-cell epitopes. The top-ranked epitopes conservancy was assessed in 31 Cfv strains. The selected epitopes were integrated to form a multi-epitope fragment of 241 amino acids, which included 2 epitopes from OmpA and 13 epitopes from FliK linked by GPGPG linkers and connected to the cholera toxin subunit B by an EAAAK linker. The vaccine candidate was predicted to be antigenic, non-toxic, non-allergenic, and soluble upon overexpression. The protein structure was predicted and optimized, and the sequence was successfully cloned in silico into a plasmid vector. Additionally, immunological simulations demonstrated the vaccine candidate's ability to stimulate an immune response. CONCLUSIONS: This study developed a novel vaccine candidate suitable for further in vitro and in vivo experimental validation, which may become a useful tool for the control of BGC.

Measuring paw preferences in dogs, cats and rats: Design requirements and innovations in methodology.

Studying behavioural lateralization in animals holds great potential for answering important questions in laterality research and clinical neuroscience. However, comparative research encounters challenges in reliability and validity, requiring new approaches and innovative designs to overcome. Although validated tests exist for some species, there is yet no standard test to compare lateralized manual behaviours between individuals, populations, and animal species. One of the main reasons is that different fine-motor abilities and postures must be considered for each species. Given that pawedness/handedness is a universal marker for behavioural lateralization across species, this article focuses on three commonly investigated species in laterality research: dogs, cats, and rats. We will present six apparatuses (two for dogs, three for cats, and one for rats) that enable an accurate assessment of paw preference. Design requirements and specifications such as zoometric fit for different body sizes and ages, reliability, robustness of the material, maintenance during and after testing, and animal welfare are extremely important when designing a new apparatus. Given that the study of behavioural lateralization yields crucial insights into animal welfare, laterality research, and clinical neuroscience, we aim to provide a solution to these challenges by presenting design requirements and innovations in methodology across species.

Rescue of mitochondrial import failure by intercellular organellar transfer.

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cell's energy supplies - particularly troublesome for energy-demanding cells of nervous tissue and muscle. Unsurprisingly then, import breakdown is implicated in disease. Here, we explore the consequences of import failure in mammalian cells; wherein, blocking the import machinery impacts mitochondrial ultra-structure and dynamics, but, surprisingly, does not affect import. Our data are consistent with a response involving intercellular mitochondrial transport via tunnelling nanotubes to import healthy mitochondria and jettison those with blocked import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial dysfunction.

Comparative genomics reveals probable adaptations for xylose use in Thermoanaerobacterium saccharolyticum.

Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels' industry.

Functional characterization and structural insights of three cutinases from the ascomycete Fusarium verticillioides.

Cutinases are serine esterases that belong to the α/ß hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.

An atlas of rational genetic engineering strategies for improved xylose metabolism in Saccharomyces cerevisiae.

Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

Efficacy of the Feliway® Classic Diffuser in reducing undesirable scratching in cats: A randomised, triple-blind, placebo-controlled study.

Scratching the environment is a natural behaviour that cats use for communication and physical maintenance purposes, however when it is carried out on household furniture it is considered unacceptable for some owners and even grounds for relinquishment of cats. The objective of this study was to investigate the efficacy of FELIWAY® Classic Diffuser in reducing undesirable scratching (scratching vertical surfaces indoors other than the scratching post) in cats. A 28 day, randomised, triple-blind, placebo-controlled study with a total of 1060 caregiver-cat dyads was conducted. The study contained two groups: the Pheromone Group consisted of caregivers who were given a pheromone diffuser (n = 546) and the Placebo Group consisted of caregivers who were given a placebo diffuser (n = 514). A questionnaire with three subsections was distributed online. The first section, completed by the respondents at day 0, inquired about the cats' daily routines, social and physical environments, behaviour, temperament, and emotional states. The second section filled on day 0, 7, 14, and 28, assessed the Frequency and the Intensity of the undesirable scratching problem and the effectiveness of the product. The last section, filled on the 28th day of the product application, related to the caregivers' opinions about the product and overall outcome. After 28 days the scratching Frequency reduced for 83.5% of the cats in the Pheromone Group and 68.5% for the Placebo Group (p<0.0001). The Intensity was significantly different between treatment groups at D7 (p = 0.0170), at D14 (p = 0.0189) and at D28 (p<0.001). The reduction of the Global Index Score, which was calculated by multiplying the Intensity with the Frequency, was significantly higher for the Pheromone Group (p<0.001). This study provides direct evidence that the use of FELIWAY® Classic diffuser significantly reduces the Frequency, Intensity and the Global Index Score of undesirable scratching.

Effect of Diet Supplementation with Oat Hay and Whole Carrot on Rabbit Growth and Productive Efficiency.

Is it possible to reduce feeding costs in rabbit meat production without compromising rabbit health and productive yield? The study tested four feeding strategies: Control group (CC) fed exclusively with concentrate feed; group CT supplemented with whole carrot; group OH supplemented with oat hay; and Group CO supplemented with oat hay and whole carrot. Each feeding strategy was tested in 20 rabbits, randomly allocated in five cages with four rabbits each. The average daily weight gain (ADG), feed conversion ratio (FCR), and the amount of concentrated feed consumed daily were estimated in all experimental groups. Group CC displayed the best ADG (37.1 g/rabbit/day), carrot had no significant influence on ADG (34.2 g/rabbit/day), but oat hay had a negative impact (p < 0.05), either used alone or in combination with carrot (33.0 and 32.6 g/rabbit/day, respectively). Supplementation with carrot, oat hay, or both increased the FCR (p < 0.001). Nevertheless, there were no significant differences in final live weight or carcass weight between the rabbits in the different experimental groups. In conclusion, supplementation with oat hay, carrot, or both can be a valid approach to reducing production costs by decreasing concentrate feed without affecting rabbit's health and meat yield. The combined supplementation with oat hay and carrot proved to be the best option in reducing the amount of concentrate feed ingested by rabbits (less than 1107 g/animal), but at current market values, supplementation exclusively with oat hay was the less expensive feeding strategy (less 14% than fed exclusively with concentrate feeding).

Characterization of circulating microRNA profiles of postpartum dairy cows with persistent subclinical endometritis.

Subclinical endometritis (SCE) is an unresolved inflammation of the endometrium of postpartum dairy cows, seriously affecting fertility. Current diagnosis, which relies on uterine cytology or even more invasive biopsy sampling, would benefit from the identification of blood-based diagnostic biomarkers. Due to the known role of microRNAs (miRNAs) in other diseases, this case-control study evaluated the cell-free circulating miRNA profiles of SCE cows, and the network of transcripts predicted to interact with those miRNAs, previously identified as differentially expressed genes (DEG) in the endometrium of the same cows. Healthy (H, n = 6) and persistent SCE (n = 11) cows characterized by endometrial cytology and biopsy were blood sampled at 21 and 44 d postpartum (DPP). Following extraction of cell-free plasma miRNAs and RNA-seq analysis, differential abundance analysis of miRNAs was performed with the DESeq2 R package (adjusted p-value of 0.05), and in silico prediction of miRNA-interacting genes on a sequence complementary basis was conducted using the miRWalk database. The principal component analysis showed a clear clustering between groups of uterine health phenotypes (H vs. SCE), although the clustering between groups was less pronounced at 44 DPP than at 21 DPP. No effect of the stage (21 vs. 44 DPP) was observed. A total of 799 known circulating miRNAs were identified, from which 34 demonstrated differential abundance between H and SCE cows (12 less abundant and 22 more abundant in SCE than in H cows). These 34 miRNAs are predicted to interact with 10,104 transcripts, among which 43, 81, and 147 were previously identified as differentially expressed in, respectively, endometrial luminal epithelial, glandular epithelial, and stromal cells of the same cows. This accounts for approximately half of the DEG identified between those H and SCE cows, including genes involved in endometrial cell proliferation, angiogenesis and immune response, whose dysregulation in SCE cows may impair pregnancy establishment. From 219 miRNAs with mean normalized read counts above 100, the presence and abundance of miR-425-3p and miR-2285z had the highest discriminatory level to differentiate SCE from H cows. In conclusion, despite apparent confinement to the endometrium, SCE is associated with a distinct circulating miRNA profile, which may represent a link between the systemic changes associated with disease and the endometrial immune response. The validation of a miRNA panel consisting of circulating cell-free miR-425-3p and miR-2285z may prove a relevant advancement for the noninvasive diagnosis of persistent SCE.

A novel insight on SARS-CoV-2 S-derived fragments in the control of the host immunity.

Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.

Behavioural responses to separation from human companion in the domestic cat: A survey-based study.

The domestic cat is known to react to social separation, though the conceptual relationship between separation-related behaviours outside of a clinical context has not been described in detail. We did an online survey on participants who had cats in their households (Nparticipants=114; Ncats=133) and asked them to evaluate the frequency of 12 behavioural elements associated with social separation from human companions on a 5- point Likert Scale. We performed two dimensionality reduction techniques (component and factor analyses) to assess whether the specified behaviours related to social separation belonged to the same axis. We found four distinct dimensions instead of one: (a) reactivity towards companion departure cues, (b) protest behaviour towards inaccessibility, (c) unusual elimination behaviour, and (d) negative responses following social separation. Our findings suggest a manifestation of different motivational states rather than a single, separation-related construct. Future studies would benefit from a careful evaluation of the separation-related behaviours in a multi-measure context to improve the accuracy of ethological classifications.

The MitoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import Within Mammalian Cells.

Mitochondrial protein import is critical for organelle biogenesis, bioenergetic function, and health. The mechanism of which is poorly understood, particularly of the mammalian system. To address this problem we have established an assay to quantitatively monitor mitochondrial import inside mammalian cells. The reporter is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a purified recombinant precursor protein destined for import. Following import the complementary fragments combine to form an active luciferase-providing a sensitive, accurate and continuous measure of protein import. This advance allows detailed mechanistic examination of the transport process in live cells, including the analysis of import breakdown associated with disease, and high-throughput drug screening. Furthermore, the set-up has the potential to be adapted for the analysis of alternative protein transport systems within different cell types, and multicellular model organisms.

Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Wide distribution of D-xylose dehydrogenase in yeasts reveals a new element in the D-xylose metabolism for bioethanol production.

D-xylose utilization by yeasts is an essential feature for improving second-generation ethanol production. However, industrial yeast strains are incapable of consuming D-xylose. Previous analyzes of D-xylose-consuming or fermenting yeast species reveal that the genomic features associated with this phenotype are complex and still not fully understood. Here we present a previously neglected yeast enzyme related to D-xylose metabolism, D-xylose dehydrogenase (XylDH), which is found in at least 105 yeast genomes. By analyzing the XylDH gene family, we brought evidence of gene evolution marked by purifying selection on codons and positive selection evidence in D-xylose-consuming and fermenting species, suggesting the importance of XylDH for D-xylose-related phenotypes in yeasts. Furthermore, although we found no putative metabolic pathway for XylDH in yeast genomes, namely the absence of three bacterial known pathways for this enzyme, we also provide its expression profile on D-xylose media following D-xylose reductase for two yeasts with publicly available transcriptomes. Based on these results, we suggest that XylDH plays an important role in D-xylose usage by yeasts, likely being involved in a cofactor regeneration system by reducing cofactor imbalance in the D-xylose reductase pathway.

The Effect of the Species and Harvesting Location on Dried Salted Cod Fatty Acid Signatures and Nutritional Quality.

The Atlantic cod was listed as 'vulnerable' by the International Union for Conservation of Nature, a condition that persists today. Fishing pressure on the Atlantic cod could be partially transferred to the Pacific cod, since the two cod species share genetic and phenotypic similarities. The aim of this study is to expand knowledge of the composition of dried salted cod obtained from Atlantic and Pacific cod species, with the Atlantic cod being from two different harvesting locations. The comparison of these cod species revealed the existence of nine significant differences among individual FAs (accountable for 63.2% of total FAs), which was at a similar level to that observed between different harvesting locations for the Atlantic cod (ten significant differences among individual FAs, accountable for 61.6% of total FAs). Canonical discriminant analysis and cross-validation achieved full discrimination of the cod's origin and 100% accuracy in the cod's origin classification. The amount of EPA plus DHA in dried salted cod reached its higher value among the Pacific cod (302.3 mg/100 g), while the Atlantic cod averaged 284.1 g/100 g of edible portion. The Pacific cod presented a higher α-tocopherol content than its Atlantic counterpart (8.04 vs. 4.94 µg/g).