Browsing by Issue Date, starting with "2022-07"
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- Diversity of microorganisms in Hyalomma aegyptium collected from spur-thighed tortoise (Testudo graeca) in North Africa and AnatoliaPublication . Norte, Ana Cláudia; Harris, David James; Silveira, Diogo; Nunes, Carolina Saramago; Núncio, Maria Sofia; Martínez, Eva Graciá; Giménez, Andrés; de Sousa, Rita; Lopes de Carvalho, Isabel; Perera, AnaTicks carry a diverse community of microorganisms including non-pathogenic symbionts, commensals, and pathogens, such as viruses, bacteria, protozoans, and fungi. The assessment of tick-borne microorganisms (TBM) in tortoises and their ticks is essential to understand their eco-epidemiology, and to map and monitor potential pathogens to humans and other animals. The aim of this study was to characterize the diversity of microorganisms found in ticks collected from the spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia. Ticks feeding on wild T. graeca were collected, and pathogens were screened by polymerase chain reaction using group-specific primers. In total, 131 adult Hyalomma aegyptium ticks were collected from 92 T. graeca in Morocco (n = 48), Tunisia (n = 2), Algeria (n = 70), and Turkey (n = 11). Bacteria and protozoa detected included Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), relapsing-fever borreliae (8.4%), Ehrlichia spp. (7.6%), Rickettsia spp. (3.4%), Borrelia burgdorferi s.l. (0.9%), Francisella spp. (0.9%), and Wolbachia spp. (0.8%). The characterization of Rickettsia included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R.africae (Morocco). Hemolivia mauritanica and Ehrlichia spp. prevalence varied significantly with the sampling region/country. We did not detect significant associations in microorganism presence within ticks, nor between microorganism presence and tick mitochondrial DNA haplogroups. This is the first report of Francisella persica-like, relapsing fever borreliae, M. mitochondrii, and Wolbachia spp. in H. aegyptium ticks collected from wild hosts from the South and Eastern Mediterranean region, and of R. sibirica mongolitimonae and R. africae in H. aegyptium from Algeria and Morocco, respectively. Given that T. graeca is a common species in commercial and non-commercial pet trade, the evaluation of the role of this species and its ticks as hosts for TBM is particularly relevant for public health.
- Development of a new multiplex PCR to detect prevalent species of house dust mites in house dustPublication . Oliveira, Ana Sofia; Gaspar, Carlos; Rolo, Joana; Pereira, Cristiana Costa; Palmeira-de-Oliveira, Rita; Teixeira, João Paulo; Martinez-de-Oliveira, José; Palmeira-de-Oliveira, AnaDermatophagoides pteronyssinus and Dermatophagoides farinae are the most common House Dust Mite (HDM) species in home environments worldwide and responsible for HDM allergy. Since the prevalence of HDM-related clinical conditions is linked to exposure to the mite itself, the detection of HDM in the human households gains importance. We aimed to develop a fast and accessible multiplex PCR to detect and distinguish two relevant HDM species in house dust. New primers were designed, and sensitivity analysis was performed. Sequencing of PCR products was also performed to confirm the method's specificity. The limit of detection of the multiplex PCR for both species was as low as 30 pg µL-1. The application of the multiplex PCR to dust samples also resulted in the identification of both species with high sensitivity. The protocol required small amount of template, reagents and a short reaction time thus presenting an alternative to classically used techniques for HDM identification.
- Assessing the in vitro toxicity of engineered and airborne nanoceramics: contribution to the safe production and use of nanomaterials in the ceramic industryPublication . Bessa, Maria João; Fraga, Sónia; Teixeira, João Paulo; Laffon, Blanca LageAdvanced ceramic technologies have a strong potential for airborne (nano)particle formation and emission, meaning that workers of those industries are at great risk of exposure to these particles. However, toxicological data of these (nano)particles is lacking, particularly for airborne particles released within sectors such as the ceramic industry. To address this relevant topic, the present work aimed to assess the toxicity of occupationally relevant doses of industrially process-generated particles emitted during two industrial thermal spraying technologies [atmospheric plasma spraying (APS) and high velocity oxy-fuel (HVOF)], as well as of four engineered nanoparticles [ENP; tin oxide (SnO2), antimony-tin oxide (ATO; Sb2O3●SnO2), cerium oxide (CeO2) and zirconium oxide (ZrO2)] used as raw materials for ceramics manufacture. Two human respiratory in vitro systems, either conventional alveolar epithelial A549 cultures under submerged or air-liquid interface (ALI) conditions, or advanced three-dimensional (3D) upper airway epithelium (MucilAirTM) cultures at ALI were exposed to the selected particles. Major toxicity endpoints including plasma membrane integrity, metabolic activity, oxidative stress, inflammatory response, and genotoxicity were assessed. Overall, the tested process-generated particles seem to be more toxic compared to the ENP, most likely due to their higher chemical complexity and composition [elevated levels of metallic elements like chromium (Cr) and nickel (Ni)]. Among the two evaluated thermal spraying processes, particles derived from HVOF were more cytotoxic than those emitted from APS. Either fine (PGFP) and ultrafine (PGNP) particles from both spraying processes were able to induce measurable genotoxic effects. While APS particles lead to increased levels of histone 2AX (H2AX) phosphorylation, HVOF particles caused 8-oxo-7,8-dihydroguanine (8-oxo-G) oxidative DNA lesions. ENP were more toxic to human alveolar epithelial cultures when aerosolised than in liquid suspension, particularly ZrO2 NP. On the other hand, advanced MucilAirTM cultures, that better mimic in vivo physiological features, such as the mucociliary defence mechanisms, were quite resistant to both HVOF-derived particles and ENP aerosols. Thus, while 3D human upper airway epithelial cultures exhibited attenuated responses, the conventional A549 cultures were more sensitive to the studied (nano)particles.The present work highlights the hazard of industrially derived (nano)particles, either intentionally used or incidentally released into the workplace air during advanced ceramic processes. Importantly, particles’ physicochemical properties alongside the testing conditions (cell model and type of exposure) played a determinant role in the observed biological responses. These findings reinforce the importance of using physiologically relevant in vitro models in (nano)particle toxicity studies, for better data extrapolation to humans.
- Optimization of A(H1N1)pdm09 vaccine seed viruses: The source of PB1 and HA vRNA as a major determinant for antigen yieldPublication . Almeida, Filipe; Santos, Luís A.; Trigueiro-Louro, João M.; Rebelo-de-Andrade, HelenaVaccination prevents and reduces the severity of influenza virus infections. Continuous evolution of influenza hemagglutinin (HA) and neuraminidase (NA) supports the virus to evade pre-existing immunity, which demands vaccines to be reformulated every year. Incorporation of polymerase basic protein 1 (PB1) viral RNA (vRNA) of the same origin of HA and NA vRNA has been observed in previous pandemic viruses and occasionally reported for influenza A vaccine prototype strains of prior seasons. At this point, it remains to be explored whether this phenomenon translates into an improved growth phenotype. In this work, we showed that the HA vRNA of A(H1N1)pdm09 is generally incorporated with the PB1 vRNA of the same origin, establishing the beneficial effect of the presence of PB1 and the pattern of the PB1-HA co-incorporation in the A(H1N1)pdm09 model. We further investigated the putative interplay between PB1 and antigenic proteins regarding the vRNA composition of the progeny and observed that vRNA segregation does not appear to be mainly determined by protein-protein interactions; while vRNA-vRNA interactions can be suggested as the main driving force. Our data also indicate an increase in the hemagglutination capacity and neuraminidase activity due to incorporation of PB1, HA and NA from A(H1N1)pdm09, in comparison with the recombinant virus incorporating only HA and NA from A(H1N1)pdm09 - which have the potential to improve current limitations regarding antigenicity and immunogenicity of influenza vaccines. Further knowledge of the complex vRNA-vRNA interaction network between PB1 and HA will additionally contribute to improve current vaccine formulation, and to gradually optimize the production of A(H1N1)pdm09 reverse genetics vaccine seed virus towards a higher cost-effectiveness.
- Effect on Broiler Production Performance and Meat Quality of Feeding Ulva lactuca Supplemented with CarbohydrasesPublication . Costa, Mónica M.; Pestana, José M.; Carvalho, Patrícia; Alfaia, Cristina M.; Martins, Cátia F.; Carvalho, Daniela; Mourato, Miguel; Gueifão, Sandra; Delgado, Inês; Coelho, Inês; Lemos, José P.C.; Lordelo, Madalena M.; Prates, José A.M.SIMPLE SUMMARY: Macroalgae have been increasingly exploited worldwide for feed, food and biofuel applications, due to their nutritive and bioactive compounds. Green seaweeds belonging to the genus Ulva have high growth rates, which makes them suitable for being cultured in sustainable algae production, such as an integrated multi-trophic aquaculture system. This is expected to increase the use of Ulva sp. as an alternative source to conventional feedstuffs (e.g., cereals and soybean meal) for poultry. The objective of the current study was to assess if the incorporation of 15% Ulva lactuca in broiler chickens’ diet, combined or not with carbohydrate-active enzymes, would enhance meat nutritional quality without compromising animal growth performance. Overall, U. lactuca led to an accumulation of antioxidant carotenoids, n-3 PUFA and macrominerals, including magnesium, potassium and phosphorus, in the breast muscle, with likely health benefits, without significantly impairing growth performance. The supplementation of macroalgae with a recombinant ulvan lyase reduced ileal viscosity with possible beneficial effects on broiler digestibility. Although dietary U. lactuca showed potential to increase meat quality, it reduced meat overall acceptability, which suggests the use of a lower algae inclusion level to prevent a negative meat sensory perception for consumers.
- Common Variation in the PIN1 Locus Increases the Genetic Risk to Suffer from Sertoli Cell-Only SyndromePublication . Cerván-Martín, Miriam; Bossini-Castillo, Lara; Guzmán-Jimenez, Andrea; Rivera-Egea, Rocío; Garrido, Nicolás; Luján, Saturnino; Romeu, Gema; Santos-Ribeiro, Samuel; Castilla, José A.; Gonzalvo, M. Carmen; Clavero, Ana; Vicente, F. Javier; Maldonado, Vicente; González-Muñoz, Sara; Rodríguez-Martín, Inmaculada; Burgos, Miguel; Jiménez, Rafael; Pinto, Maria Graça; Pereira, Isabel; Nunes, Joaquim; Sánchez-Curbelo, Josvany; López-Rodrigo, Olga; Pereira-Caetano, Iris; Marques, Patricia Isabel; Carvalho, Filipa; Barros, Alberto; Bassas, Lluís; Seixas, Susana; Gonçalves, João; Larriba, Sara; Lopes, Alexandra M.; Carmona, F. David; Palomino-Morales, Rogelio J.We aimed to analyze the role of the common genetic variants located in the PIN1 locus, a relevant prolyl isomerase required to control the proliferation of spermatogonial stem cells and the integrity of the blood-testis barrier, in the genetic risk of developing male infertility due to a severe spermatogenic failure (SPGF). Genotyping was performed using TaqMan genotyping assays for three PIN1 taggers (rs2287839, rs2233678 and rs62105751). The study cohort included 715 males diagnosed with SPGF and classified as suffering from non-obstructive azoospermia (NOA, n = 505) or severe oligospermia (SO, n = 210), and 1058 controls from the Iberian Peninsula. The allelic frequency differences between cases and controls were analyzed by the means of logistic regression models. A subtype specific genetic association with the subset of NOA patients classified as suffering from the Sertoli cell-only (SCO) syndrome was observed with the minor alleles showing strong risk effects for this subset (ORaddrs2287839 = 1.85 (1.17-2.93), ORaddrs2233678 = 1.62 (1.11-2.36), ORaddrs62105751 = 1.43 (1.06-1.93)). The causal variants were predicted to affect the binding of key transcription factors and to produce an altered PIN1 gene expression and isoform balance. In conclusion, common non-coding single-nucleotide polymorphisms located in PIN1 increase the genetic risk to develop SCO.
- Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22Publication . Guerra Leal, Bárbara; Barros-Barbosa, Aurora; Ferreirinha, Fátima; Chaves, João; Rangel, Rui; Santos, Agostinho; Carvalho, Cláudia; Martins-Ferreira, Ricardo; Samões, Raquel; Freitas, Joel; Lopes, João; Ramalheira, João; Lobo, Maria Graça; Martins da Silva, António; Costa, Paulo P.; Correia-de-Sá, PauloObjective: ATP-gated ionotropic P2X7 receptors (P2X7R) actively participate in epilepsy and other neurological disorders. Neocortical nerve terminals of patients with Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS) express higher P2X7R amounts. Overexpression of P2X7R bolsters ATP signals during seizures resulting in glial cell activation, cytokines production, and GABAergic rundown with unrestrained glutamatergic excitation. In a mouse model of status epilepticus, increased expression of P2X7R has been associated with the down-modulation of the non-coding micro RNA, miR-22. MiR levels are stable in biological fluids and normally reflect remote tissue production making them ideal disease biomarkers. Here, we compared P2X7R and miR-22 expression in epileptic brains and in the serum of patients with MTLE-HS, respectively. Methods: Quantitative RT-PCR was used to evaluate the expression of P2X7R in the hippocampus and anterior temporal lobe of 23 patients with MTLE-HS and 10 cadaveric controls. Confocal microscopy and Western blot analysis were performed to assess P2X7R protein amounts. MiR-22 expression was evaluated in cell-free sera of 40 MTLE-HS patients and 48 healthy controls. Results: Nerve terminals of the hippocampus and neocortical temporal lobe of MTLE-HS patients overexpress (p < 0.05) an 85 kDa P2X7R protein whereas the normally occurring 67 kDa receptor protein dominates in the brain of the cadaveric controls. Contrariwise, miR-22 serum levels are diminished (p < 0.001) in MTLE-HS patients compared to age-matched control blood donors, a situation that is more evident in patients requiring multiple (>3) anti-epileptic drug (AED) regimens. Conclusion: Data show that there is an inverse relationship between miR-22 serum levels and P2X7R expression in the hippocampus and neocortex of MTLE-HS patients, which implies that measuring serum miR-22 may be a clinical surrogate of P2X7R brain expression in the MTLE-HS. Moreover, the high area under the ROC curve (0.777; 95% CI 0.629-0.925; p = 0.001) suggests that low miR-22 serum levels may be a sensitive predictor of poor response to AEDs among MTLE-HS patients. Results also anticipate that targeting the miR-22/P2X7R axis may be a good strategy to develop newer AEDs.
- The Type III Secretion Effector CteG Mediates Host Cell Lytic Exit of Chlamydia trachomatisPublication . Pereira, Inês Serrano; Pais, Sara Vilela; Borges, Vítor; Borrego, Maria José; Gomes, João Paulo; Mota, Luís JaimeChlamydia trachomatis is an obligate intracellular bacterium causing ocular and urogenital infections in humans that are a significant burden worldwide. The completion of its characteristic infectious cycle relies on the manipulation of several host cell processes by numerous chlamydial type III secretion effector proteins. We previously identified the C. trachomatis CteG effector and showed it localizes at the host cell plasma membrane at late stages of infection. Here, we showed that, from 48 h post-infection, mammalian cells infected by wild-type C. trachomatis contained more infectious chlamydiae in the culture supernatant than cells infected by a CteG-deficient strain. This phenotype was CteG-dependent as it could be complemented in cells infected by the CteG-deficient strain carrying a plasmid encoding CteG. Furthermore, we detected a CteG-dependent defect on host cell cytotoxicity, indicating that CteG mediates chlamydial lytic exit. Previous studies showed that Pgp4, a global regulator of transcription encoded in the C. trachomatis virulence plasmid, also mediates chlamydial lytic exit. However, by using C. trachomatis strains encoding or lacking Pgp4, we showed that production and localization of CteG are not regulated by Pgp4. A C. trachomatis strain lacking both CteG and Pgp4 was as defective in promoting host cell cytotoxicity as mutant strains lacking only CteG or Pgp4. Furthermore, CteG overproduction in a plasmid suppressed the host cell cytotoxic defect of CteG- and Pgp4-deficient chlamydiae. Overall, we revealed the first chlamydial type III secretion effector involved in host cell lytic exit. Our data indicates that CteG and Pgp4 participate in a single cascade of events, but involving multiple layers of regulation, leading to lysis of host cells and release of the infectious chlamydiae.
- Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water SamplesPublication . Nácher-Vázquez, Montserrat; Barbosa, Ana; Armelim, Inês; Azevedo, Andreia Sofia; Almeida, Gonçalo Nieto; Pizarro, Cristina; Azevedo, Nuno Filipe; Almeida, Carina; Cerqueira, LauraLegionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires' disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents' autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3-4 h) and accurate detection of Legionella.
- New Alternatives to Milk From Pulses: Chickpea and Lupin Beverages With Improved Digestibility and Potential Bioactivities for Human HealthPublication . Duarte, Carla Margarida; Mota, Joana; Assunção, Ricardo; Martins, Carla; Ribeiro, Ana Cristina; Lima, Ana; Raymundo, Anabela; Nunes, Maria Cristiana; Ferreira, Ricardo Boavida; Sousa, IsabelThere is a strong demand for plant-based milk substitutes, often low in protein content (<1.5% w/v). Protein-rich pulse seeds and the right processing technologies make it possible to make relevant choices. The major objective of this study was to assess the impact of processing on the nutritional characteristics of beverages with a high impact on health, in particular on digestibility and specific bioactivities. The results suggest that pulse beverages are as high in protein content (3.24% w/v for chickpea and 4.05% w/v for lupin) as cow's milk. The anti-nutrient level characteristics of pulses have been considerably reduced by strategic processing. However, when present in small quantities, some of these anti-nutritional factors may have health benefits. Controlling processing conditions play a crucial role in this fine balance as a tool to take advantage of their health benefits. There is evidence of protein hydrolysis by in vitro digestion and limited bioaccessibility of minerals. In addition to being highly digestible, lupin and chickpea beverages have anti-inflammatory and anti-carcinogenic potential evaluated through the inhibition of metalloproteinase MMP-9.