Silver and Antimicrobial Polymer Nanocomplexes to Enhance Biocidal Effects.

Antimicrobial resistance has become a major problem over the years and threatens to remain in the future, at least until a solution is found. Silver nanoparticles (Ag-NPs) and antimicrobial polymers (APs) are known for their antimicrobial properties and can be considered an alternative approach to fighting resistant microorganisms. Hence, the main goal of this research is to shed some light on the antimicrobial properties of Ag-NPs and APs (chitosan (CH), poly-L-lysine (PLL), ε-poly-L-lysine (ε-PLL), and dopamine (DA)) when used alone and complexed to explore the potential enhancement of the antimicrobial effect of the combination Ag-NPs + Aps. The resultant nanocomplexes were chemically and morphologically characterized by UV-visible spectra, zeta potential, transmission electron microscopy, and Fourier-transform infrared spectroscopy. Moreover, the Ag-NPs, APs, and Ag-NPs + APs nanocomplexes were tested against Gram-positive Staphylococcus aureus (S. aureus) and the Gram-negative Escherichia coli (E. coli) bacteria, as well as the fungi Candida albicans (C. albicans). Overall, the antimicrobial results showed potentiation of the activity of the nanocomplexes with a focus on C. albicans. For the biofilm eradication ability, Ag-NPs and Ag-NPs + DA were able to significantly remove S. aureus preformed biofilm, and Ag-NPs + CH were able to significantly destroy C. albicans biofilm, with both performing better than Ag-NPs alone. Overall, we have proven the successful conjugation of Ag-NPs and APs, with some of these formulations showing potential to be further investigated for the treatment of microbial infections.

Metallic Structures: Effective Agents to Fight Pathogenic Microorganisms.

The current worldwide pandemic caused by coronavirus disease 2019 (COVID-19) had alerted the population to the risk that small microorganisms can create for humankind's wellbeing and survival. All of us have been affected, directly or indirectly, by this situation, and scientists all over the world have been trying to find solutions to fight this virus by killing it or by stop/decrease its spread rate. Numerous kinds of microorganisms have been occasionally created panic in world history, and several solutions have been proposed to stop their spread. Among the most studied antimicrobial solutions, are metals (of different kinds and applied in different formats). In this regard, this review aims to present a recent and comprehensive demonstration of the state-of-the-art in the use of metals, as well as their mechanisms, to fight different pathogens, such as viruses, bacteria, and fungi.

Molecular identification of clinical and environmental avian Aspergillus isolates.

Aspergillosis causes high morbidity and mortality in avian species. The main goal of this study was to use molecular techniques to identify Aspergillus species collected from different avian species with aspergillosis. A subsample of those isolates was also screened for resistance to itraconazole. Over a 2-year period, clinical samples were recovered from 44 birds with clinical signs of the disease, clinical pathology results suspicious of aspergillosis, or from birds that died from Aspergillus spp. infection. Environmental sampling was also performed in seabird rehabilitation centers and natural seabird environments. Seventy-seven isolates (43 clinical and 34 environmental) were identified as Aspergillus fumigatus sensu stricto. No cryptic species from the Fumigati section were detected. Two environmental isolates were identified as Aspergillus nidulans var. dentatus and Aspergillus spinulosporus. None of the Aspergillus isolates tested were resistant to itraconazole. Our study emphasizes the dominant association of Aspergillus fumigatus sensu stricto in avian mycoses and shows the lack of itraconazole resistance in the studied isolates.

Individual exposure to air pollutants in a Portuguese urban industrialized area.

Industrial development from the second half of the 20th century coupled with population growth and concentration in urban areas has accentuated the concern for potential effects and impacts from air pollutant emissions on environmental and human health. This study examined the Estarreja region, an urban area that has one of the largest chemical complexes in Portugal, a complex that was recently under expansion. In the scope of the INSPIRAR project, individual daily exposure of a group of individuals to particulate matter of 10 µm aerodynamic diameter (PM10) and nitrogen dioxide (NO2) in two phases was determined using a microenvironmental approach. In this context, personal daily activity profiles of individuals were established for their normal routine by personal interviews. These profiles enabled determination of where each individual was at each moment of the day. Utilizing this information with hourly air quality maps simulated with URBAIR air quality model and indoor/outdoor relationships, it was possible to calculate personal daily exposure of each individual to air pollutants. Results from the analysis of daily activity profiles showed a high level of sedentariness of this population and long durations spent indoors. The studied individuals displayed high personal PM10 and NO2 exposure variability. Data demonstrated possible error when a single concentration measurement was assumed as a proxy of exposure. In general, no significant differences were found between the two population groups, indicating that workers of the chemical complex were not exposed to a greater extent to PM10 and NO2 than the general population working in the same area.

First report of Thelazia callipaeda in red foxes (Vulpes vulpes) from Portugal.

The first cases of infection with the eyeworm Thelazia callipaeda (Spirurida, Thelaziidae) in red foxes (Vulpes vulpes) from Portugal are described. Worms were collected from 1 fox (7 worms) in the north and from 2 foxes (10 worms) in the central region of the country. Partial molecular amplification of mitochondrial cythocrome c oxidase subunit 1 gene and sequencing revealed a 100% homology with T. callipaeda haplotype 1, which is the sequence type circulating in Europe. Data suggest that wildlife participate in maintaining this endemic infection in dog and cat populations from the studied areas. Furthermore, due to the zoonotic potential of T. callipaeda, there is a risk of infection in humans residing in areas where thelaziosis is present in wild and domestic animals.

Metal-based approaches to fight cervical cancer.

Cervical cancer (CC) is one of the leading causes of death among women worldwide. The current treatments for this cancer consist of invasive methods such as chemotherapeutic drugs, radiation, immunotherapy and surgery, which could lead to severe side effects and hinder the patient's life quality. Although metal-based therapies, including cisplatin and ruthenium-based compounds, offer promising alternatives, they lack specificity and harm healthy cells. Combining metal nanoparticles with standard approaches has demonstrated remarkable efficacy and safety in the fight against CC. Overall, this review is intended to show the latest advancements and insights into metal-based strategies, creating a promising path for more effective and safer treatments in the battle against CC.

Ocean acidification will not affect the shell strength of juveniles of the commercial clam species Chamelea gallina: Implications of the local alkalinization of seawater.

Ocean acidification (OA) is expected to decrease the strength of bivalves' shells, especially during the early stages of development, with negative consequences to the resilience of natural populations and the economy. The objectives of the present study were to assess the long-term effect of increasing pCO2 after 217 days of exposure under controlled conditions of pH of ∼8.2, 8.0, and 7.7 on the strength and integrity of shells of juveniles of the commercial striped venus clam Chamelea gallina. Shell strength was estimated through compression tests and integrity through scanning electron microscopy (SEM) and dispersive X-ray analyses (EDX). The results showed that under increasing pCO2 the shell strength of juveniles is unaffected, which could be related to the locally elevated total alkalinity of seawater with respect to other parts of the coastal lagoon. However, despite this, it was also observed that the juvenile clams exposed to elevated pCO2 decreased their shell thickness and increased the porosity of their prismatic layer. Under future OA conditions, these changes could eventually compromise the integrity of the shells, becoming more vulnerable to the attack of predators and breakable during fishing operations. Future studies should address the plasticity of the organisms and the effect of the alkalinization of seawater on the resilience of shellfish juveniles under global change conditions.

Wildland smoke exposure values and exhaled breath indicators in firefighters.

Smoke from forest fires contains significant amounts of gaseous and particulate pollutants. Firefighters exposed to wildland fire smoke can suffer from several acute and chronic adverse health effects. Consequently, exposure data are of vital importance for the establishment of cause/effect relationships between exposure to smoke and firefighter health effects. The aims of this study were to (1) characterize the relationship between wildland smoke exposure and medical parameters and (2) identify health effects pertinent to wildland forest fire smoke exposure. In this study, firefighter exposure levels of carbon monoxide (CO), nitrogen dioxide (NO2), and volatile organic compounds (VOC) were measured in wildfires during three fire seasons in Portugal. Personal monitoring devices were used to measure exposure. Firefighters were also tested for exhaled nitric oxide (eNO) and CO before and after their firefighting activities. Data indicated that exposure levels during firefighting activities were beyond limits recommended by the Occupational Exposure Standard (OES) values. Medical tests conducted on the firefighters also indicated a considerable effect on measured medical parameters, with a significant increase in CO and decrease in NO in exhaled air of majority of the firefighters.

Editorial for Special Issue: "Additive Manufacturing Approaches to Produce Drug Delivery Systems".

Cutting-edge technologies such as additive manufacturing (AM) have had an enormous impact in a multitude of sectors [...].

Arginine-Affinity Chromatography for Nucleic Acid (DNA and RNA) Isolation.

Nucleic acid-based therapy has been emerging as a new strategy with great potential for the treatment of numerous diseases, especially those caused by gene defects. In this context, biotechnology plays a critical role on establishing suitable processes for biopharmaceuticals manufacturing, while the purification step still imposes a major burden. Affinity chromatography using amino acids as specific ligands has been successfully applied for plasmid DNA purification. In this protocol, we describe the process for nucleic acids production and extraction, as well as the chromatographic matrix synthesis for separation between DNA and RNA. This novel arginine-macroporous support presents excellent binding capacity and great robustness for nucleic acids isolation.

Arginine-Modified 3D-Printed Chromatographic Supports.

The increasing progression of biopharmaceutical-based therapies highlights the demand for efficient chromatographic methods that can be used to purify the desired biomolecules (e.g., nucleic acids, enzymes, or monoclonal antibodies) which are presently under consideration in clinical trials or approved by the Food and Drug Administration. These molecules present distinct chemical and structural properties, which are critical cues for the development and production of adequate chromatographic supports. Until now, it has not been possible to fully control the characteristics of the chromatographic matrices to assure the total reproducibility of their structure and packing. Meanwhile, three-dimensional printing (3DP) is in the early stage of its use in the production of chromatographic supports as a fast, very precise, and reproducible methodology. Although 3DP can provide excellent performance properties to the chromatographic structures, it cannot, per se, lead to high-quality pharmaceutical products. However, the association of affinity ligands, such as amino acids, which is possible in 3DP, could enable the attainment of high-purity yields of the desired molecules. Beyond the amino acids most widely studied as chromatographic ligands, arginine has been successfully immobilized on different chromatographic supports (namely, agarose bead matrices, macroporous matrices, and monoliths) to achieve extra-pure gene therapy products. In this research, we studied the immobilization of arginine on 3DP chromatographic supports, evaluating the stability of the ligand/chromatographic support linkage under different chromatographic conditions to determine the robustness of these new prototypes. Moreover, we also applied plasmid DNA samples to these supports to observe the practical behaviour of the developed arginine 3DP chromatographic structures.

Electrospun-Based Membranes as a Key Tool to Prevent Respiratory Infections.

The use of electrospun meshes has been proposed as highly efficient protective equipment to prevent respiratory infections. Those infections can result from the activity of micro-organisms and other small dust particles, such as those resulting from air pollution, that impair the respiratory tract, induce cellular damage and compromise breathing capacity. Therefore, electrospun meshes can contribute to promoting air-breathing quality and controlling the spread of such epidemic-disrupting agents due to their intrinsic characteristics, namely, low pore size, and high porosity and surface area. In this review, the mechanisms behind the pathogenesis of several stressors of the respiratory system are covered as well as the strategies adopted to inhibit their action. The main goal is to discuss the performance of antimicrobial electrospun nanofibers by comparing the results already reported in the literature. Further, the main aspects of the certification of filtering systems are highlighted, and the expected technology developments in the industry are also discussed.

Advanced Face Mask Filters Based on PCL Electrospun Meshes Dopped with Antimicrobial MgO and CuO Nanoparticles.

The pandemic situation caused by coronavirus clearly demonstrated the need for alternatives able to protect the respiratory tract and inactivate the infectious agents. Based on this, antibacterial face-mask filters of polycaprolactone (PCL) dopped with magnesium oxide (MgO) and copper oxide (CuO) nanoparticles (NPs) were produced using an electrospinning technique. A morphological analysis of electrospun meshes evaluated the success of nanoparticles' incorporation as well as the average fibers' diameters (481 ± 272 nm). The performance of electrospun nanofibers was also assessed in terms of tensile strength (0.88 ± 0.25 MPa), water vapor permeability (11,178.66 ± 35.78 g·m-2·day-1), stability under wet conditions and antibacterial activity according to the standard guidelines. The filters showed structural stability up to 2 h of washing and improved antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for optimized concentrations of MgO and CuO NPs. Overall, electrospun meshes with antibacterial activity were successfully developed for advanced filtering applications.

Assessment of 3D-Printed Polycaprolactone, Hydroxyapatite Nanoparticles and Diacrylate Poly(ethylene glycol) Scaffolds for Bone Regeneration.

Notwithstanding the advances achieved in the last decades in the field of synthetic bone substitutes, the development of biodegradable 3D-printed scaffolds with ideal mechanical and biological properties remains an unattained challenge. In the present work, a new approach to produce synthetic bone grafts that mimic complex bone structure is explored. For the first time, three scaffolds of various composition, namely polycaprolactone (PCL), PCL/hydroxyapatite nanoparticles (HANp) and PCL/HANp/diacrylate poly(ethylene glycol) (PEGDA), were manufactured by extrusion. Following the production and characterisation of the scaffolds, an in vitro evaluation was carried out using human dental pulp stem/stromal cells (hDPSCs). Through the findings, it was possible to conclude that, in all groups, the scaffolds were successfully produced presenting networks of interconnected channels, adequate porosity for migration and proliferation of osteoblasts (approximately 50%). Furthermore, according to the in vitro analysis, all groups were considered non-cytotoxic in contact with the cells. Nevertheless, the group with PEGDA revealed hydrophilic properties (15.15° ± 4.06) and adequate mechanical performance (10.41 MPa ± 0.934) and demonstrated significantly higher cell viability than the other groups analysed. The scaffolds with PEGDA suggested an increase in cell adhesion and proliferation, thus are more appropriate for bone regeneration. To conclude, findings in this study demonstrated that PCL, HANp and PEGDA scaffolds may have promising effects on bone regeneration and might open new insights for 3D tissue substitutes.

A Case of XX Disorder of Sexual Development in a Female-Phenotype Roe Deer (Capreolus capreolus L.) Associated with Antlers Growth with Retained Velvet.

A 3-to-4-year-old roe deer (Capreolus capreolus L.) was admitted to the Veterinary Hospital. Although it showed well-developed antlers with retained velvet, an external female appearance and genitalia were evident. External biometrical measurements were taken for the antlers, and a computed tomography was performed. Molecular studies targeting the SRY gene were performed, and a PIS (polled intersex syndrome) mutation diagnosis was implemented. The gonads consisted of a right testicle paired with a left ovotestis. Histologically, the ovary-like structures in the ovotestis were functional, but the testis, as the testis-like structure in the ovotestis, did not show active spermatogenesis. No evidence of SRY gene was detected by PCR, suggesting an XX-chromosome constitution. Additionally, polled intersex syndrome (PIS) deletion was not detected in the case under study. The clinical and histopathological findings confirmed the DSD with the presence of a testicle and a contralateral ovotestis.

Impact of Deficit Irrigation on Grapevine cv. 'Touriga Nacional' during Three Seasons in Douro Region: An Agronomical and Metabolomics Approach.

The introduction of irrigation in vineyards of the Mediterranean basin is a matter of debate, in particular in those of the Douro Demarcated Region (DDR), due to the limited number of available studies. Here, we aimed to perform a robust analysis in three consecutive vintages (2018, 2019, and 2020) on the impact of deficit irrigation on the yield, berry quality traits, and metabolome of cv. 'Touriga Nacional'. Results showed that in the peaks of extreme drought, irrigation at 30% crop evapotranspiration (ETc) (R30) was able to prevent a decay of up to 0.4 MPa of leaf predawn water potential (ΨPd), but irrigation at 70% ETc (R70) did not translate into additional protection against drought stress. Following three seasons of irrigation, the yield was significantly improved in vines irrigated at R30, whereas irrigation at R70 positively affected the yield only in the 2020 season. Berry quality traits at harvest were not significantly changed by irrigation, except for Total Soluble Solids (TSS) in 2018. A UPLC-MS-based targeted metabolomic analysis identified eight classes of compounds, amino acids, phenolic acids, stilbenoid DP1, stilbenoid DP2, flavonols, flavan-3-ols, di-OH- and tri-OH anthocyanins, and showed that anthocyanins and phenolic acids did not change significantly with irrigation. The present study showed that deficit irrigation partially mitigated the severe summer water deficit conditions in the DDR but did not significantly change key metabolites.

An Unusual Case of Feline Leishmaniosis With Involvement of the Mammary Glands.

We report an unusual case of leishmaniosis with the involvement of mammary glands in an old cat with what seems to be a concurrent regressive feline leukemia virus infection. Leishmania donovani complex parasites were identified for the first time in inflammatory breast fluid during a clinical recurrence manifested about 4 years after the first diagnosis of feline leishmaniosis. Combined treatment with allopurinol and meglumine antimoniate resulted in clinical cure of mammary lesion and a concurrent uveitis.

The clinical use of exhaled nitric oxide in wheezing children.

The body of published work on the role of exhaled nitric oxide (FENO) in the study of bronchial inflammation allows it to be classed as a simple, non-invasive measurement that is very useful in evaluating asthmatic patients. During a prospective study into the effects of air pollution on the health of the population of Viseu (Saud'AR Project), children with a clinical history of wheezing were identified through using the International Study of Asthma and Allergy in Childhood (ISAAC) questionnaire. Children later filled in a new standardised questionnaire and underwent skin-prick-tests, spirometry and FENO measurement. Their mean age was 7.8+/-1.1 years. Comparing those who wheezed in the 6 months before evaluation (n=27) with those who didn't, statistical differences for DeltaFEV1 (8% median versus 4.5%, p=0.0399) and for FENO (23 ppb median versus 12 ppb, p=0.0195, respectively) were observed. Concerning children who needed a bronchodilator in the six previous months (n=19) and those who didn't, there was also a statistically significant difference in FENO: 27 ppb median versus 11 ppb median, respectively; p<0.0001. When comparing children who needed an unscheduled medical appointment in the six months previous to the evaluation (n=9) and those who didn't, there was also significant differences for FE NO: 28 ppb median versus 13 ppb median, p=0.0029. In conclusion, the existence of symptoms seems to be better related to FE NO than spirometry.

p53 as the Focus of Gene Therapy: Past, Present and Future.

BACKGROUND: Several gene deviations can be responsible for triggering oncogenic processes. However, mutations in tumour suppressor genes are usually more associated to malignant diseases, with p53 being one of the most affected and studied element. p53 is implicated in a number of known cellular functions, including DNA damage repair, cell cycle arrest in G1/S and G2/M and apoptosis, being an interesting target for cancer treatment. OBJECTIVE: Considering these facts, the development of gene therapy approaches focused on p53 expression and regulation seems to be a promising strategy for cancer therapy. RESULTS: Several studies have shown that transfection of cancer cells with wild-type p53 expressing plasmids could directly drive cells into apoptosis and/or growth arrest, suggesting that a gene therapy approach for cancer treatment can be based on the re-establishment of the normal p53 expression levels and function. Up until now, several clinical research studies using viral and non-viral vectors delivering p53 genes, isolated or combined with other therapeutic agents, have been accomplished and there are already in the market, therapies based on the use of this gene. CONCLUSION: This review summarizes the different methods used to deliver and/or target the p53 as well as the main results of therapeutic effect obtained with the different strategies applied. Finally, the ongoing approaches are described, also focusing on the combinatorial therapeutics to show increased therapeutic potential of combining gene therapy vectors with chemo or radiotherapy.

Affinity analysis and application of dipeptides derived from l-tyrosine in plasmid purification.

The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivated the search and improvement of optimized purification processes. In this context, dipeptides l-tyrosine-l-tyrosine and l-tyrosine-l-arginine are synthetized to explore their application as affinity ligands for supercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N-Boc-l-tyrosine, followed by condensation with l-tyrosine or l-arginine methyl esters in the presence of dicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides. The supports are then obtained by coupling l-tyrosine, l-tyrosine-l-tyrosine and l-tyrosine-l-arginine to epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS) NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor is used to establish the promising ligand to be used in the chromatographic experiments and ascertain experimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln) pDNA, being the open circular (oc) the one that promoted the lowest affinity to l-tyrosine-l-arginine. Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobic with the majority of the 5'-mononucleotides, except for 5'-GMP with l-tyrosine-l-arginine Sepharose that is mainly electrostatic. The support l-tyrosine Sepharose used in chromatographic experiments promotes the separation of native pVAX1-LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing the salt concentration. The results suggest that it is possible to purify different plasmids with the l-tyrosine Sepharose, with slight adjustments in the gradient conditions.