Assessment of Douro and Ave River (Portugal) lower basin water quality focusing on physicochemical and trace element spatiotemporal changes.

Water quality of Douro and Ave lower basin was evaluated regarding physicochemical parameters (pH, conductivity, dissolved oxygen and temperature), nutrient compounds (nitrates, nitrites, ammonium and orthophosphates), chlorophyll a and occurrence of trace elements (Li, Be, Al, Ti, V, Cr, Co, Ni, Cu, Zn, Se, Mo, Ag, Cd, Sb, Ba, Tl, Pb, Th and U). To study spatiotemporal variations and possible anthropogenic sources, estuarine samples were collected at nine sampling sites in Douro and five in Ave distributed along the estuaries at four sampling campaigns (spring, summer, fall and winter). According to the water quality standards for aquatic life and recreation, Douro and Ave river water quality was found out of safe limits regarding several parameters. Nitrate levels were systematically high (> 50 mg L-1 in a significant number of samples) and mean levels of trace elements were higher than the established values of Canadian Environmental Quality Guidelines for aquatic life protection for Al, Cu, Se, Ag, Cd and Pb in Douro and Ave, and also Zn in Ave. Significant spatial differences were found in Ave river estuary for trace elements with a clear trend for higher values from upstream to downstream found. Seasonal differences were also observed particularly in Douro river estuary with higher levels in spring for most elements.

How gamma-rays and electron-beam irradiation would affect the antimicrobial activity of differently processed wild mushroom extracts?

AIMS: The effects of irradiation (gamma-rays and electron-beams), up to 10 kGy, in the antimicrobial activity of mushroom species (Boletus edulis, Hydnum repandum, Macrolepiota procera and Russula delica) differently processed (fresh, dried, freeze) were evaluated. METHODS AND RESULTS: Clinical isolates with different resistance profiles from hospitalized patients in Local Health Unit of Mirandela, Northeast of Portugal, were used as target micro-organisms. The mushrooms antimicrobial activity did not suffer significant changes that might compromise applying irradiation as a possible mushroom conservation technology. CONCLUSIONS: Two kGy dose (independently of using gamma-rays or electron-beams) seemed to be the most suitable choice to irradiate mushrooms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides important results in antimicrobial activity of extracts prepared from irradiated mushroom species.

First cytogenetic study of Cavernicola pilosa Barber, 1937 (Hemiptera, Triatominae).

Cavernicola pilosa is a triatomine species that lives in caves and feeds on bat blood. This vector has a wide geographical distribution, and is found in Brazil, Colombia, Panama, Peru, and Venezuela. Little is known about the reproductive biology of this species, because most previous studies have only characterized its morphology, morphometry, ecology, and epidemiology. Therefore, this study aimed to obtain preliminary data related to spermatogenesis in C. pilosa by conducting cytogenetic analysis. Analysis of the heterochromatic pattern of C. pilosa during the initial prophases revealed that heterochromatic blocks are only present in the sex chromosomes. Based on the analyses of the meiotic metaphase and prophases, we found that the sex determination system of C. pilosa is XY and the chromosomes are holocentric. C. pilosa spermatids are filamentous and have long flagella. It was not possible to detect corpuscle or filament heteropycnosis in spermatids of this species. The initial cytogenetic data presented in this study are important in characterizing the spermatogenesis and heterochromatic patterns of C. pilosa. Our results suggest that adaptation to troglodytism did not result in differences in spermatogenesis in this vector.

Microfluidic-based skin-on-chip systems for safety assessment of nanomaterials.

The skin is the body's largest organ, continuously exposed to and affected by natural and anthropogenic nanomaterials (materials with external and internal dimensions in the nanoscale range). This broad spectrum of insults gives rise to irreversible health effects (from skin corrosion to cancer). Organ-on-chip systems can recapitulate skin physiology with high fidelity and potentially revolutionize the safety assessment of nanomaterials. Here, we review current advances in skin-on-chip models and their potential to elucidate biological mechanisms. Further, strategies are discussed to recapitulate skin physiology on-chip, improving control over nanomaterials exposure and transport across cells. Finally, we highlight future opportunities and challenges from design and fabrication to acceptance by regulatory bodies and industry.

Iodine and selenium supplementation increased survival and changed thyroid hormone status in Senegalese sole (Solea senegalensis) larvae reared in a recirculation system.

To test how iodine and both iodine and selenium supplementation affected the thyroid status as well as growth and survival in Senegalese sole, larvae were reared in a recirculation system from 15 to 34 DAH. Sets of three tanks were assigned to each of the following three diets: control (C), iodine (I) and iodine and selenium (I + Se). Samples were collected at 15, 27 and 34 DAH to determine dry weight, iodine and selenium levels, GPx and ORD activities, thyroid hormone levels and thyroid follicles histology. At 34 DAH, fish from the control (C) treatment suffered from hyperplasia of the thyroid follicles (goitre), whereas iodine-treated larvae did not (I and I + Se). Lower survival rates in the C groups were probably a consequence of the hyperplasia. Moreover, there was an improvement in thyroid hormone status in I- and I + Se-treated larvae, showing that further supplementation of live feed with iodine can be crucial for fish at early life stages, as it seems to sustain normal larval development, when reared in a recirculation system. Selenium did not affect the results. Together with previous results, this indicates selenium supplement is more important at younger life stages.

TiO2 bioactive implant surfaces doped with specific amount of Sr modulate mineralization.

One of the main problems that remain in the implant industry is poor osseointegration due to bioinertness of implants. In order to promote bioactivity, calcium (Ca), phosphorus (P) and strontium (Sr) were incorporated into a TiO2 porous layer produced by micro-arc oxidation. Ca and P as bioactive elements are already well reported in the literature, however, the knowledge of the effect of Sr is still limited. In the present work, the effect of various amounts of Sr was evaluated and the morphology, chemical composition and crystal structure of the oxide layer were investigated. Furthermore, in vitro studies were carried out using human osteoblast-like cells. The oxide layer formed showed a triplex structure, where higher incorporation of Sr increased Ca/P ratio, amount of rutile and promoted the formation of SrTiO3 compound. Biological tests revealed that lower concentrations of Sr did not compromise initial cell adhesion neither viability and interestingly improved mineralization. However, higher concentration of Sr (and consequent higher amount of rutile) showed to induce collagen secretion but with compromised mineralization, possibly due to a delayed mineralization process or induced precipitation of deficient hydroxyapatite. Ca-P-TiO2 porous layer with less concentration of Sr seems to be an ideal candidate for bone implants.

Toxic element concentrations in the Razorbill Alca torda (Charadriiformes, Alcidae) in Portugal.

The present study provides the first data on inorganic element levels (As, Cd, Co, Cr, Cu, Hg, Mn, Pb, Se, and Zn) in juvenile, immature, and adult razorbills (Alca torda) collected along the central coast of Portugal. Element concentrations were assessed by ICP-MS in kidney, liver, muscle, and feathers of 28 razorbills, including 4 juveniles, 17 subadults, and 7 adults. The effect of age and tissue on element accumulation was also assessed. The detected levels in razorbills may indicate a possible contamination risk by Hg and Cr. With respect to bird tissues, higher accumulation of Se and Cd was detected in kidney, Zn and Pb in feathers, and As and Mn in liver. Age was found to affect the accumulation of Cd, Cr, Cu, Hg, and Mn, juveniles presenting higher levels of Cu and Mn than older individuals. In razorbill kidney, Zn-Hg constituted the most significant relationship among metal concentrations. Liver presented the highest number of significant relationships (mostly involving Zn and Co). With regard to feathers, the most significant relationships involved Se, Zn, Cr and Cu concentrations. Positive linear relationships were detected among kidney, liver, and muscle, with emphasis on relationships involving Se and Hg, which may be indicative of similar accumulation/regulation mechanisms in those organs. Element concentrations are discussed in view of possible detoxification mechanisms in seabirds.

Continuous ozonation of urban wastewater: Removal of antibiotics, antibiotic-resistant Escherichia coli and antibiotic resistance genes and phytotoxicity.

This work evaluated the removal of a mixture of eight antibiotics (i.e. ampicillin (AMP), azithromycin (AZM), erythromycin (ERY), clarithromycin (CLA), ofloxacin (OFL), sulfamethoxazole (SMX), trimethoprim (TMP) and tetracycline (TC)) from urban wastewater, by ozonation operated in continuous mode at different hydraulic retention times (HRTs) (i.e. 10, 20, 40 and 60 min) and specific ozone doses (i.e. 0.125, 0.25, 0.50 and 0.75 gO3 gDOC- 1). As expected, the efficiency of ozonation was highly ozone dose- and contact time-dependent. The removal of the parent compounds of the selected antibiotics to levels below their detection limits was achieved with HRT of 40 min and specific ozone dose of 0.125 gO3 gDOC- 1. The effect of ozonation was also investigated at a microbiological and genomic level, by studying the efficiency of the process with respect to the inactivation of Escherichia coli and antibiotic-resistant E. coli, as well as to the reduction of the abundance of selected antibiotic resistance genes (ARGs). The inactivation of total cultivable E. coli was achieved under the experimental conditions of HRT 40 min and 0.25 gO3 gDOC-1, at which all antibiotic compounds were already degraded. The regrowth examinations revealed that higher ozone concentrations were required for the permanent inactivation of E. coli below the Limit of Quantification (

Rutile nano-bio-interactions mediate dissimilar intracellular destiny in human skin cells.

The use of nanoparticles (NPs) in the healthcare market is growing exponentially, due to their unique physicochemical properties. Titanium dioxide nanoparticles (TiO2 NPs) are used in the formulation of sunscreens, due to their photoprotective capacity, but interactions of these particles with skin cells on the nanoscale are still unexplored. In the present study we aimed to determine whether the initial nano-biological interactions, namely the formation of a nano-bio-complex (other than the protein corona), can predict rutile internalization and intracellular trafficking in primary human fibroblasts and keratinocytes. Results showed no significant effect of NPs on fibroblast and keratinocyte viability, but cell proliferation was possibly compromised due to nano-bio-interactions. The bio-complex formation is dependent upon the chemistry of the biological media and NPs' physicochemical properties, facilitating NP internalization and triggering autophagy in both cell types. For the first time, we observed that the intracellular traffic of NPs is different when comparing the two skin cell models, and we detected NPs within multivesicular bodies (MVBs) of keratinocytes. These structures grant selected input of molecules involved in the biogenesis of exosomes, responsible for cell communication and, potentially, structural equilibrium in human tissues. Nanoparticle-mediated alterations of exosome quality, quantity and function can be another major source of nanotoxicity.

The two faces of titanium dioxide nanoparticles bio-camouflage in 3D bone spheroids.

Titanium (Ti) and its alloys are widely used in dental implants and hip-prostheses due to their excellent biocompatibility. Growing evidence support that surface degradation due to corrosion and wear processes, contribute to implant failure, since the release of metallic ions and wear particles generate local tissue reactions (peri-implant inflammatory reactions). The generated ions and wear debris (particles at the micron and nanoscale) stay, in a first moment, at the interface implant-bone. However, depending on their size, they can enter blood circulation possibly contributing to systemic reactions and toxicities. Most of the nanotoxicological studies with titanium dioxide nanoparticles (TiO2 NPs) use conventional two-dimensional cell culture monolayers to explore macrophage and monocyte activation, where limited information regarding bone cells is available. Recently three-dimensional models have been gaining prominence since they present a greater anatomical and physiological relevance. Taking this into consideration, in this work we developed a human osteoblast-like spheroid model, which closely mimics bone cell-cell interactions, providing a more realistic scenario for nanotoxicological studies. The treatment of spheroids with different concentrations of TiO2 NPs during 72 h did not change their viability significantly. Though, higher concentrations of TiO2 NPs influenced osteoblast cell cycle without interfering in their ability to differentiate and mineralize. For higher concentration of TiO2 NPs, collagen deposition and pro-inflammatory cytokine, chemokine and growth factor secretion (involved in osteolysis and bone homeostasis) increased. These results raise the possible use of this model in nanotoxicological studies of osseointegrated devices and demonstrate a possible therapeutic potential of this TiO2 NPs to prevent or reverse bone resorption.

Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function.

Delayed graft function (DGF) often occurs in kidney transplants from deceased donors. We wanted to provide studies giving more accurate non-invasive tests for acute rejection (AR). Using real-time PCR, we examined the expression of cytolytic molecules such as perforin, granzyme B, and fas-ligand along with serpin proteinase inhibitor-9. We also measured the expression of FOXP3, a characteristic gene of T-regulatory cells known to be involved in AR. These studies were conducted on peripheral blood monocytes, urinary cells, and 48 surveillance kidney biopsies taken from a total of 35 patients with DGF. Of these patients, 20 had a histopathological diagnosis of AR, whereas other 28 had characteristics of acute tubular necrosis (ATN). Expression of cytolytic and apoptotic-associated genes in the biopsy tissue, peripheral blood leukocytes, and urinary cells was significantly higher in patients with AR than that in patients with ATN. Diagnostic parameters associated with FOXP3 gene expression were most accurate in peripheral blood leukocytes and urine cells with sensitivity, specificity, positive and negative predictive values, and accuracy between 94 and 100%. Our study shows that quantification of selected genes in peripheral blood leukocytes and urinary cells from renal transplant patients with DGF may provide a useful and accurate non-invasive diagnosis of AR.

Association between the presence of anti-HLA antibodies with acute rejection and chronic allograft nephropathy in the first year after kidney transplantation.

The clinical relevance of anti-HLA antibodies following kidney transplantation has been a recent focus of research. Patients who present anti-HLA antibodies in the posttransplantation period have shown higher incidences of acute rejection episodes (ARE) and chronic allograft nephropathy (CAN). The objective of this study was to evaluate the presence of anti-HLA antibodies during the first year after kidney transplantation and their association with the occurrence of ARE and CAN. Eighty-eight kidney transplant recipients were evaluated for the presence of IgG anti-HLA antibodies using an enzyme-linked immunosorbent assay (LAT-M and LAT-1240, One Lambda Inc, Calif, United States). Protocol kidney biopsies were performed in consenting patients. ARE and CAN were diagnosed by clinical, laboratory, and histopathological criteria. Anti-HLA antibodies were observed in 20 (22.7%) patients. At 1 year follow-up, 26.1% presented ARE and 51.2% developed CAN. Nine patients (45%) with antibodies developed ARE as opposed to 20.6% without antibodies and 64.7% developed CAN as opposed to 47.8% of those without antibodies. In the histological analysis, the anti-HLA antibodies were associated with Banff IIA ARE (P = .001) and Banff grade II CAN (P = .012). Routine posttransplantation search for antibodies may identify cases at higher risk for acute and chronic rejection, and perhaps help to tailor the immunosuppressive regimen.

Distribution and environmental assessment of trace elements contamination of water, sediments and flora from Douro River estuary, Portugal.

The present study evaluated the content and distribution of several trace elements (Li, Be, Al, V, Cr, Co, Ni, Cu, Zn, Se, Mo, Ag, Cd, Sb, Ba, Tl, Pb, and U) in the Douro River estuary. For that, three matrices were collected (water, sediments and native local flora) to assess the extent of contamination by these elements in this estuarine ecosystem. Results showed their occurrence in estuarine water and sediments, but significant differences were recorded on the concentration levels and pattern of distribution among both matrices and sampling points. Generally, the levels of trace elements were higher in the sediments than in the respective estuarine water. Nonetheless, no correlation among trace elements was determined between water and sediments, except for Cd. Al was the trace element found at highest concentration at both sediments and water followed by Zn. Pollution indices such as geo-accumulation (Igeo), enrichment factor (EF) and contamination factor (CF) were determined to understand the levels and sources of trace elements pollution. Igeo showed strong contamination by anthropogenic activities for Li, Al, V, Cr, Ni, Cu, Zn, Ba and Pb at all sampling points while EF and CF demonstrated severe enrichment and contamination by Se, Sb and Pb. Levels of trace elements were compared to acceptable values for aquatic organisms and Sediment Quality Guidelines. The concentration of some trace elements, namely Al, Pb and Cu, were higher than those considered acceptable, with potential negative impact on local living organisms. Nevertheless, permissible values for all trace elements are still not available, demonstrating that further studies are needed in order to have a complete assessment of environmental risk. Furthermore, the occurrence and possible accumulation of trace elements by local plant species and macroalgae were investigated as well as their potential use as bioindicators of local pollution and for phytoremediation purposes.

Basiliximab improves graft survival in renal transplant recipients with delayed graft function.

The aim of this study was to evaluate the use of basiliximab in first renal transplant recipients with delayed graft function, defined by the need for dialysis in the first week posttransplantation. Among 148 patients in the study, 90 received basiliximab (60.8%) with 58 comprising the control group. There were no significant differences between the 2 groups related to the evaluated variables, except that the control group received more blood transfusions pretransplantation. There was a lower incidence of steroid-resistant rejection (6% vs 20.9%; P = .017) and humoral rejections (0% vs 7%; P = .038) in the basiliximab group. Also, graft survival was significantly higher in basiliximab group compared with the control one (92.8% vs 80.4%; P = .028). There were no significant differences in the other outcomes. In conclusion, this study confirmed the beneficial effects of addition of basiliximab to the immunosuppressive schema of patients with delayed graft function.

Utility of biopsy in kidney transplants with delayed graft function and acute dysfunction.

Renal biopsy is currently the gold standard to assess the causes of renal allograft dysfunction. In the present study, we prospectively assessed the role of the renal allograft biopsy in the diagnosis and treatment of renal allograft dysfunction. Seven hundred and fifteen biopsies were performed in 399 patients. The anatomopathological results in group 1 (delayed graft function) were: 60.4% acute tubular necrosis, 17.6% acute rejection, 4.3% calcineurin inhibitor toxicity, and 17.7% other diagnoses; in group 2 (acute graft dysfunction): 42.3% acute rejection, 22% acute tubular necrosis, 8.4% calcineurin inhibitor toxicity, and 27.3% other diagnoses. Among patients with delayed graft function, 42.2% of biopsies led to a change in the treatment. In 60.5%, the biopsy of patients with acute dysfunction led to a change in the patient management. In our series, the result of the biopsy disagreed with the clinical diagnosis in 39.6% and 57.7% of cases, respectively. These results demonstrated that renal graft biopsy remains an indispensable tool for the accurate management of kidney transplant patients.

High throughput toxicity screening and intracellular detection of nanomaterials.

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety-preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read-across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter-experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM-cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose- and time-dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label-free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance-based monitoring, Multiplex analysis of secreted products, and genotoxicity methods-namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells.

Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of 'Trojan-horse' internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications.

Titanium (Ti) is commonly used in dental implant applications. Surface modification strategies are being followed in last years in order to build Ti oxide-based surfaces that can fulfill, simultaneously, the following requirements: induced cell attachment and adhesion, while providing a superior corrosion and tribocorrosion performance. In this work micro-arc oxidation (MAO) was used as a tool for the growth of a nanostructured bioactive titanium oxide layer aimed to enhance cell attachment and adhesion for dental implant applications. Characterization of the surfaces was performed, in terms of morphology, topography, chemical composition and crystalline structure. Primary human osteoblast adhesion on the developed surfaces was investigated in detail by electronic and atomic force microscopy as well as immunocytochemistry. Also an investigation on the early cytokine production was performed. Results show that a relatively thick hybrid and graded oxide layer was produced on the Ti surface, being constituted by a mixture of anatase, rutile and amorphous phases where calcium (Ca) and phosphorous (P) were incorporated. An outermost nanometric-thick amorphous oxide layer rich in Ca was present in the film. This amorphous layer, rich in Ca, improved fibroblast viability and metabolic activity as well as osteoblast adhesion. High-resolution techniques allowed to understand that osteoblasts adhered less in the crystalline-rich regions while they preferentially adhere and spread over in the Ca-rich amorphous oxide layer. Also, these surfaces induce higher amounts of IFN-γ cytokine secretion, which is known to regulate inflammatory responses, bone microarchitecture as well as cytoskeleton reorganization and cellular spreading. These surfaces are promising in the context of dental implants, since they might lead to faster osseointegration.

Anterior tuberculous scleritis: A diagnostic challenge.

CASE REPORT: We present the case of a 26 year-old Pakistani male, who after the presumptive diagnosis of anterior tuberculous scleritis (by an atypical clinical appearance and positive epidemiological link), was diagnosed with pulmonary tuberculosis. DISCUSSION: Nodular anterior scleritis is an uncommon presentation of tuberculosis (TB). It is considered a diagnostic challenge because of the difficulty to extract bacilli from the ocular tissue. However, a detailed medical history and eye examination can be the key to an accurate diagnosis and appropriate treatment of the TB.

Intrahepatic distribution of vitamin A in humans and rats.

Liver vitamin A was determined in 10 different regions of the liver from 13 rats and 17 human individuals deceased of various causes. Linear regression analysis and comparison of the values obtained from the different anatomical regions showed that, despite a high degree of heterogeneity in distribution and lack of a consistent pattern, samples from any of the sites analyzed ensure representativeness of the whole liver concentration, taken as the average of the values from the 10 samples of each liver. This means that properly trained personnel, but not necessarily specialized, could draw the liver samples, thus significantly reducing the cost of surveys of vitamin A status based on liver reserves of the vitamin determined at autopsy.