Extraction of Antioxidants from Brown Macroalgae Fucus spiralis.

In this study, different extraction methods and conditions were used for the extraction of antioxidants from brown macroalgae Fucus spiralis. The extraction methodologies used were ultrasound-assisted extraction (ultrasonic bath and ultrasonic probe), extraction with a vortex, extraction with an Ultra-Turrax® homogenizer, and high-pressure-assisted extraction. The extracts were analyzed for their total phenolic content (TPC) and their antioxidant activity, and evaluated through the 2,2-difenil-1-picrilhidrazil (DPPH) free radical scavenging method and ferric reducing antioxidant power (FRAP) assay. Ultrasonic probe-assisted extraction yielded the highest values of TPC (94.78-474.16 mg gallic acid equivalents/g extract). Regarding the antioxidant activity, vortex-assisted extraction gave the best DPPH results (IC50 1.89-16 µg/mL), while the highest FRAP results were obtained using the Ultra-Turrax® homogenizer (502.16-1188.81 µmol ascorbic acid equivalents/g extract). For each extraction method, response surface methodology was used to analyze the influence of the experimental conditions "extraction time" (t), "biomass/solvent ratio" (R), "solvent" (S, water % in water/ethanol mixture), and "pressure" (P) on TPC, DPPH, and FRAP of the F. spiralis extracts. In general, higher TPC content and higher antioxidant capacity (lower IC50 and higher FRAP) were obtained with higher R, t, and P, and lower S (higher ethanol %). The model regarding the combined effects of independent variables t, R, and S on the FRAP response values for vortex-assisted extractions best fitted the experimental data (R2 0.957), with optimal extraction conditions of t = 300 s, R = 50 g, and S = 25%.

Radiation exposure in very low birth weight infants.

BACKGROUND: The survival of very low birth weight (VLBW) infants increased in the past few decades. These neonates often require multiple diagnostic and management image procedures that involve ionizing radiation, which can have long term implications. The aim of our study was to evaluate the level of radiation exposure in VLBW infants during their stay in the Neonatal Intensive Care Unit (NICU). METHODS: We collected demographic and medical data of 149 VLBW who were admitted to our NICU between January 2011 and December 2014. All radiographic procedures were reviewed retrospectively. Absorbed ionizing radiation was calculated according to literature reference values. RESULTS: A total of 1496 images were obtained. Infants underwent 10.0±11.3 examinations, and the maximum of images registered per patient was 65. Four babies (2.7%) received more than 1000 µSv, the recommended maximum of ionizing radiation exposure. Infants of lower birth weight, who needed invasive ventilation, with bronchopulmonary dysplasia, sepsis, and surgical pathology required significantly more radiographs (P<0.001). CONCLUSIONS: In this study, lower birth weight, need of invasive ventilation, bronchopulmonary dysplasia and sepsis were associated with the need of more X-ray studies. In order to protect the vulnerable population of severely-ill newborns, guidelines for radiation exposure in newborns should be issued and implemented.

Inhibition of TGF-ß signaling promotes expansion of human epidermal keratinocytes in feeder cell co-culture.

Cultured epidermal autografts have been used worldwide since 1981 for patients with extensive third-degree burn wounds and limited skin donor sites. Despite significant progress in techniques toward improving clinical outcome of skin grafts, the long in vitro preparation time of cultured autografts has remained a major factor limiting its widespread use. Here, we show that pharmacological inhibition of TGF-ß signaling promotes the expansion of human epidermal keratinocytes (HEKs) with high proliferative potential in co-cultures with both murine 3T3-J2 cells and human feeder cells, including dermal fibroblasts and preadipocytes. In contrast, TGF-ß signaling inhibition does not enhance the growth of HEKs in a serum- and feeder-free condition, an alternative approach to propagate HEKs for subsequent autograft production. Our results have important implications for the use of TGF-ß signaling inhibition as a viable therapeutic strategy for improving Green's methodology and for more efficient production of customized skin autografts with human feeder cells.

Effect of Heavy Metals in Plants of the Genus Brassica.

Several species from the Brassica genus are very important agricultural crops in different parts of the world and are also known to be heavy metal accumulators. There have been a large number of studies regarding the tolerance, uptake and defense mechanism in several of these species, notably Brassica juncea and B. napus, against the stress induced by heavy metals. Numerous studies have also been published about the capacity of these species to be used for phytoremediation purposes but with mixed results. This review will focus on the latest developments in the study of the uptake capacity, oxidative damage and biochemical and physiological tolerance and defense mechanisms to heavy metal toxicity on six economically important species: B. juncea, B. napus, B. oleracea, B. carinata, B. rapa and B. nigra.

Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persist for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.

Inhibition of TGF-ß signaling enables long-term proliferation of mouse primary epithelial stem/progenitor cells of the tympanic membrane and the middle ear mucosa.

The surface of the middle ear is composed of the tympanic membrane (TM) and the middle ear mucosa (MEM). A number of diseases and conditions such as otitis media, middle ear cholesteatoma, and perforation of the TM have been reported to cause dysfunction of the middle ear, ultimately leading to high-frequency hearing loss. Despite its importance in repairing the damaged tissues, the stem/progenitor cells of the TM and the MEM epithelia remains largely uncharacterized due, in part, to the lack of an optimal methodology to expand and maintain stem/progenitor cells long-term. Here, we show that suppression of TGF-ß signaling in a low Ca2+ condition enables long-term proliferation of p63-positive epithelial stem/progenitor cells of the TM and the MEM while avoiding their malignant transformation. Indeed, our data show that the expanded TM and MEM stem/progenitor cells respond to Ca2+ stimulation and differentiate into the mature epithelial cell lineages marked by cytokeratin (CK) 1/8/18 or Bpifa1, respectively. These results will allow us to expand epithelial stem/progenitor cells of the TM and MEM in quantity for large-scale analyses and will enhance the use of mouse models in developing stem cell-mediated therapeutic strategies for the treatment of middle ear diseases and conditions.

Valorization of the Red Seaweed Gracilaria gracilis Through a Biorefinery Approach.

The interest in seaweeds as an abundant feedstock to obtain valuable and multitarget bioactive ingredients is continuously growing. In this work, we explore the potential of Gracilaria gracilis, an edible red seaweed cultivated worldwide for its commercial interest as a source of agar and other ingredients for cosmetic, pharmacological, food, and feed applications. G. gracilis growth conditions were optimized through vegetative propagation and sporulation while manipulating the physicochemical conditions to achieve a large biomass stock. Green extraction methodologies with ethanol and water were performed over the seaweed biomass. The bioactive potential of extracts was assessed through a set of in vitro assays concerning their cytotoxicity, antioxidant, and antimicrobial properties. Additionally, dried seaweed biomass was incorporated into pasta formulations to increase food's nutritional value. Pigments extracted from G. gracilis have also been incorporated into yogurt as a natural colorant, and their stability was evaluated. Both products were submitted to the appreciation of a semi-trained sensorial panel aiming to achieve the best final formulation before reaching the market. Results support the versatility of G. gracilis whether it is applied as a whole biomass, extracts and/or pigments. Through implementing several optimized protocols, this work allows the development of products with the potential to profit the food, cosmetic, and aquaculture markets, promoting environmental sustainability and a blue circular economy. Moreover, and in line with a biorefinery approach, the residual seaweed biomass will be used as biostimulant for plant growth or converted to carbon materials to be used in water purification of the in-house aquaculture systems of MARE-Polytechnic of Leiria, Portugal.

Adrenal carcinoma as the first manifestation of a Li­Fraumeni syndrome in three paediatric patients.

Not required for clinical vignette.

Seasonal study of the nutritional composition of unexploited and low commercial value fish species from the Portuguese coast.

Target species diversification is essential for fisheries sustainability and fish market revitalization. Fish discards are a widely recognized problem resulting from fisheries worldwide, and are of major concern for all sector players, from administrations, to fishermen, and scientists. However, non-target species are seldom studied, and information on nutritional profiles and seasonal changes in nutritional properties is generally lacking. This study assessed the seasonal nutritional composition of two unexploited (Serranus cabrilla, Capros aper) and three low commercial value fish species (Trachurus picturatus, Spondyliosoma cantharus, and Trigla lyra), captured on the Portuguese coast over 1 year. Significant seasonal variations were observed in the nutritional composition of all the species studied. Moisture and ash contents varied from 70% to 81% and from 5% to 13%, respectively. The maximum fat contents were 5% for C. aper and 4% for T. picturatus, allowing to classify all studied fishes as lean. The highest protein contents were recorded for C. aper (25%) and S. cantharus (20%). The unexploited and low commercial value fish species studied were shown to be good fat and protein sources, comparable to commonly consumed species, such as cod and salmon, having a great potential to become commonly consumed fish in Portugal.

Annual variations in the mineral element content of five fish species from the Portuguese coast.

The most consumed fish species in Portugal do not correspond to the most caught fish species on the Portuguese coast. From the point of view of ocean sustainability and economics, it is important to study unexploited or underexploited fish species that have the potential to be introduced into the food market. This potential can be reflected by their nutritional value, where mineral elements are often overlooked despite their importance in human health. Therefore, it is important to study the elemental concentration of those fish species, which should also include seasonal variations of essential mineral elements such as Ca, P, Mg, Na, K, Fe, Cu, Mn, Zn, and I, and non-essential elements such as As, Ni, Cd, and Pb. This work is part of a project that studied five fish species with the potential to be brought to the market: three species with low commercial value (Trachurus picturatus, Spondyliosoma cantharus and Trigla lyra) and two species with no commercial value (Serranus cabrilla and Capros aper), caught over a year in the coast of Portugal. Mineral elements were quantified by ICP-OES (ICP-MS for I). The results showed that the studied fish species present significant elemental concentrations, similar to those found in the most caught fish species in Portugal. Trachurus picturatus was found to be a source of Fe, and all species are sources of I. Overall, Spondyliosoma cantharus proved to be the most element-rich species. Sensorially, Cu was related to "metallic odour" and Fe to "metallic flavour", and I was related to "seaweed odour" and "seawater flavour". Additionally, toxic elements such as Cd, Pb, Ni and As were not detected. The corresponding Hazard Quotients (HQ) show the safety of the consumption of these fish species concerning the content of potentially toxic elements.

Fabry Disease: A Atypical Presentation.

Fabry Disease (FD) is a rare X-linked recessive disease caused by mutations in the GLA gene that lead to a decrease or lack of activity of the enzyme alpha galactosyl A. This lysosomal storage disorder results in progressive damage and dysfunction of several organs and, depending on the type of mutation and gender of the patient, and it may have different manifestations. As FD is a multisystem disease with a progressive character and varying severity, the diagnosis can be challenging, especially when it comes to non-classical forms. As this is a hereditary disease, its diagnosis impacts not only the patient but also his family, making an accurate and timely diagnosis even more important. We present the case of a 59-years-old man diagnosed with non-classical FD, with previous neurological and psychiatric complaints, who was admitted to the Emergency Department (ED) with a generalized tonic-clonic seizure that required orotracheal intubation for airway protection and transferred to an Intensive Care Unit (ICU).

Effects of sitagliptin treatment on dysmetabolism, inflammation, and oxidative stress in an animal model of type 2 diabetes (ZDF rat).

The purpose of this paper is to evaluate the chronic effect of sitagliptin on metabolic profile, inflammation, and redox status in the Zucker Diabetic Fatty (ZDF) rat, an animal model of obese type 2 diabetes. Diabetic and obese ZDF (fa/fa) rats and their controls (ZDF +/+) were treated during 6 weeks with vehicle (control) and sitagliptin (10 mg/kg/bw). Glucose, HbA1c, insulin, Total-c, TGs, IL-1beta, TNF-alpha, CRPhs, and adiponectin were assessed in serum and MDA and TAS in serum, pancreas, and heart. Pancreatic histology was also evaluated. Sitagliptin in diabetic rats promoted a decrease in glucose, HbA1c, Total-c, and TGs accompanied by a partial prevention of insulinopenia, together, with a decrease in CRPhs and IL-1beta. Sitagliptin also showed a positive impact on lipid peroxidation and hypertension prevention. In conclusion, chronic sitagliptin treatment corrected the glycaemic dysmetabolism, hypertriglyceridaemia, inflammation, and hypertension, reduced the severity of the histopathological lesions of pancreatic endocrine and exocrine tissues, together with a favourable redox status, which might be a further advantage in the management of diabetes and its proatherogenic comorbidities.

The Simplest Protocol for Rapid and Long-Term Culture of Primary Epidermal Keratinocytes from Human and Mouse.

Although mouse models have been used as an essential tool for studying the physiology and diseases of the skin, propagation of mouse primary epidermal keratinocytes remains challenging. In this chapter, we introduce the simplest, at least to our knowledge, protocol that enables long-term expansion of p63+ mouse epidermal keratinocytes in low Ca2+ media without the need of progenitor cell-purification steps or support by a feeder cell layer. Pharmacological inhibition of TGF-ß signaling in crude preparations of mouse epidermis robustly increases proliferative capacity of p63+ epidermal progenitor cells, while preserving their ability to differentiate. Suppression of TGF-ß signaling also permits p63+ epidermal keratinocytes to form macroscopically large clones in 3T3-J2 feeder cell co-culture. Suppression of TGF-ß signaling also enhances the clonal growth of human keratinocytes in co-culture with a variety of feeder cells. This simple and efficient approach will not only facilitate the use of mouse models by providing p63+ primary epidermal keratinocytes in quantity but also significantly reduce the time needed for preparing the customized skin grafts in Green method.

Quality Assessment of Chilled and Frozen Fish-Mini Review.

Fish is a very perishable food and therefore several storage strategies need to be employed to increase its shelf-life, guaranteeing its safety and quality from catch to consumption. Despite the advances in modern fish storage technologies, chilling and freezing are still the most common preservation methods used onboard. The present review aims to summarize strategies to increase the shelf-life of fresh (chilled) and frozen fish, as whole, gutted, or fillet, involving the assessment of different traditional cooling and freezing conditions of different fish species caught in different locations. Although there are other factors that influence the fish shelf-life, such as the fish species and the stress suffered during catch, storage time and temperature and the amount of ice are some of the most important. In addition, the way that fish is stored (whole, fillet, or gutted) also contributes to the final quality of the product. In most studies, whole chilled and frozen fish present longer shelf-life than those preserved as gutted and filleted. However, it should be noted that other factors related to the organism, capture method, and transport to the preparation/processing industry should be considered for shelf-life extension.

Seasonal Sensory Evaluation of Low Commercial Value or Unexploited Fish Species from the Portuguese Coast.

Overfishing is increasing over time, and according to FAO (Food and Agriculture Organization), about one third of the world's fish stocks are now overfished. Thus, diversifying the target species is essential for fisheries sustainability contributing to improve resource-efficient processes. Non-target species can be valuable resources for the development of new food products. However, those species are scarcely studied, and it is of high importance to trace their seasonal sensory profile as a first step towards their valorisation. Therefore, in this study, seasonal influence on sensory properties of five low commercial value or unexploited fish species, namely Trachurus picturatus (blue jack mackerel), Spondyliosoma cantharus (black seabream), Trigla lyra (piper gurnard), Serranus cabrilla (comber) and Capros aper (boarfish), was assessed in order to identify the most favourable season for catching each species. Fish samples were assessed by a panel of 16 semi-trained assessors for sensory attributes previously identified. The evaluation takes place every 2 months. Statistical differences were reported between attributes and seasons for all species, except for T. lyra, which did not present any difference in its sensory attributes throughout the year.

Adding Value to Bycatch Fish Species Captured in the Portuguese Coast-Development of New Food Products.

We live in a world of limited biological resources and ecosystems, which are essential to feed people. Consequently, diversifying target species and considering full exploitation are essential for fishery sustainability. The present study focuses on the valorization of three low commercial value fish species (blue jack mackerel, Trachurus picturatus; black seabream, Spondyliosoma cantharus; and piper gurnard, Trigla lyra) and of two unexploited species (comber, Serranus cabrilla and boarfish, Capros aper) through the development of marine-based food products with added value. A preliminary inquiry with 155 consumers from Região de Lisboa e Vale do Tejo (Center of Portugal) was conducted to assess fish consumption, the applicability of fish product innovation, and the importance of valorizing discarded fish. Five products (black seabream ceviche, smoked blue jack mackerel pâté, dehydrated piper gurnard, fried boarfish, and comber pastries) were developed and investigated for their sensory characteristics and consumer liking by hedonic tests to 90 consumers. The most important descriptors were identified for each product (texture, flavor, color, and appearance). Comber pastries had the highest purchase intention (88%), followed by black seabream ceviche (85%) and blue jack mackerel pâté (76%). Sensory evaluations showed a clear tendency of consumers to accept reformulated products, with the introduction of the low-value and unexploited species under study.

Promotion of Human Epidermal Keratinocyte Expansion in Feeder Cell Co-culture.

Co-culture of human epidermal keratinocytes with mouse 3T3-J2 feeder cells, developed by Green and colleagues, has been used worldwide to generate skin autografts since the early 1980s. In addition, co-culture with 3T3-J2 cells has served as a fundamental tool in skin stem cell biology as it allows the evaluation of self-renewal capacity of epidermal stem cells. This chapter describes a recent improvement in the Green method to promote further the expansion of human epidermal keratinocytes utilizing a small-molecule inhibitor of TGF-ß signaling. This new protocol enables more rapid expansion of human epidermal keratinocytes in co-culture with not only 3T3-J2 cells but also other feeder cells including human dermal fibroblasts and human preadipocytes, two major alternatives to 3T3-J2 cells, with a long-term goal of developing customized skin autografts.

Long-Term Expansion of Mouse Primary Epidermal Keratinocytes Using a TGF-ß Signaling Inhibitor.

Mouse models have been used to study the physiology and pathogenesis of the skin. However, propagation of mouse primary epidermal keratinocytes remains challenging. In this chapter, we introduce a newly developed protocol that enables long-term expansion of p63+ mouse epidermal keratinocytes in low-Ca2+ media without the need of progenitor cell purification steps or support by a feeder cell layer. Pharmacological inhibition of TGF-ß signaling in crude preparations of mouse epidermis robustly increases proliferative capacity of p63+ epidermal progenitor cells while preserving their ability to differentiate. Suppression of TGF-ß signaling also permits p63+ epidermal keratinocytes to form macroscopically large clones in 3T3-J2 feeder cell co-culture. This simple and efficient approach will facilitate the use of mouse models by providing p63+ primary epidermal keratinocytes in quantity.

Inhibition of TGF-ß signaling supports high proliferative potential of diverse p63+ mouse epithelial progenitor cells in vitro.

Mouse models have been used to provide primary cells to study physiology and pathogenesis of epithelia. However, highly efficient simple approaches to propagate mouse primary epithelial cells remain challenging. Here, we show that pharmacological inhibition of TGF-ß signaling enables long-term expansion of p63+ epithelial progenitor cells in low Ca2+ media without the need of progenitor cell-purification steps or support by a feeder cell layer. We find that TGF-ß signaling is operative in mouse primary keratinocytes in conventional cultures as determined by the nuclear Smad2/3 localization. Accordingly, TGF-ß signaling inhibition in crude preparations of mouse epidermis robustly increases proliferative capacity of p63+ epidermal progenitor cells, while preserving their ability of differentiation in response to Ca2+ stimulation. Notably, inhibition of TGF-ß signaling also enriches and expands other p63+ epithelial progenitor cells in primary crude cultures of multiple epithelia, including the cornea, oral and lingual epithelia, salivary gland, esophagus, thymus, and bladder. We anticipate that this simple and efficient approach will facilitate the use of mouse models for studying a wide range of epithelia by providing highly enriched populations of diverse p63+ epithelial progenitor cells in quantity.

Optimal isolation and xeno-free culture conditions for limbal stem cell function.

PURPOSE: To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions. METHODS: Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype. RESULTS: Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2. CONCLUSIONS: The LSC function can be maintained in vitro under appropriate Xeno-free conditions.