Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Da superfície para a profundidade: Curva de aprendizado em lifting facial profundo estendido - meus primeiros 100 casos / From Surface to Depth: Learning Curve in Extended Deep Facelift ­ my First 100 Cases

Introdução: O envelhecimento facial é um processo gradual, complexo e multifatorial. É o resultado de mudanças na qualidade, volume e posicionamento dos tecidos. Cirurgiões plásticos têm modificado sua abordagem na cirurgia do rejuvenescimento facial optando pelo plano subaponeurótico (SMAS). O objetivo deste estudo é analisar 100 casos de pacientes operados pela técnica de SMAS profundo, avaliando sua aplicabilidade e eficácia. Método: Foram avaliados 100 pacientes, submetidos a cirurgia plástica facial pela técnica de SMAS profundo - "Deep Smas", e acompanhados por 6 meses. Observou-se a satisfação dos pacientes, número de complicações, número de reoperações, riscos e vantagens da técnica. Resultados: Foram operados 100 pacientes, num período de 3 anos. A idade variou de 41 a 79 anos, sendo 95% sexo feminino. As complicações foram 8 casos (8%) de lesões de ramos do nervo facial, sendo: 4 casos lesão do zigomático, 3 casos de lesão do mandibular e 1 caso de lesão do bucal; houve 1 caso (1%) de queloide retroauricular; 1 caso (1%) de hematoma. Em relação às revisões cirúrgicas, houve 8 casos (8%) de complementação cirúrgica por insatisfação das pacientes. Houve 15% de lesões nervosas entre a 1ª e a 40ª cirurgia, 5% entre a 41ª e a 80ª, e nenhuma lesão entre o 81º e o 100º paciente. Conclusão: O lifting facial profundo ou subSMAS mostrou ser efetivo, proporcionando bons resultados estéticos. Apresenta baixa taxa de recidiva e baixa taxa de morbidade, porém, necessita de uma longa curva de aprendizagem.

Introduction: Facial aging is a gradual, complex, and multifactorial process. It is the result of changes in the quality, volume, and positioning of tissues. Plastic surgeons have modified their approach to facial rejuvenation surgery, opting for the subaponeurotic plane (SMAS). The objective of this study is to analyze 100 cases of patients operated on using the deep SMAS technique, evaluating its applicability and effectiveness. Method: 100 patients were evaluated, undergoing facial plastic surgery using the deep SMAS technique - "Deep Smas", and followed up for 6 months. Patient satisfaction, number of complications, number of reoperations, risks, and advantages of the technique were observed. Results: 100 patients were operated on over 3 years. Age ranged from 41 to 79 years, with 95% being female. The complications were 8 cases (8%) of injuries to branches of the facial nerve, of which 4 cases of zygomatic injury, 3 cases of mandibular injury, and 1 case of buccal injury; there was 1 case (1%) of post-auricular keloid; 1 case (1%) of hematoma. Regarding surgical revisions, there were 8 cases (8%) of surgical completion due to patient dissatisfaction. There were 15% of nerve injuries between the 1st and 40th surgery, 5% between the 41st and 80th, and no injuries between the 81st and 100th patient. Conclusion: Deep facial lifting or subSMAS has proven to be effective, providing good aesthetic results. It has a low recurrence rate and low morbidity rate; however, it requires a long learning curve.

Tratamento cirúrgico de ginecomastia grau III com a técnica de cicatriz horizontal e transposição do complexo areolopapilar - "No vertical scar" / Surgical treatment of grade III gynecomastia with the horizontal scar technique and transposition of the nipple-areolar complex - "No vertical scar"

Introdução: A demanda por cirurgia para correção de ginecomastia tem aumentado em função da grande quantidade de pacientes submetidos a tratamentos bariátricos. A maioria destes pacientes requerem grande remoção do excesso de pele, tecido gorduroso e reposicionamento do complexo areolopapilar, assim como adequação do volume da aréola. O objetivo deste estudo é descrever a técnica de correção de ginecomastia grau III através de cicatriz horizontal sem cicatriz vertical, com reposicionamento do complexo areolopapilar. Método: Estudo prospectivo de 27 pacientes masculinos com diagnóstico de ginecomastia grau III da classificação de Simon, operados em clínica particular, entre janeiro de 2013 e agosto de 2020, pela técnica de cicatriz horizontal com transposição do complexo areolopapilar e sem cicatriz vertical. Resultados: Foram operados 27 pacientes, num período de 7,5 anos, todos ex-obesos, submetidos previamente a tratamento de perda de peso. A idade variou de 17 a 74 anos, média etária de 52 anos. O peso das peças removidas variou de 175 a 758 gramas, média de 376 gramas. As complicações foram: 1 caso de hematoma tardio (3,7%), 1 caso de seroma (3,7%) e 2 casos de cicatriz hipertrófica (7,4%). Conclusão: O tratamento cirúrgico da ginecomastia grau III através da técnica de cicatriz horizontal, sem cicatriz vertical e com reposicionamento do complexo areolopapilar demonstrou ser efetivo, com baixa morbidade e bons resultados estéticos.

Introduction: The demand for surgery to correct gynecomastia has increased due to the many patients undergoing bariatric treatments. Most patients require extensive removal of excess skin, and fatty tissue, repositioning the nipple-areolar complex, and adjusting the areola volume. This study aims to describe the correction technique for grade III gynecomastia using a horizontal scar without a vertical scar, with repositioning of the nipple-areolar complex. Method: Prospective study of 27 male patients diagnosed with Simon grade III gynecomastia, operated in a private clinic between January 2013 and August 2020, using the horizontal scar technique with transposition of the nipple-areola complex and without a vertical scar. Results: 27 patients underwent surgery over 7.5 years, all ex-obese, previously submitted to weight loss treatment. Age ranged from 17 to 74 years, mean age of 52 years. The weight of the removed parts ranged from 175 to 758 grams, an average of 376 grams. Complications were 1 case of late hematoma (3.7%), 1 case of seroma (3.7%), and 2 cases of hypertrophic scarring (7.4%). Conclusion: Surgical treatment of grade III gynecomastia using the horizontal scar technique, without a vertical scar and with repositioning of the nippleareolar complex, proved to be effective, with low morbidity and good aesthetic results.

Tendon Aging.

Tendons are mechanosensitive connective tissues responsible for the connection between muscles and bones by transmitting forces that allow the movement of the body, yet, with advancing age, tendons become more prone to degeneration followed by injuries. Tendon diseases are one of the main causes of incapacity worldwide, leading to changes in tendon composition, structure, and biomechanical properties, as well as a decline in regenerative potential. There is still a great lack of knowledge regarding tendon cellular and molecular biology, interplay between biochemistry and biomechanics, and the complex pathomechanisms involved in tendon diseases. Consequently, this reflects a huge need for basic and clinical research to better elucidate the nature of healthy tendon tissue and also tendon aging process and associated diseases. This chapter concisely describes the effects that the aging process has on tendons at the tissue, cellular, and molecular levels and briefly reviews potential biological predictors of tendon aging. Recent research findings that are herein reviewed and discussed might contribute to the development of precision tendon therapies targeting the elderly population.

Platelet-Derived Extracellular Vesicles Promote Tenogenic Differentiation of Stem Cells on Bioengineered Living Fibers.

Tendon mimetic scaffolds that recreate the tendon hierarchical structure and niche have increasing potential to fully restore tendon functionality. However, most scaffolds lack biofunctionality to boost the tenogenic differentiation of stem cells. In this study, we assessed the role of platelet-derived extracellular vesicles (EVs) in stem cells' tenogenic commitment using a 3D bioengineered in vitro tendon model. First, we relied on fibrous scaffolds coated with collagen hydrogels encapsulating human adipose-derived stem cells (hASCs) to bioengineer our composite living fibers. We found that the hASCs in our fibers showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted the hASCs' tenogenic commitment, prevented phenotypic drift, enhanced the deposition of the tendon-like extracellular matrix, and induced lower collagen matrix contraction. In conclusion, our living fibers provided an in vitro system for tendon tissue engineering, allowing us to study not only the tendon microenvironment but also the influence of biochemical cues on stem cell behavior. More importantly, we showed that platelet-derived EVs are a promising biochemical tool for tissue engineering and regenerative medicine applications that are worthy of further exploration, as paracrine signaling might potentiate tendon repair and regeneration.

S-Adenosylmethionine (SAM)-Dependent Methyltransferase MftM is Responsible for Methylation of the Redox Cofactor Mycofactocin.

Mycobacteria produce several unusual cofactors that contribute to their metabolic versatility and capability to survive in different environments. Mycofactocin (MFT) is a redox cofactor involved in ethanol metabolism. The redox-active core moiety of mycofactocin is derived from the short precursor peptide MftA, which is modified by several maturases. Recently, it has been shown that the core moiety is decorated by a ß-1,4-glucan chain. Remarkably, the second glucose moiety of the oligosaccharide chain was found to be 2-O-methylated in Mycolicibacterium smegmatis. The biosynthetic gene responsible for this methylation, however, remained elusive, and no methyltransferase gene was part of the MFT biosynthetic gene cluster. Here, we applied reverse genetics to identify the gene product of MSMEG_6237 (mftM) as the SAM-dependent methyltransferase was responsible for methylation of the cofactor in M. smegmatis. According to metabolic analysis and comparative genomics, the occurrence of methylated MFT species was correlated with the presence of mftM homologues in the genomes of mycofactocin producers. This study revealed that the pathogen Mycobacterium tuberculosis does not methylate mycofactocins. Interestingly, mftM homologues co-occur with both mycofactocin biosynthesis genes as well as the putative mycofactocin-dependent alcohol dehydrogenase Mdo. We further showed that mftM knock-out mutants of M. smegmatis suffer from a prolonged lag phase when grown on ethanol as a carbon source. In addition, in vitro digestion of the glucose chain by cellulase suggested a protective function of glucan methylation. These results close an important knowledge gap and provide a basis for future studies into the physiological functions of this unusual cofactor modification.

Controlling the fate of regenerative cells with engineered platelet-derived extracellular vesicles.

Extracellular vesicles (EVs) have emerged as cell-free nanotherapeutic agents for the potential treatment of multiple diseases and for tissue engineering and regenerative medicine strategies. Nevertheless, the field has typically relied on EVs derived from stem cells, the production of which in high quantities and high reproducibility is still under debate. Platelet-derived EVs were produced by a freeze-thaw method of platelet concentrates, a highly available clinical waste material. The aim of this study was to produce and thoroughly characterize platelet-derived EVs and understand their effects in adipose-tissue derived stem cells (hASCs), endothelial cells (HUVECs) and macrophages. Two different EV populations were obtained after differential centrifugation, namely small EVs (sEVs) and medium EVs (mEVs), which showed different size distributions and unique proteomic signatures. EV interaction with hASCs resulted in the modulation of the gene expression of markers related to their commitment toward different lineages. Moreover, mEVs showed higher angiogenic potential than sEVs, in a tube formation assay with HUVECs. Also, the EVs were able to modulate macrophage polarization. Altogether, these results suggest that platelet-derived EVs are promising candidates to be used as biochemical signals or therapeutic tools in tissue engineering and regenerative medicine approaches.

Therapeutic Effects of Platelet-Derived Extracellular Vesicles in a Bioengineered Tendon Disease Model.

Tendon injuries represent over 30-50% of musculoskeletal disorders worldwide, yet the available therapies do not provide complete tendon repair/regeneration and full functionality restoring. Extracellular vesicles (EVs), membrane-enclosed nanoparticles, have emerged as the next breakthrough in tissue engineering and regenerative medicine to promote endogenous tissue regeneration. Here, we developed a 3D human in vitro model mimicking the signature of pathological tendon and used it to evaluate the influence that different platelet-derived EVs might have in tendon tissue repair mechanisms. For this, different EV populations isolated from platelets, small EVs (sEVs) and medium EVs (mEVs), were added to the culture media of human tendon-derived cells (hTDCs) cultured on isotropic nanofibrous scaffolds. The platelet-derived EVs increased the expression of tenogenic markers, promoted a healthy extracellular matrix (ECM) remodeling, and the synthesis of anti-inflammatory mediators. These findings suggest that platelet EVs provided relevant biochemical cues that potentiated a recovery of hTDCs phenotype from a diseased to a healthy state. Thus, this study opens new perspectives for the translation of platelet-derived EVs as therapeutics.

Frontoplastia endoscópica subperiostal com fixação temporal por fios de sutura / Subperiosteal endoscopic frontoplasty with temporal suture threads fixation

Introdução: O envelhecimento do terço superior da face se caracteriza por ptose da sobrancelha, rugas glabelares e rugas transversais frontais. O tratamento pode ser realizado através da frontoplastia coronal, da frontoplastia temporal com incisões limitadas e da frontoplastia endoscópica. O objetivo deste estudo é descrever a técnica de frontoplastia endoscópica subperiostal com miotomia dos músculos da região glabelar e fixação do retalho na fáscia temporal profunda, avaliando sua aplicabilidade e eficácia. Métodos: Foram avaliadas 24 pacientes, do sexo feminino, submetidas a frontoplastia endoscópica associada a blefaroplastia superior, com idade variando entre 37 e 72 anos, em um período de 10 anos. Medidas entre a distância da linha interpupilar e a porção superior da sobrancelha foram realizadas através de análise fotográfica com uso do sistema digital de imagem Mirror 6,0, na região medial, central e lateral de cada lado, no pré-operatório, e no pós-operatório de 6 meses. Resultados: A média de idade foi de 52 anos. Houve significância estatística (p<0,05) em todas as áreas da sobrancelha avaliadas, a média de foi de 0,3 cm mais alta. As complicações foram: 1 caso de extrusão fio, 1 caso de assimetria, 2 casos de correção insuficiente da sobrancelha e 2 casos de recidiva de rugas glabelares. Conclusão: A frontoplastia endoscópica subperiostal com miotomia dos músculos da região glabelar e flxação do retalho na fáscia temporal profunda com pontos demonstrou ser efetiva no tratamento do envelhecimento do terço superior da face, com resultados estatisticamente comprovados, baixa morbidade e bons resultados estéticos.

Introduction: The aging of the upper third of the face is characterized by ptosis of the eyebrow, glabellar wrinkles and frontal transverse wrinkles. Treatment can be performed through coronal frontoplasty, temporal frontoplasty with limited incisions and endoscopic frontoplasty. The aim of this study is to describe the technique of subperiosteal endoscopic frontoplasty with myotomy of the muscles of the glabellar region and fixation of the flap in the deep temporal fascia, evaluating its applicability and effectiveness. Methods: Twenty-four female patients who underwent endoscopic frontoplasty associated with upper blepharoplasty, aged between 37 and 72 years old, over a 10-year period were evaluated. Measurements between the distance from the inter-pupillary line and the upper portion of the eyebrow were performed through photographic analysis using the Mirror 6.0 digital image system, in the medial, central and lateral regions on each side, in the preoperative period, and in the 6 month postoperative period. Results: The average age was 52 years. There was statistical significance (p<0.05) in all evaluated eyebrow areas, the mean was 0.3 cm higher. Complications were: 1 case of wire extrusion, 1 case of asymmetry, 2 cases of insufficient correction of the eyebrow and 2 cases of recurrence of glabellar wrinkles. Conclusion: Subperiosteal endoscopic frontoplasty with myotomy of the muscles of the glabellar region and fixation of the flap in the deep temporal fascia with stitches, proved to be effective in the treatment of aging of the upper third of the face, with statistically proven results, low morbidity and good aesthetic results.

On the Relationship Between Authentic Leadership, Flourishing, and Performance in Healthcare Teams: A Job Demands-Resources Perspective.

This study integrates the job demands-resources model and authentic leadership theory to test the general hypothesis that authentic leadership is a job resource that enables flourishing and performance in healthcare teams. Furthermore, this article tests the hypothesis that the daily bed occupancy is a job demand that weakens this relationship. Participants were 106 nurses that were distributed across 33 teams from two hospitals. The results suggest that the authentic leadership of team leaders is positively related with subjective and objective team performance, but only when daily bed occupancy is low. Authentic leadership had no relationship with team flourishing, regardless of the daily bed occupancy. Our findings suggest that the extent to which authentic leadership is adequate to promote the performance of teams working in a hospital setting is sensitive to contextual boundary conditions. Leading authentically might only be effective under specific circumstances.

Natural-Based Hydrogels for Tissue Engineering Applications.

In the field of tissue engineering and regenerative medicine, hydrogels are used as biomaterials to support cell attachment and promote tissue regeneration due to their unique biomimetic characteristics. The use of natural-origin materials significantly influenced the origin and progress of the field due to their ability to mimic the native tissues' extracellular matrix and biocompatibility. However, the majority of these natural materials failed to provide satisfactory cues to guide cell differentiation toward the formation of new tissues. In addition, the integration of technological advances, such as 3D printing, microfluidics and nanotechnology, in tissue engineering has obsoleted the first generation of natural-origin hydrogels. During the last decade, a new generation of hydrogels has emerged to meet the specific tissue necessities, to be used with state-of-the-art techniques and to capitalize the intrinsic characteristics of natural-based materials. In this review, we briefly examine important hydrogel crosslinking mechanisms. Then, the latest developments in engineering natural-based hydrogels are investigated and major applications in the field of tissue engineering and regenerative medicine are highlighted. Finally, the current limitations, future challenges and opportunities in this field are discussed to encourage realistic developments for the clinical translation of tissue engineering strategies.

Structure elucidation of the redox cofactor mycofactocin reveals oligo-glycosylation by MftF.

Mycofactocin (MFT) is a redox cofactor belonging to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and is involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. A preliminary biosynthetic model had been established by bioinformatics and in vitro studies, while the structure of natural MFT and key biosynthetic steps remained elusive. Here, we report the discovery of glycosylated MFT by 13C-labeling metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Extensive structure elucidation including NMR revealed that MFT is decorated with up to nine ß-1,4-linked glucose residues including 2-O-methylglucose. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the redox cofactor function of glycosylated MFTs by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results will guide future studies into the biochemical functions and physiological roles of MFT in bacteria.

Escherichia coli and Cronobacter sakazakii in 'Tommy Atkins' minimally processed mangos: Survival, growth and effect of UV-C and electrolyzed water.

These studies were aimed at assessing the growing capacity of Escherichia coli and Cronobacter sakazakii and the effectiveness of Ultraviolet-C (UV-C) radiation, acidic electrolyzed (AEW) and neutral electrolyzed (NEW) waters in the inhibition of these bacteria on minimally processed 'Tommy Atkins' mangoes (MPM). The fruits were contaminated by dip inoculation and kept 10 days at 4, 8, 12 and 20 °C while enumerating bacteria. Contaminated mangoes were disinfected using UV-C (2.5, 5, 7.5 and 10 kJ/m2), AEW, NEW and sodium hypochlorite (SH) and the microorganisms were monitored. None of the enterobacteria grew at 4, 8 and 12 °C regardless of having persisted during the 10-day period. At 20 °C, E. coli and C. sakazakii grew, after adaption phases of 48 h and 24 h, to values of 8.7 and 8.5 log cfu/g at day eight, respectively. E. coli showed the highest reduction counts on the MPM washed with NEW and SH (2.2 log cfu/g). UV-C was more effective in reducing C. sakazakii (2.4-2.6 log cfu/g), when compared to AEW, NEW and SH (1.2-1.8 log cfu/g). The efficacy of decontamination technologies depends on microorganisms, highlighting the importance of preventing contamination at the primary production and of combining different methods to increase the safety of fresh-cut fruits.

Planctomycetes as Novel Source of Bioactive Molecules.

Marine environments are a fruitful source of bioactive compounds some of which are the newest leading drugs in medicinal therapeutics. Of particular importance are organisms like sponges and macroalgae and their associated microbiome. Planctomycetes, abundant in macroalgae biofilms, are promising producers of bioactive compounds since they share characteristics, like large genomes and complex life cycles, with the most bioactive bacteria, the Actinobacteria. Furthermore, genome mining revealed the presence of secondary metabolite pathway genes or clusters in 13 analyzed Planctomycetes genomes. In order to assess the antimicrobial production of a large and diverse collection of Planctomycetes isolated from macroalgae from the Portuguese coast, molecular, and bioactivity assays were performed in 40 bacteria from several taxa. Two genes commonly associated with the production of bioactive compounds, nonribosomal peptide synthetases (NRPS), and polyketide synthases (PKS) genes were screened. Molecular analysis revealed that 95% of the planctomycetes potentially have one or both secondary bioactive genes; 85% amplified with PKS-I primers and 55% with NRPS primers. Some of the amplified genes were confirmed to be involved in secondary metabolite pathways. Using bioinformatic tools their biosynthetic pathways were predicted. The secondary metabolite genomic potential of strains LF1, UC8, and FC18 was assessed using in silico analysis of their genomes. Aqueous and organic extracts of the Planctomycetes were evaluated for their antimicrobial activity against an environmental Escherichia coli, E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and a clinical isolate of Candida albicans. The screening assays showed a high number of planctomycetes with bioactive extracts revealing antifungal (43%) and antibacterial (54%) activity against C. albicans and B. subtilis, respectively. Bioactivity was observed in strains from Rhodopirellula lusitana, R. rubra, R. baltica, Roseimaritima ulvae, and Planctomyces brasiliensis. This study confirms the bioactive capacity of Planctomycetes to produce antimicrobial compounds and encourages further studies envisaging molecule isolation and characterization for the possible discovery of new drugs.

Survival and growth of Cronobacter sakazakii on fresh-cut fruit and the effect of UV-C illumination and electrolyzed water in the reduction of its population.

Cronobacter sakazakii, found in foods such as powdered infant formula and plant origin ready-to-eat food, is an opportunistic pathogen to infants, neonates and vulnerable adults. The objective of this study was to monitor the growth of C. sakazakii in fresh-cut 'Royal gala' apple, 'Rocha' pear, and 'Piel de sapo' melon, and the effect of UV-C illumination, acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW) in the reduction of its population. Fresh-cut fruits were inoculated and incubated at different temperatures during 10days while monitoring C. sakazakii. The inhibitory activity of different doses of UV-C (0-10kJ.m(2)), electrolyzed water and sodium hypochlorite (SH) (100ppm chlorine) was evaluated on the fruits inoculated with C. sakazakii. The bacterium showed a significant growth in the fruits at 12 and 20°C, but did not grow at 4°C, despite having survived for 10days. At 8°C, adaptation phases of 0.6-3.9days were estimated in the fruits before exponential growth. The UV-C 7.5 and 10kJ/m(2) produced greater C. sakazakii population decreases (2-2.4logcfu/g) than AEW (1.3-1.8logcfu/g), NEW (1-1.2logcfu/g) and SH (0.8-1.4logcfu/g). The UV-C decontamination system and refrigeration at 4°C, may contribute to the product's safety and quality. The results help better understand the behavior of C. sakazakii on fresh-cut fruit alerting producers of the necessity to respect the high hygienic practices, adequate refrigerating temperature maintenance and caution with the tendency to prolong the validity of this kind of ready-to-eat food.

Low epinephrine levels and selective deficiency of ß2-adrenoceptor vasodilation at birth.

AIMS: Epinephrine is unique among biogenic catecholamines as a potent agonist of ß2-adrenoceptors. The ß2-adrenoceptor mediated effects during development might be linked to the increase of epinephrine synthesis. Our purpose was to characterize ß-adrenoceptor-mediated relaxation in the aorta of newborn and young rabbits (3 to 4months old), and to relate those responses with the epinephrine content of the adrenal gland. MAIN METHODS: The epinephrine levels and the tyrosine hydroxylase activity were determined in adrenal glands of newborn and young rabbits. Also, concentration-response curves to phenylephrine (selective α1-adrenoceptor agonist), dobutamine (selective ß1-adrenoceptor agonist), terbutaline (selective ß2-adrenoceptor agonist), and CL 316243 (selective ß3-adrenoceptor agonist) were determined in isolated aortic rings obtained from both groups. KEY FINDINGS: The adrenal gland content and the plasma concentrations of epinephrine were lower in newborn than in young rabbits. In contrast, the tyrosine hydroxylase activity was higher in newborn than in young rabbits. On the other hand, the maximal response to phenylephrine was lower in newborn than in young rabbits. Terbutaline at concentrations selective for ß2-adrenoceptors had no relaxing effects in neonates, in contrast to young rabbits. The potency and the maximal response of neither dobutamine nor CL 316243 were significantly different between the two groups. SIGNIFICANCE: In rabbits, as well as in humans, ß2-adrenoceptor-mediated responses and epinephrine synthesis are both immature at birth. On the other hand, the ß1 and ß3-adrenoceptor-mediated responses are fully developed. We conclude that epinephrine may influence the development of the ß2-adrenoceptor-mediated responses at birth and the rabbit is an excellent model to study these issues.

Evaluation of microbial quality and yeast diversity in fresh-cut apple.

The present work's aim was to study the microbial quality of minimally processed apples commercialized in Portugal. Sixty eight samples of fresh-cut apple were analyzed before their best-before date in 2011 and 2012 for aerobic mesophilic and psychrotrophic microorganisms, total coliforms, lactic-acid bacteria (LAB), coagulase-positive staphylococci and fungi. The parameters of food safety studied were Cronobacter sakazakii, Salmonella spp. and Listeria sp. Samples were analyzed according to standard methodologies and using Chromocult Agar for coliforms and Escherichia coli. The yeasts were identified by restriction analysis of the ITS-5.8S rDNA-region and 26S rDNA partial sequencing. The mesophilic and psychrotrophic microorganisms ranged from 3.3 to 8.9 and from 4.9 to 8.4 log CFU/g, respectively. Coliforms were detected in all the samples and staphylococci in 5.8% of them. LAB numbers varied from 2.8 to 8.7 and fungi (yeast and molds) from 3.6 to 7.1 log CFU/g. The most common yeasts were Candida sake and Pichia fermentans followed by Hanseniaspora spp., Candida spp., Meyerozyma guilliermondii, Metschnikowia pulcherrima, Cryptococcus spp. and the psychrotrophic Cystofilobasidium infirmominiatum. Foodborne bacteria and opportunistic pathogenic yeasts were not detected in the apples studied. The results obtained respected the European Commission regulation regarding criteria of food hygiene and safety.

The antimicrobial activity of heterotrophic bacteria isolated from the marine sponge Erylus deficiens (Astrophorida, Geodiidae).

Interest in the study of marine sponges and their associated microbiome has increased both for ecological reasons and for their great biotechnological potential. In this work, heterotrophic bacteria associated with three specimens of the marine sponge Erylus deficiens, were isolated in pure culture, phylogenetically identified and screened for antimicrobial activity. The isolation of bacteria after an enrichment treatment in heterotrophic medium revealed diversity in bacterial composition with only Pseudoalteromonas being shared by two specimens. Of the 83 selected isolates, 58% belong to Proteobacteria, 23% to Actinobacteria and 19% to Firmicutes. Diffusion agar assays for bioactivity screening against four bacterial strains and one yeast, revealed that a high number of the isolated bacteria (68.7%) were active, particularly against Candida albicans and Vibrio anguillarum. Pseudoalteromonas, Microbacterium, and Proteus were the most bioactive genera. After this preliminary screening, the bioactive strains were further evaluated in liquid assays against C. albicans, Bacillus subtilis and Escherichia coli. Filtered culture medium and acetone extracts from three and 5 days-old cultures were assayed. High antifungal activity against C. albicans in both aqueous and acetone extracts as well as absence of activity against B. subtilis were confirmed. Higher levels of activity were obtained with the aqueous extracts when compared to the acetone extracts and differences were also observed between the 3 and 5 day-old extracts. Furthermore, a low number of active strains was observed against E. coli. Potential presence of type-I polyketide synthases (PKS-I) and non-ribosomal peptide synthetases (NRPSs) genes were detected in 17 and 30 isolates, respectively. The high levels of bioactivity and the likely presence of associated genes suggest that Erylus deficiens bacteria are potential sources of novel marine bioactive compounds.

Attenuated aortic vasodilation and sympathetic prejunctional facilitation in epinephrine-deficient mice: selective impairment of ß2-adrenoceptor responses.

It has been suggested that there is a link between epinephrine synthesis and the development of ß2-adrenoceptor-mediated effects, but it remains to be determined whether this development is triggered by epinephrine. The aim of this study was to characterize ß-adrenoceptor-mediated relaxation and facilitation of norepinephrine release in the aorta of phenylethanolamine-N-methyltransferase-knockout (Pnmt-KO) mice. Catecholamines were quantified by reverse-phase high-performance liquid chromatography-electrochemical detection. Aortic rings were mounted in a myograph to determine concentration-response curves to selective ß1- or ß2-adrenoceptor agonists in the absence or presence of selective ß1- or ß2-adrenoceptor antagonists. Aortic rings were also preincubated with [(3)H]norepinephrine to measure tritium overflow elicited by electrical stimulation in the presence of increasing concentrations of nonselective ß- or selective ß2-adrenoceptor agonists. ß2-Adrenoceptor protein density was evaluated by Western blotting and ß2-adrenoceptor localization by immunohistochemistry. Epinephrine is absent in Pnmt-KO mice. The potency and the maximal effect of the ß2-adrenoceptor agonist terbutaline were lower in Pnmt-KO than in wild-type (WT) mice. The selective ß2-adrenoceptor antagonist ICI 118,551 [(±)-erythro-(S*,S*)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride] antagonized the relaxation caused by terbutaline in WT but not in Pnmt-KO mice. Isoproterenol and terbutaline induced concentration-dependent increases in tritium overflow in WT mice only. ß2-Adrenoceptor protein density was decreased in membrane aorta homogenates of Pnmt-KO mice, and this finding was supported by immunofluorescence confocal microscopy. In conclusion, epinephrine is crucial for ß2-adrenoceptor-mediated vasodilation and facilitation of norepinephrine release. In the absence of epinephrine, ß2-adrenoceptor protein density was decreased in aorta cell membranes, thus potentially hindering its functional activity.