Zostera noltei response to transplantation into historically Hg-contaminated sediments (A mesocosm experiment): Growth, bioaccumulation and photosynthetic performance.

The present study aimed to evaluate the effectiveness of seagrass recolonization as a nature-based solution for the recovery of a coastal area historically contaminated by mercury (Laranjo Bay, Ria de Aveiro, Portugal). A mesocosm approach was employed to assess the resistance of Zostera noltei to transplantation into contaminated sediments collected in-situ (0.5-20 mg kg-1 Hg). At each sampling time (15, 30, 60, 120 and 210 days), the resistance of transplanted Z. noltei was evaluated through growth parameters (biomass and coverage area), photosynthetic performance and elemental composition. Although some significant differences (p ≤ 0.05) were observed between treatments, essentially associated with the elemental composition of plant tissues, the most relevant variations were associated with seasonality. Overall, plants were found to not be affected by sediment contamination, under the tested concentrations, suggesting that recolonization with Z. noltei can be an effective restoration strategy for historically contaminated coastal areas.

Biological properties and potential of compounds extracted from red seaweeds.

Macroalgae have been recently used for different applications in the food, cosmetic and pharmaceutical industry since they do not compete for land and freshwater against other resources. Moreover, they have been highlighted as a potential source of bioactive compounds. Red algae (Rhodophyta) are the largest group of seaweeds, including around 6000 different species, thus it can be hypothesized that they are a potential source of bioactive compounds. Sulfated polysaccharides, mainly agar and carrageenans, are the most relevant and exploited compounds of red algae. Other potential molecules are essential fatty acids, phycobiliproteins, vitamins, minerals, and other secondary metabolites. All these compounds have been demonstrated to exert several biological activities, among which antioxidant, anti-inflammatory, antitumor, and antimicrobial properties can be highlighted. Nevertheless, these properties need to be further tested on in vivo experiments and go in-depth in the study of the mechanism of action of the specific molecules and the understanding of the structure-activity relation. At last, the extraction technologies are essential for the correct isolation of the molecules, in a cost-effective way, to facilitate the scale-up of the processes and their further application by the industry. This manuscript is aimed at describing the fundamental composition of red algae and their most studied biological properties to pave the way to the utilization of this underused resource.

Bioinspired gelatin/bioceramic composites loaded with bone morphogenetic protein-2 (BMP-2) promote osteoporotic bone repair.

There are currently several commercialized products approved by the Food and Drug Administration and the European Medicines Agency based on the use of recombinant human BMP-2 for the treatment of non-unions long fractures and spinal fusion. However, the adverse effects recorded with the use of BMPs suggest the need for drug delivery carriers that allow reducing the required doses and improve their cost-effectiveness. Herein, we have developed a new osteoconductive scaffold that reduces the required doses of BMP-2 for promoting bone regeneration in an osteoporotic defect model. The composite is, in brief, a gelatin-based 3D scaffold reinforced with either calcium sulfate or hydroxyapatite as an inorganic osteoconductive biomaterial. To this end, the organic/inorganic composite systems showed high hydration capacity and good in vitro degradability. The incorporation of 7.5% (m/v) ceramic compounds resulted in scaffolds with stiffer Young modulus (179 and 75 kPa for CaSO4_7 and HA_7, respectively) than bare gelatin hydrogels (48 kPa). Studies with human bone-marrow derived mesenchymal stem cells (hBM-MSCs) revealed that the 3D scaffolds promote cell adhesion and proliferation along with osteogenic differentiation capabilities. Specifically, downregulation of stemness (Nanog, Oct4) genes and upregulation of osteogenic markers (ALP, Col1a1, Fmod) by two fold were observed over 10 days under basal culture conditions. Promisingly, the sustained in vitro release of BMP-2 observed from the porous reinforced scaffolds allowed us to address the critical-sized osteoporotic mice calvarial defects with a relatively low growth factor doses (600 ng BMP-2/scaffold) compared to conventional doses at 2-15 micrograms. Overall, this study demonstrates the promising potential of osteoconductive gelatin/calcium bioceramics composites as osteogenic growth factors delivery carriers for bone-regeneration via ultra-low growth factor doses.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells.

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Expression of spindle assembly checkpoint proteins BubR1 and Mad2 expression as potential biomarkers of malignant transformation of oral leukoplakia: an observational cohort study.

BACKGROUND: The Spindle Assembly Checkpoint (SAC) is a surveillance mechanism essential to ensure the accuracy of chromosome segregation during mitosis. Our aim was to evaluate the expression of SAC proteins in oral carcinogenesis, and to assess their potential in predicting malignant transformation of oral leukoplakia. MATERIAL AND METHODS: We analysed the immunoexpression of BubR1, Mad2, Bub3, and Spindly proteins in 64 oral biopsies from 52 oral leukoplakias and 12 normal tissues. Univariate and multivariate analysis were performed to evaluate predictive factors for malignant transformation (MT). RESULTS: We observed that BubR1 and Mad2 were more highly expressed in high dysplasia grade lesions than in low grade or normal tissues (P<0.05). High expression of Spindly was significantly correlated with a high Ki-67 score (P=0.004). Six (11.5%) oral leukoplakias underwent malignant transformation. In univariate analysis, the binary dysplasia grade (high grade) (P<0.001) was associated with a higher risk of malignant transformation as well as high BubR1 (P<0.001) and high Mad2 (P=0.013) expression. In multivariate analysis, high expression of BubR1 and Mad2 when combined showed an increased risk for malignant transformation (P=0.013; HR of 4.6, 95% CI of 1.4-15.1). CONCLUSIONS: Our findings reveal that BubR1 and Mad2 were associated with an increased risk for malignant transformation independently of histological grade and could be potential and useful predictive risk markers of malignant transformation in oral leukoplakias.

Risk factors for developmental defects of enamel in children from southeastern Brazil.

INTRODUCTION: Developmental Defects of Enamel (DDEs) comprise qualitative and/or quantitative changes to the enamel during amelogenesis. The aetiology of DDE remains inconclusive. AIM: To determine the association of pre, peri, and postnatal factors with the presence of DDE. DESIGN: Cross-sectional study with 353 children (8 to 11 years-old) in a Brazilian town. METHODS: One calibrated dentist assessed DDE using the Developmental Defects of Enamel Index and a questionnaire collected medical and sociodemographic data. MAIN OUTCOMES: Children with at least one type of DDE were categorized into the DDE group. Subtypes of DDE were also recorded. RESULTS: 63.1% of children had at least one type of DDE. Diffuse opacity was present in 36.7%, demarcated opacity in 14.8%, and hypoplasia in 5.83% of the children. In multivariate analysis, demarcated opacities and hypoplasia were associated with birth weight ⟨ 2500g (OR = 4.82; 95% CI 1.23-1.95). CONCLUSION: Low birth weight predicted DDE.

Double plating technique for fixing tibial plateau leveling osteotomy and modified cranial closing wedge ostectomy of the tibia in a dog with cranial cruciate ligament disease and excessive plateau angle: case report / [Técnica de placa dupla para fixação concomitante da osteotomia de nivelamento do platô tibial e ostectomia modificada em cunha de fechamento cranial da tíbia em um cão com doença do ligamento cruzado cranial e ângulo de platô tibial excessivo: relato de caso]

Tibial plateau leveling osteotomy (TPLO) associated to cranial wedge closing ostectomy (CCWO) has been one of the best options to manage cranial cruciate ligament (CCL) disease and excessive tibial plateau angle (TPA) in large dogs, however, the complication rate is potentially high. It is believed that a more robust fixation is necessary to stabilize them and decrease the risk of implant failure. A 6-year-old male American Pit Bull, weighing 36kg, with 90-day history of right hind limb lameness, was diagnosed with CCL disease. Due to the excessive tibial plateau angle (42°), TPLO was associated with a modified CCWO using a double plating technique. A final TPA of 12° was accomplished, and a restricted level of exercises and physiotherapy were recommended. The patient was followed monthly until the fifth month postoperatively, when radiographic bone consolidation and no lameness were observed. By the date of this submission, 3 years after the procedure, the owner has reported no complications. The double plating technique for fixing TPLO and modified CCWO proved to be effective for the treatment of CrCL deficiency in a large dog with an excessive TPA.(AU)

A osteotomia de nivelamento do platô tibial (TPLO) associada à ostectomia modificada em cunha de fechamento cranial da tíbia (CCWO) tem sido uma das melhores opções para tratamento de cães grandes com doença do ligamento cruzado cranial (DLCCr) e ângulo excessivo do platô tibial, mas o índice de complicações é alto. Acredita-se haver necessidade de fixação mais robusta para reduzir as chances de falha nos implantes. Um cão macho, seis anos, da raça American Pit Bull, 36kg, com histórico de claudicação em membro pélvico direito há 90 dias, foi diagnosticado com DLCCr. Devido ao ângulo excessivo do platô tibial (42°), a osteotomia de nivelamento do platô tibial foi associada à ostectomia modificada em cunha de fechamento cranial da tíbia (CCWO) por meio da técnica de placa dupla. No pós-operatório imediato, identificou-se TPA de 12°; exercícios controlados e fisioterapia foram recomendados e a evolução do quadro foi analisada mensalmente até o quinto mês pós-cirurgia. Com 150 dias de evolução, não houve alterações de locomoção e havia ocorrido completa consolidação radiográfica das osteotomias. Até o momento da submissão deste artigo, três anos pós-procedimento, o tutor relata ausência de complicações, via contato telefônico. Portanto, a técnica modificada mostrou-se eficaz no tratamento da DLCCr.(AU)

Non-lactose-fermenting uropathogenic Escherichia coli from dogs: virulence profile characterization.

Non-lactose-fermenting Escherichia coli (NLFEC) has a few descriptive studies restricted to human infections. In the present study, isolates of NLFEC obtained from urine samples of dogs with hyperadrenocorticism were characterized regarding their virulence ability, biofilm formation capacity and antimicrobial susceptibility profile. Escherichia coli lactose-fermenting strains from urinary infection in dogs with the same conditions were analysed to provide comparisons. The non-lactose-fermenting E. coli strains were classified as belonging to clade I E. coli, whereas the lactose-fermenting strains were classified in phylogroup B2. All strains presented virulence markers to adhesion, iron acquisition, toxins, colicin and cytotoxin production, and biofilm regulation. Components of the extracellular matrix in addition to the in vitro biofilm formation ability were observed in the strains. Multidrug resistance (MDR) profiles were observed by in vitro susceptibility tests to all NLFEC strains. In summary, non-lactose-fermenting uropathogenic E. coli from dogs behaves similar to lactose-fermenting E. coli, exhibiting MDR profile, and pathogenic potential of promote animal infections.

Main bioactive phenolic compounds in marine algae and their mechanisms of action supporting potential health benefits.

Given the growing tendency of consumers to choose products with natural ingredients, food industries have directed scientific research in this direction. In this regard, algae are an attractive option for the research, since they can synthesize a group of secondary metabolites, called phenolic compounds, associated with really promising properties and bioactivities. The objective of this work was to classify the major phenolic compounds, compare the effectiveness of the different extractive techniques used for their extraction, from traditional systems (like heat assisted extraction) to the most advance ones (such as ultrasound, microwave or supercritical fluid extraction); the available methods for identification and quantification; the stability of the enriched extract in phenolic compounds and the main bioactivities described for these secondary metabolites, to offer an overview of the situation to consider if it is possible and/or convenient an orientation of phenolic compounds from algae towards an industrial application.

Evaluation of the biological properties of two experimental calcium silicate sealers: an in vivo study in rats.

AIM: To evaluate the biological properties of experimental sealers based on tricalcium silicate and dicalcium silicate, manipulated with polyethylene glycol (CE-1) and with the addition of calcium hypochlorite (CE-2) compared to AH Plus (AHP) and TotalFill BC Sealer (TBC). METHODOLOGY: The tissue reaction caused by the materials in the subcutaneous tissue of rats was evaluated after implantation of polyethylene tubes filled with the materials at 7, 15, 30 and 60 days. Sections were stained with haematoxylin and eosin (HE) for morphological analysis and to evaluated number of inflammatory cells/mm2 (ICs). Sections were used for immunohistochemical detection of interleukin-6 (IL-6) and osteocalcin (OC). The von Kossa method was used to identify calcium precipitation in the capsules. The data were submitted to anova and Tukey's tests, with 5% significance level. RESULTS: At 7 days, CE-1, CE-2 and AHP had higher numbers of ICs. AHP presented higher immunolabelling for IL-6. After 15 days, regarding IL-6, there was no difference between CE-2 and the control group. At 30 days, AHP exhibited the highest number of IC (P < 0.05) and CE-2 and the control group presented the lowest ICs and IL-6-positive cells. After 60 days, all materials exhibited decreases in ICs. CE-2, TBC and the control had the lowest values (P < 0.05). No significant difference was detected between CE-1 and TBC, and between CE-2 and control considering the immunoexpression of IL-6. In this period, AHP had the greatest number of IC and IL-6 (P < 0.05). In all periods, CE-1, CE-2 and TBC sealers had von Kossa-positive structures and OC-immunolabelled cells. CE-2 had higher number of OC-positive cells than the CE-1 and TBC sealers (P < 0.05), in all periods. OC immunolabelling was not observed in the capsules of AH Plus and the control groups. CONCLUSIONS: The experimental sealer and its association with calcium hypochlorite, in addition to TotalFill BC Sealer, were biocompatible and had bioactive potential.

An observational retrospective study of odontogenic cyst´s and tumours over an 18-year period in a Portuguese population according to the new WHO Head and Neck Tumour classification.

BACKGROUND: Odontogenic cysts and tumours of the jaws represent one of the most prevalent groups of oral-maxillofacial lesions. We aimed to evaluate the clinical and pathological characteristics of a cohort of odontogenic cysts (OC) and odontogenic tumours (OT) of the jaws in a Portuguese population. MATERIAL AND METHODS: This observational retrospective study analysed patients diagnosed with either an OC or OT of the jaws at a central hospital of Oporto, Portugal, between 1988 and 2006. Data collected from patients' files included demographic, clinical, radiological and histopathological information. Recurrence was evaluated using univariate and multivariate analysis. RESULTS: The sample consisted of 397 patients, 231 males (58.2%) and 166 females (41.8%), with a mean-age of 36.7±17 years. Twenty-seven patients (6.8%) presented with more than one lesion providing a total of 433 lesions. There were 396 (91.5%) OC, mostly represented by radicular cysts (n=257;59.4%), dentigerous cysts (n=79;18.2%), or odontogenic keratocysts (n=50;11.5%). There were 37 (8.5%) OT, mostly represented by ameloblastomas (n=16;3.7%), and odontomas (n=9;2.1%). The most common initial clinical manifestation was swelling (n=224;51.7%). Recurrence was observed in 30 cases (6.9%), mostly in ameloblastomas (n=6;37.5%) and odontogenic keratocysts (n=12;24%). In the multivariate analysis the diagnosis classification of the lesion was the only independent and significant variable related with the recurrence (P=0.04). CONCLUSIONS: Radicular cysts were the most commonly occurring type of OC and ameloblastomas the most commonly occurring OT. Amelobastomas and odontogenic keratocysts were the lesions with the highest rates of recurrence. This large sample provides useful information about the frequency profile and characteristics of OC and OT over a period of 18 years, allowing valuable comparison with data from other countries.

Melanoma oral amelanótico metastático com acometimento neurológico e gonadal em um cão fêmea - relato de caso / [Metastatic oral amelanotic melanoma with neurologic and gonadal involvement in a female dog - case report]

Relata-se um caso de melanoma oral disseminado em uma cadela de dois anos, com protrusão de bulbo ocular unilateral e quadro convulsivo progressivo. Os exames de imagem revelaram aumento de volume nas regiões submandibular, maxilar e cerebral, padrão nodular pulmonar e aumento das dimensões ovarianas. A citologia da massa submandibular indicou proliferação epitelial maligna, enquanto a biópsia excisional foi sugestiva de melanoma amelanótico. Na necropsia, havia uma massa gengival localmente infiltrativa e nodulações brancas nos linfonodos, nos rins, no pulmão, no cérebro e nos ovários, indicativas de metástase. O diagnóstico histopatológico consistiu de neoplasia maligna metastática indiferenciada, indicativo de melanoma amelanótico. Células caracterizadas por núcleo com cromatina espessa, múltiplos nucléolos bem evidentes, mitoses atípicas e multinucleações consistiram nos principais critérios de malignidade. No espaço peritrabecular ósseo facial, havia rara diferenciação pigmentar melanocítica, confirmada histoquimicamente pela técnica de Fontana-Massom e Giemsa. Algumas células foram positivas pela imuno-histoquímica para PNL-2 e Melan-A, e o diagnóstico de melanoma amelanótico disseminado foi firmado. A indiferenciação neoplásica marcante, com disseminação metastática multissistêmica e acometimento mútuo de sítios anatômicos pouco comuns, conjuntamente com a ampla variação dos padrões celulares, foi responsável pelo desafio diagnóstico do presente caso, ressaltando o papel decisivo da imuno-histoquímica para confirmação diagnóstica. A importância clínica deste trabalho consiste ainda em alertar a comunidade clínica e científica acerca da dificuldade diagnóstica, devendo-se considerar o melanoma amelanótico como diferencial mesmo em casos de lesões orais menos perceptíveis e/ou desprovidas de pigmentação.(AU)

A case of disseminated oral melanoma in a two year old female dog with unilateral protuberance of the eye bulb and progressive seizure is described. Imaging exams revealed increase of the submandibular, maxillary and cerebral regions, nodular pattern in lungs and increased ovarian dimensions. The cytology of the submandibular mass indicated a malignant epithelial proliferation, whereas the excisional biopsy indicated an amelanotic melanoma. At necropsy, a locally infiltrating gingival mass and white nodules in the lymph nodes, kidneys, lung, brain and ovaries were observed, indicative of metastases. Histopathological diagnosis consisted of an undifferentiated malignant metastatic neoplasm. Nucleus with coarse chromatin, prominent nucleoli, bizarre mitotic figures and multinucleated cells were the major malignant features. There was a poor melanocytic pigment differentiation in the peritrabecular space of facial bones, confirmed by Fontana-Masson and Giemsa histochemical techniques. Only a few cells were immunohistochemically positive for PNL-2 and Melan-A and the diagnosis of a disseminated amelanotic melanoma was performed. The diagnostic challenge was based on marked neoplastic undifferentiation, with multisystemic metastasis and mutual involvement of uncommon anatomic sites, associated with a large variability of cellular patterns, highlighting the decisive role of immunohistochemistry for diagnostic confirmation. Therefore, the clinical importance of this study is to warn the clinical and scientific community about the diagnostic challenge, considering the amelanotic melanoma as a differential even in cases of poorly apparent and/or nonpigmented oral lesions.(AU)