Case report: Equine metacarpophalangeal joint partial and full thickness defects treated with allogenic equine synovial membrane mesenchymal stem/stromal cell combined with umbilical cord mesenchymal stem/stromal cell conditioned medium.

Here, we describe a case of a 5-year-old show-jumping stallion presented with severe lameness, swelling, and pain on palpation of the left metacarpophalangeal joint (MCj). Diagnostic imaging revealed full and partial-thickness articular defects over the lateral condyle of the third metacarpus (MC3) and the dorsolateral aspect of the first phalanx (P1). After the lesion's arthroscopic curettage, the patient was subjected to an innovative regenerative treatment consisting of two intra-articular injections of equine synovial membrane mesenchymal stem/stromal cells (eSM-MSCs) combined with umbilical cord mesenchymal stem/stromal cells conditioned medium (UC-MSC CM), 15 days apart. A 12-week rehabilitation program was accomplished, and lameness, pain, and joint effusion were remarkably reduced; however, magnetic resonance imaging (MRI) and computed tomography (CT) scan presented incomplete healing of the MC3's lesion, prompting a second round of treatment. Subsequently, the horse achieved clinical soundness and returned to a higher level of athletic performance, and imaging exams revealed the absence of lesions at P1, fulfillment of the osteochondral lesion, and cartilage-like tissue formation at MC3's lesion site. The positive outcomes suggest the effectiveness of this combination for treating full and partial cartilage defects in horses. Multipotent mesenchymal stem/stromal cells (MSCs) and their bioactive factors compose a novel therapeutic approach for tissue regeneration and organ function restoration with anti-inflammatory and pro-regenerative impact through paracrine mechanisms.

Issue 2 - "Update on adverse respiratory effects of indoor air pollution" Part 1): Indoor air pollution and respiratory diseases: A general update and a Portuguese perspective.

OBJECTIVE: To quantify the impact of different air pollutants on respiratory health based on robust estimates based on international data and to summarise the evidence of associations between indoor exposure to those pollutants and respiratory morbidity in the Portuguese population. RESULTS: Several systematic reviews and meta-analyses (MA) at the world level demonstrate the impact of indoor air quality on respiratory health, with indoor particulate matter and gasses exerting a significant effect on the airways. Volatile organic compounds (VOC) have been related to asthma and lung cancer. However, only meta-analyses on biomass use allowed documentation of long-term respiratory effects. While early publications concerning Portuguese-based populations mainly focused on indoor exposure to environmental tobacco smoke, later studies relocated the attention to relevant exposure environments, such as day care buildings, schools, residences and nursing homes. Looking at the pooled effects from the reviewed studies, high levels of carbon dioxide and particulate matter in Portuguese buildings were significantly associated with asthma and wheezing, with VOC and fungi showing a similar effect in some instances. CONCLUSIONS: Despite the significant reduction of indoor air pollution effects after the 2008 indoor smoking prohibition in public buildings, studies show that several indoor air parameters are still significantly associated with respiratory health in Portugal. The country shares the worldwide necessity of standardisation of methods and contextual data to increase the reach of epidemiological studies on household air pollution, allowing a weighted evaluation of interventions and policies focused on reducing the associated respiratory morbidity.

Issue 1 - "Update on adverse respiratory effects of outdoor air pollution" Part 2): Outdoor air pollution and respiratory diseases: Perspectives from Angola, Brazil, Canada, Iran, Mozambique and Portugal.

OBJECTIVE: To analyse the GARD perspective on the health effects of outdoor air pollution, and to synthesise the Portuguese epidemiological contribution to knowledge on its respiratory impact. RESULTS: Ambient air pollution has deleterious respiratory effects which are more apparent in larger, densely populated and industrialised countries, such as Canada, Iran, Brazil and Portugal, but it also affects people living in low-level exposure areas. While low- and middle-income countries (LMICs), are particularly affected, evidence based on epidemiological studies from LMICs is both limited and heterogeneous. While nationally, Portugal has a relatively low level of air pollution, many major cities face with substantial air pollution problems. Time series and cross-sectional epidemiological studies have suggested increased respiratory hospital admissions, and increased risk of respiratory diseases in people who live in urban areas and are exposed to even a relatively low level of air pollution. CONCLUSIONS: Adverse respiratory effects due to air pollution, even at low levels, have been confirmed by epidemiological studies. However, evidence from LMICs is heterogeneous and relatively limited. Furthermore, longitudinal cohort studies designed to study and quantify the link between exposure to air pollutants and respiratory diseases are needed. Worldwide, an integrated approach must involve multi-level stakeholders including governments (in Portugal, the Portuguese Ministry of Health, which hosts GARD-Portugal), academia, health professionals, scientific societies, patient associations and the community at large. Such an approach not only will garner a robust commitment, establish strong advocacy and clear objectives, and raise greater awareness, it will also support a strategy with adequate measures to be implemented to achieve better air quality and reduce the burden of chronic respiratory diseases (CRDs).

The application of Bonelike® Poro as a synthetic bone substitute for the management of critical-sized bone defects - A comparative approach to the autograft technique - A preliminary study.

The effective treatment of non-unions and critical-sized defects remains a challenge in the orthopedic field. From a tissue engineering perspective, this issue can be addressed through the application bioactive matrixes to support bone regeneration, such as Bonelike®, as opposed to the widespread autologous grafting technique. An improved formulation of Bonelike® Poro, was assessed as a synthetic bone substitute in an ovine model for critical-sized bone defects. Bone regeneration was assessed after 5 months of recovery through macro and microscopic analysis of the healing features of the defect sites. Both the application of natural bone graft or Bonelike® Poro resulted in bridging of the defects margins. Untreated defect remained as fibrous non-unions at the end of the study period. The characteristics of the newly formed bone and its integration with the host tissue were assessed through histomorphometric and histological analysis, which demonstrated Bonelike® Poro to result in improved healing of the defects. The group treated with synthetic biomaterial presented bone bridges of increased thickness and bone features that more closely resembled the native spongeous and cortical bone. The application of Bonelike® Poro enabled the regeneration of critical-sized lesions and performed comparably to the autograph technique, validating its octeoconductive and osteointegrative potential for clinical application as a therapeutic strategy in human and veterinary orthopedics.

Alternative substrates for conidiogenesis of the entomopathogenic fungus Beauveria bassiana (Bals) Vuillemin (Deuteromycotina: Hyphomycetes) / Substratos alternativos para conidiogênese do fungo entomopatogênico Beauveria bassiana (Bals) Vuillemin (Deuteromycotina: Hyphomycetes)

Abstract Beauveria bassiana is a promising fungus for the biological control of insect pests. The growing costs of conidia production have raised the need to ascertain the efficiency of some low cost substrates. The aim of this study was to analyze the potential use of different raw substrates without nutritional supplement for B. bassiana conidiogenesis. Growth and sporulation were evaluated using 30 g of substrate and 0.3 μL of a conidia suspension (1 x 106 conidia/mL). After 10 days of incubation (70 ± 10% humidity and temperature (T) = 29 ± 1 °C), rice (2.00 x 106 conidia/g substrate), algaroba (2.36 x 106 conidia/g), malt A (1.22 x 106 conidia/g) and malt B (1.75 x 106 conidia/g) showed the highest levels of conidia production. The resulting conidia showed insecticidal activity higher than 80% on coconut termites. These new raw substrates may represent viable alternatives for the production of entomopathogenic fungi for use in the biological control of various insect pests.

Resumo Beauveria bassiana é um fungo promissor no controle biológico de insetos-praga. As crescentes despesas na produção de conídios levantam a necessidade de averiguar a eficiência de alguns substratos de baixo custo. O objetivo deste trabalho foi analisar o potencial de utilização de diferentes substratos brutos para a conidiogênese de B. bassiana. O crescimento e esporulação foram realizados utilizando 30 g do substrato e 0,3 µL da suspensão de conídios (1 x 106 conídios/mL). Após 10 dias de incubação (umidade 70 ± 10% e temperatura T = 29 ± 1° C), o arroz (2,00 x 106 conídios/g de substrato), algaroba (2,36 x 106 conídios/g), malte A (1,22 x 106 conídios/g) e B (1,75 x 106 conídios/g), apresentaram maior produção de conídios. Os conídios produzidos mostraram atividade inseticida sobre o cupim do coqueiro acima de 80% de mortalidade. Estes novos substratos brutos podem representar uma alternativa viável para produção de fungos entomopatogênicos para uso no controle biológico de vários insetos praga.

Alternative substrates for conidiogenesis of the entomopathogenic fungus Beauveria bassiana (Bals) Vuillemin (Deuteromycotina: Hyphomycetes).

Beauveria bassiana is a promising fungus for the biological control of insect pests. The growing costs of conidia production have raised the need to ascertain the efficiency of some low cost substrates. The aim of this study was to analyze the potential use of different raw substrates without nutritional supplement for B. bassiana conidiogenesis. Growth and sporulation were evaluated using 30 g of substrate and 0.3 µL of a conidia suspension (1 x 106 conidia/mL). After 10 days of incubation (70 ± 10% humidity and temperature (T) = 29 ± 1 °C), rice (2.00 x 106 conidia/g substrate), algaroba (2.36 x 106 conidia/g), malt A (1.22 x 106 conidia/g) and malt B (1.75 x 106 conidia/g) showed the highest levels of conidia production. The resulting conidia showed insecticidal activity higher than 80% on coconut termites. These new raw substrates may represent viable alternatives for the production of entomopathogenic fungi for use in the biological control of various insect pests.

Detection of rare and novel EGFR mutations in NSCLC patients: Implications for treatment-decision.

OBJECTIVES: Mutations in the gene that encodes epidermal growth factor receptor (EGFR) are biomarkers that predict how non-small cell lung cancer (NSCLC) patients respond to EGFR-targeted therapies collectively known as tyrosine kinase inhibitors (TKIs). Thus, EGFR genotyping provides crucial information for treatment decision. Both Sanger sequencing and real-time PCR methodologies are used for EGFR genotyping. However, methods based on real-time PCR have limitations, as they may not detect rare or novel mutations. The aim of this study was to determine the prevalence of rare mutations in the tyrosine kinase domain (exons 18-21) of the EGFR gene not targeted by the most frequently used real-time PCR approaches, i.e., the cobas® EGFR Mutation Test, and the Idylla™ EGFR Mutation Assay. METHODS: A total of 1228 NSCLC patients were screened for mutations in exons 18-21 of the EGFR gene using Sanger sequencing. RESULTS: We observed that 252 patients (∼20%) had at least one mutation in the EGFR gene, and 38 (∼3%) carried uncommon genetic alterations that would not be identified by the cobas® or the Idylla™ tests. We further found six new single mutations and seven previously unreported compound mutations. Clinical information and patient outcome are presented for these cases. CONCLUSIONS: This study highlights the value of sequencing-based approaches to identify rare mutations. Our results add to the inventory of known EGFR mutations, thus contributing to improved lung cancer precision treatment.

Dental pulp stem cells and Bonelike® for bone regeneration in ovine model.

Development of synthetic bone substitutes has arisen as a major research interest in the need to find an alternative to autologous bone grafts. Using an ovine model, the present pre-clinical study presents a synthetic bone graft (Bonelike®) in combination with a cellular system as an alternative for the regeneration of non-critical defects. The association of biomaterials and cell-based therapies is a promising strategy for bone tissue engineering. Mesenchymal stem cells (MSCs) from human dental pulp have demonstrated both in vitro and in vivo to interact with diverse biomaterial systems and promote mineral deposition, aiming at the reconstruction of osseous defects. Moreover, these cells can be found and isolated from many species. Non-critical bone defects were treated with Bonelike® with or without MSCs obtained from the human dental pulp. Results showed that Bonelike® and MSCs treated defects showed improved bone regeneration compared with the defects treated with Bonelike® alone. Also, it was observed that the biomaterial matrix was reabsorbed and gradually replaced by new bone during the healing process. We therefore propose this combination as an efficient binomial strategy that promotes bone growth and vascularization in non-critical bone defects.

Alternative substrates for conidiogenesis of the entomopathogenic fungus Beauveria bassiana (Bals) Vuillemin (Deuteromycotina: Hyphomycetes)

Abstract Beauveria bassiana is a promising fungus for the biological control of insect pests. The growing costs of conidia production have raised the need to ascertain the efficiency of some low cost substrates. The aim of this study was to analyze the potential use of different raw substrates without nutritional supplement for B. bassiana conidiogenesis. Growth and sporulation were evaluated using 30 g of substrate and 0.3 L of a conidia suspension (1 x 106 conidia/mL). After 10 days of incubation (70 ± 10% humidity and temperature (T) = 29 ± 1 °C), rice (2.00 x 106 conidia/g substrate), algaroba (2.36 x 106 conidia/g), malt A (1.22 x 106 conidia/g) and malt B (1.75 x 106 conidia/g) showed the highest levels of conidia production. The resulting conidia showed insecticidal activity higher than 80% on coconut termites. These new raw substrates may represent viable alternatives for the production of entomopathogenic fungi for use in the biological control of various insect pests.

Resumo Beauveria bassiana é um fungo promissor no controle biológico de insetos-praga. As crescentes despesas na produção de conídios levantam a necessidade de averiguar a eficiência de alguns substratos de baixo custo. O objetivo deste trabalho foi analisar o potencial de utilização de diferentes substratos brutos para a conidiogênese de B. bassiana. O crescimento e esporulação foram realizados utilizando 30 g do substrato e 0,3 µL da suspensão de conídios (1 x 106 conídios/mL). Após 10 dias de incubação (umidade 70 ± 10% e temperatura T = 29 ± 1° C), o arroz (2,00 x 106 conídios/g de substrato), algaroba (2,36 x 106 conídios/g), malte A (1,22 x 106 conídios/g) e B (1,75 x 106 conídios/g), apresentaram maior produção de conídios. Os conídios produzidos mostraram atividade inseticida sobre o cupim do coqueiro acima de 80% de mortalidade. Estes novos substratos brutos podem representar uma alternativa viável para produção de fungos entomopatogênicos para uso no controle biológico de vários insetos praga.

Genetic diversity of the forage peanut in the Jequitinhonha, São Francisco, and Paranã River valleys of Brazil.

Arachis pintoi and A. repens are legumes with a high forage value that are used to feed ruminants in consortium systems. Not only do they increase the persistence and quality of pastures, they are also used for ornamental and green cover. The objective of this study was to analyze microsatellite markers in order to access the genetic diversity of 65 forage peanut germplasm accessions in the section Caulorrhizae of the genus Arachis in the Jequitinhonha, São Francisco and Paranã River valleys of Brazil. Fifty-seven accessions of A. pintoi and eight of A. repens were analyzed using 17 microsatellites, and the observed heterozygosity (HO), expected heterozygosity (HE), number of alleles per locus, discriminatory power, and polymorphism information content were all estimated. Ten loci (58.8%) were polymorphic, and 125 alleles were found in total. The HE ranged from 0.30 to 0.94, and HO values ranged from 0.03 to 0.88. By using Bayesian analysis, the accessions were genetically differentiated into three gene pools. Neither the unweighted pair group method with arithmetic mean nor a neighbor-joining analysis clustered samples into species, origin, or collection area. These results reveal a very weak genetic structure that does not form defined clusters, and that there is a high degree of similarity between the two species.

Association of ADAMTS7 gene polymorphism with cardiovascular survival in coronary artery disease.

Recent genetic studies have revealed an association between polymorphisms at the ADAMTS7 gene locus and coronary artery disease (CAD) risk. Functional studies have shown that a CAD-associated polymorphism (rs3825807) affects ADAMTS7 maturation and vascular smooth muscular cell (VSMC) migration. Here, we tested whether ADAMTS7 (A/G) SNP is associated with cardiovascular (CV) survival in patients with established CAD. A cohort of 1,128 patients with angiographic proven CAD, who were followed up prospectively for a mean follow-up period of 63 (range 6-182) mo, were genotyped for rs3825807 A/G. Survival statistics (Cox regression) compared heterozygous (AG) and wild-type (AA) with the reference homozygous GG. Kaplan-Meier (K-M) survival curves were performed according to ADAMTS7 genotypes for CV mortality. Results showed that 47.3% of patients were heterozygous (AG), 36.5% were homozygous for the wild-type allele (AA) and only 16.2% were homozygous for the GG genotype. During the follow-up period, 109 (9.7%) patients died, 77 (6.8%) of CV causes. Survival analysis showed that AA genotype was an independent risk factor for CV mortality compared with reference genotype GG (HR = 2.7, P = 0.025). At the end of follow-up, the estimated survival probability (K-M) was 89.8% for GG genotype, 82.2% for AG and 72.3% for AA genotype (P = 0.039). Carriage of the mutant G allele of the ADAMTS7 gene was associated with improved CV survival in patients with documented CAD. The native overfunctional ADAMTS7 allele (A) may accelerate VSMC migration and lead to neointimal thickening, atherosclerosis progression and acute plaque events. ADAMTS7 gene should be further explored in CAD for risk prediction, mechanistic and therapeutic goals.

Laryngoscope blades and handles as sources of cross-infection: an integrative review.

The lack of standardization of efficient procedures to clean and disinfect laryngoscope blades and handles, which may be important sources of infection during their clinical use, has been reported previously, revealing contamination with blood, body fluids and micro-organisms. This paper aimed to evaluate the evidence available in the literature regarding the risk of laryngoscope blades and handles as a source of patient contamination. An integrative review of the literature was performed using databases such as Medline, LILACS, SciELO, Cochrane Library, BDENF and PubMed, and keywords in Medical Subject Headings (MeSH). The sample comprised 20 articles published between 1994 and 2012. The studies demonstrated risk of cross-infection and no consensus in current guidelines regarding cleaning and disinfection of this equipment. It was concluded that there are important gaps to be filled and urgent investigations required in order to facilitate standardization of efficient procedures to clean and disinfect laryngoscope blades and handles, and in turn to reduce the potential risk to which the patient and/or health team is exposed.

Different VO2max time-averaging intervals in swimming.

We aimed to determine the effect of sampling interval strategy on VO(2max) assessment to establish a standard time averaging method that allows a better identification of the VO(2) plateau incidence in swimming. To this end, 3 incremental protocols utilizing different step lengths for each sampling interval were used to compare VO(2max)measurements. 11 trained male swimmers performed 3 repetitions of a front crawl intermittent incremental protocol until exhaustion (increments of 0.05 m.s(-1), with 30 s and 24-48 h intervals between steps and tests, respectively) with 200, 300 and 400-m step lengths. VO(2) was directly measured, and 6 sampling intervals were compared: bxb and averages of 5, 10, 15, 20 and 30 s. Shorter sampling intervals (≤ 15 s) allowed the highest incidence of the VO(2) plateau, independent of the step lengths used; the 200 and 300-m step protocols accounted for higher percentage of VO(2) plateau incidence, and higher VO(2max) values, compared to the 400-m step protocol. As an optimal sampling interval should be used for the validation of the research findings, and considering that swimmers and coaches prefer less time-consuming protocols, the use of the 10 s time-average interval (once bxb and 5 s samplings present high variability) in a 200-m step incremental protocol for VO(2max) assessment in swimming is suggested.

Genetic diversity of Metrodorea nigra (Rutaceae) from a small forest remnant in Brazil assessed with microsatellite markers.

Metrodorea nigra (Rutaceae) is an endemic Brazilian tree of great ecological importance, frequently found in the submontane regions of ombrophilous dense and semideciduous forests. This tree is useful for reforesting degraded areas and the wood can be employed in construction. We developed 12 microsatellite markers from a genomic library enriched for GA/CA repeats, for this species. Polymorphisms were assessed in 40 trees of a highly fragmented population found in Cravinhos, State of São Paulo, in southeastern Brazil. Among the 12 loci, 8 were polymorphic and only one had fixed alleles in this population. The number of alleles per locus and expected heterozygosity ranged from 2 to 11 and from 0.190 to 0.889, respectively. These results revealed moderate levels of genetic variation in M. nigra population when compared to other tropical species. Additionally, transferability of the 12 primers was tested in seven other Brazilian Rutaceae tree species (endemics: M. stipularis, Galipea jasminiflora, Esenbeckia leiocarpa and non-endemics: E. febrifuga, E. grandiflora, Balfourodendron riedelianum, Zanthoxylum riedelianum). Transferability ranged among species, but at least 8 loci (~67%) amplified in M. stipularis, demonstrating a high potential for transferring microsatellite markers between species of the same genus in the Rutaceae family.

VO2 kinetics in 200-m race-pace front crawl swimming.

Studies that aim to characterize oxygen uptake kinetics in efforts above maximal oxygen consumption intensity are scarce. Our aim was to analyze the oxygen kinetics in a maximal 200-m front crawl, all measurements being conducted in swimming pool conditions. 10 high-level male swimmers performed a maximal 200-m bout and oxygen uptake was directly measured through breath-by-breath gas analysis. Mean (±SD) peak oxygen uptake was 68.58 (±5.79) ml.kg(-1).min(-1), evidencing a fast component phase. As expected, peak oxygen uptake presented a direct relationship with mean swimming speed of the first 50-m lap and with the 200-m effort, and was also correlated with the amplitude of the fast component (r=0.75, r=0.72, r=0.73, p<0.05, respectively). The observed mean amplitude value was higher than those observed in the literature for other exercise intensity domains. However, the time for its onset, as well as the duration for attaining the steady state, was shorter, as the peak oxygen uptake was not correlated with these 2 components. Moreover, as previously described for swimming at high intensities, the slow component phenomenon was not observed. Aerobic metabolic pathway accounted for 78.6%, confirming the high aerobic contribution in middle distance swimming events.

Pathogenicity for onion and genetic diversity of isolates of the pathogenic fungus Colletotrichum gloeosporioides (Phyllachoraceae) from the State of Pernambuco, Brazil.

Onion anthracnose, caused by Colletotrichum gloeosporioides, is one of the main diseases of onions in the State of Pernambuco. We examined the pathogenicity of 15 C. gloeosporioides strains and analyzed their genetic variability using RAPDs and internal transcribed spacers (ITS) of the rDNA region. Ten of the strains were obtained from substrates and hosts other than onion, including chayote (Sechium edule), guava (Psidium guajava), pomegranate (Punica granatum), water from the Capibaribe River, maracock (Passiflora sp), coconut (Cocus nucifera), surinam cherry (Eugenia uniflora), and marine soil; five isolates came from onions collected from four different regions of the State of Pernambuco and one region of the State of Amazonas. Pathogenicity tests were carried out using onion leaves and bulbs. All strains were capable of causing disease in leaves, causing a variable degree of lesions on the leaves; four strains caused the most severe damage. In the onion bulb tests, only three of the above strains caused lesions. Seven primers of arbitrary sequences were used in the RAPD analysis, generating polymorphic bands that allowed the separation of the strains into three distinct groups. The amplification products generated with the primers ITS1 and ITS4 also showed polymorphism when digested with three restriction enzymes, DraI, HaeIII and MspI. Only the latter two demonstrated genetic variations among the strains. These two types of molecular markers were able to differentiate the strain from the State of Amazonas from those of the State of Pernambuco. However, there was no relationship between groups of strains, based on molecular markers, and degree of pathogenicity for onion leaves and bulbs.

Genetic diversity and population structure analysis of the tropical pasture grass Brachiaria humidicola based on microsatellites, cytogenetics, morphological traits, and geographical origin.

Brachiaria humidicola (Rendle) Schweick. is a warm-season grass commonly used as forage in the tropics. Accessions of this species were collected in eastern Africa and massively introduced into South America in the 1980s. Several of these accessions form a germplasm collection at the Brazilian Agricultural Research Corporation. However, apomixis, ploidy, and limited knowledge of the genetic basis of this germplasm collection have constrained breeding activities. The objectives of this work were to identify genetic variability in the Brazilian B. humidicola germplasm collection using microsatellite markers and to compare the results with information on the following: (1) collection sites of the accessions; (2) reproductive mode and ploidy levels; and (3) genetic diversity revealed by morphological traits. The evaluated germplasm population is highly structured into four major groups. The sole sexual accession did not group with any of the clusters. Genetic dissimilarities did not correlate with either geographic distances or genetic distances inferred from morphological descriptors. Additionally, the genetic structure identified in this collection did not correspond to differences in ploidy level. Alleles exclusive to either sexual or apomictic accessions were identified, suggesting that further evaluation of the association of these loci with apospory should be carried out.