Abstract Several Piper species accumulate piperamides as secondary metabolites, and although they have relevant biological importance, many details of their biosynthetic pathways have not yet been described experimentally. Experiments involving enzymatic reactions and labeled precursor feeding were performed using the species Piper tuberculatum and Piper arboreum. The activities of the phenylalanine ammonia lyase (PAL) enzymes, which are involved in the general phenylpropanoid pathway, were monitored by the conversion of the amino acid L-phenylalanine to cinnamic acid. The activity of the 4-hydroxylase (C4H) enzyme was also observed in P. tuberculatum by converting cinnamic acid to p-coumaric acid. L-[UL-14C]-phenylalanine was fed into the leaves of P. tuberculatum and incorporated into piperine (1), 4,5-dihydropiperine (2), fagaramide (4), trans-piplartine (7), and dihydropiplartine (9). In P. arboreum, it was only incorporated into the piperamide 4,5-dihydropiperiline (3). L-[UL-14C]-lysine was successfully incorporated into the 4,5-dihydropiperine piperidine group (2), dihydropyridinone, and trans- (7) and cis-piplartine (8). These data corroborate the proposal of mixed biosynthetic origin of piperamides with the aromatic moiety originating from cinnamic acid (shikimic acid pathway) and key amide construction with amino acids as precursors.

Resumo Diversas espécies do gênero Piper acumulam piperamidas como metabólitos secundários e, embora tenham relevante importância biológica, detalhes de suas vias biossintéticas ainda não foram descritos experimentalmente. Experimentos envolvendo reações enzimáticas e incorporação de precursores marcados foram realizados utilizando as espécies Piper tuberculatum e Piper arboreum. As atividades das enzimas fenilalanina amônia liase (PAL), que estão envolvidas na via geral dos fenilpropanoides, foram monitoradas pela conversão do aminoácido L-fenilalanina em ácido cinâmico. A atividade da enzima 4-hidroxilase (C4H) também foi observada em P. tuberculatum pela conversão do ácido cinâmico em ácido p-cumárico. L-[UL-14C]-fenilalanina foi administrada em folhas de P. tuberculatum e incorporada em piperina (1), 4,5-di-hidropiperina (2), fagaramida (4), trans-piplartina (7) e diidropiplartina (9). Em P. arboreum, foi incorporado apenas à piperamida 4,5-dihidropiperilina (3). L-[UL-14C]-lisina foi incorporada com sucesso em 4,5-diidropiperina (2), trans-piplartina (7) e cis-piplartina (8). Esses dados corroboram a proposta de origem biossintética mista de piperamidas com a porção aromática originada do ácido cinâmico (via do ácido chiquímico) e construção de amida chave com aminoácidos como precursores.

Maytenus ilicifolia (Celastraceae) is a medicinal plant that is native to southern Brazil and is popularly known as “espinheira-santa”. From a biosynthesis perspective, this species accumulates quinonemethide triterpenes and sesquiterpene pyridine alkaloids as major secondary metabolites that exhibit interesting biological properties, with antitumoral and antiprotozoal activities, respectively, being the most frequently reported. Additionally, the restricted accumulation of such compounds in the roots raises questions about the expression of proteins involved in such compartmentalization and their possible biological and/or ecological role in M. ilicifolia. Thus, this article describes the use of shotgun proteomics and histochemical studies for the characterization of the main biosynthetic pathways involved in the regulation of the metabolism in M. ilicifolia roots. This combined approaches also resulted in the identification of a series of proteins involved in the quinonemethide triterpenes and sesquiterpene pyridine alkaloids, providing evidences of their differential compartmentalization.

RESUMO Estudos indicam, por meio de infecção experimental, que primatas não humanos são susceptíveis à infecção por Neospora caninum. Relata-se um caso de um macaco-da-noite (Aotus azarae infulatus), que apresentou sinais inespecíficos e não respondeu à terapêutica clínica de suporte, evoluindo a óbito, encaminhado em seguida para exame anatomopatológico. Amostras de tecidos foram coletadas e processadas rotineiramente para confecção de lâminas histológicas. Microscopicamente, a principal lesão foi observada no coração e consistia em miocardite necrótica multifocal por protozoário, com a presença de estruturas compatíveis com o estágio de taquizoítos de protozoários dos gêneros Neospora sp. ou Toxoplasma sp. No sistema nervoso central, predominantemente no tronco encefálico, havia estruturas semelhantes às descritas no coração. Os resultados da reação em cadeia pela polimerase (PCR) foram positivos para N. caninum e negativos para Toxoplasma gondii, usando DNA extraído do sangue e dos tecidos. Este relato de caso fornece evidências histológicas e moleculares de que o primata em questão foi susceptível a uma infecção natural, porém estudos devem ser realizados para investigar o real papel dos primatas no ciclo de vida de N. caninum.

ABSTRACT Studies indicate through experimental infection that non-human primates are susceptible to infection by Neospora caninum. This report is of a case of a night monkey (Aotus azarae infulatus) that presented nonspecific signs and did not respond to supportive clinical therapy evolving to death, followed by a pathology examination. Tissue specimens were routinely collected and processed for the preparation of histological slides. Microscopically, the main lesion was observed in the heart and consisted of multifocal necrotic myocarditis by protozoa, with the presence of structures compatible with the stage of protozoan tachyzoites of the genus Neospora sp. or Toxoplasma sp. In the central nervous system, predominantly in the brainstem there were structures similar to those described in the heart. Polymerase chain reaction (PCR) results were positive for N. caninum and was negative for Toxoplasma gondii using DNA extracted from blood and tissues. This case report provides histological and molecular evidence that the primate in question was susceptible to a natural infection, but studies should be conducted to investigate the real role of primates in the life cycle of N. caninum.

The known kavalactones (E)-4-methoxy-6-styryl-2H-pyran-2-one, 4-methoxy-6-(3-phenyloxiran-2-yl)-2H-pyran-2-one, 6-(1,2-dihydroxy-2-phenylethyl)-4-methoxy-2H-pyran-2-one, the three benzoic acid derivatives methyl-4-methoxy-3-(3'-methyl-2'-butenyl)benzoate and methyl 2,2-dimethyl-4-oxochroman-6-carboxylate, and a new methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate were isolated from the ethanolic extract of Piper fuligineum. The structures of these compounds were determined by using a combination of spectroscopic methods, including 1D- and 2D-nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. This is the first report of the chemical study of P. fuligineum, and the methyl 4-methoxy-3-(3-methylbut-2-enoyl)benzoate is described as a new natural product.

Piper gaudichaudianum Kunth (Piperaceae) accumulates gaudichaudianic acid, a prenylated benzopyran, as its major component. Interestingly, this trypanocidal compound occurs as a racemic mixture. Herein, transcriptomic investigations of Piper gaudichaudianum using the RNA-seq approach are reported, and from the analysis of the transcripts expressed it was possible to propose a complete biosynthetic pathway for the production of gaudichaudianic acid, including the steps that originate its precursor, p-hydroxybenzoic acid. Peperomia obtusifolia (L.) A. Dietr. (Piperaceae) also accumulates racemic benzopyrans, however, its chromanes originate from the polyketide pathway, while the chromenes from Piper derives from the shikimate pathway. Recent transcriptomic and proteomic studies of the former species did not identify polyketide synthases involved in the production of the benzopyran moiety, but revealed the expression of tocopherol cyclase, which may be responsible for the cyclization of the 3,4-dihydro-2H-pyran ring. The analysis of the enzymes involved in the secondary metabolism of Piper gaudichaudianum and the comparison with the data previously obtained from Peperomia obtusifolia can provide valuable information on how these compounds are biosynthesized.

RESUMO Dentre as técnicas utilizadas pela biotecnologia para multiplicação de mudas de orquídea, o cultivo in vitro destaca-se pelo elevado potencial de produção de novas plântulas. A presença de algumas substâncias químicas utilizadas no meio de cultura, dependendo da concentração, pode favorecer ou não o crescimento e o desenvolvimento da planta. Diante do exposto, objetivou-se, com este trabalho, avaliar o efeito do glifosato no cultivo in vitro da orquídea Cattleya nobilior Rchb. F. O delineamento experimental utilizado foi inteiramente casualizado, com seis tratamentos e dez repetições. Os tratamentos foram constituídos de seis concentrações de glifosato (0,0; 8,5; 17,0; 25,5; 34,0 e 42,5 mg L-1 e.a.), acrescidas ao meio de cultura MS modificado, com metade das concentrações de macronutrientes. As plântulas submetidas às diferentes concentrações de glifosato foram obtidas de sementes germinadas in vitro. As avaliações de comprimento e de biomassas secas de parte aérea e de raízes, de número de folhas, de comprimento da folha, de largura da folha, do número de brotos dos teores de clorofilas a, b e de carotenoides foram realizadas 200 dias após o transplantio das plântulas. Dentre os componentes fitométricos analisados, apenas o comprimento, a biomassa seca de raízes e o teor de carotenoides diminuíram com o incremento das doses do herbicida glifosato. Baixas concentrações de glifosato adicionadas ao meio de cultura resultam em efeito hormese, com aumento do crescimento da parte aérea de Cattleya nobilior Rchb. F.

ABSTRACT Among the techniques used by biotechnology for multiplication of orchid seedlings, the in vitro cultivation stands out due to the high production potential of new seedlings. The presence of some chemical substances used in the culture medium, depending on the concentration, may or may not favor the growth and development of the plant. Therefore, the objective of this work was to evaluate the effect of glyphosate on the in vitro cultivation of the orchid Cattleya nobilior Rchb. F. The experimental design was completely randomized, with six treatments and ten replications. The treatments consisted of six concentrations of glyphosate (0.0, 8.5, 17.0, 25.5, 34.0, and 42.5 mg L-1 a.i.), added to the modified MS culture medium with half the concentrations of macronutrients. Seedlings subjected to different glyphosate concentrations were obtained from seeds germinated in vitro. The length and dry biomass measurements of the shoot and root, number of leaves, leaf length, leaf width, number of shoots, chlorophyll a, b, and carotenoid contents were performed 200 days after transplanting of the seedlings. Among the phytometric components analyzed, only the length and dry biomass of root and the carotenoid content decreased with increasing doses of the glyphosate herbicide. Low concentrations of glyphosate added to the culture medium result in hormese effect, with increased growth of Cattleya nobilior Rchb. F. shoot.

Resumo Piper tuberculatum (Piperaceae) é uma espécie que acumula especialmente amidas como metabólitos secundários e diversas atividades biológicas dessa espécie foram relatadas anteriormente. No presente artigo, relatamos um estudo proteômico dessa espécie. Eletroforese bidimensional (2D SDS-PAGE) e espectrometria de massas (ESI-Q-TOF) foram utilizadas nesse estudos. Mais de cem spots e vários peptídeos foram identificados nesta espécie e as funções putativas desses peptídeos relacionadas a mecanismo de defesa como estresse biótico e abiótico foram atribuídos. As informações apresentadas ampliam a gama de informações moleculares dessa espécie.

Abstract Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.

ABSTRACT Aims The use of electromagnetic waves by phototherapy to skeletal muscle presents potential ergogenic effects. The aim of this study was to analyze the effect of using bioceramic clothes on performance, heart rate (HR) and rating of perceived exertion (RPE) during a 10 km race. Our hypothesis is that the use of such clothes modifies these variables. Methods Participants were 10 runners (27.9 ± 4.2 years) who performed two 10 km performances on track under different intervention conditions: bioceramic garments (CER) and placebo garments (PLA). The mean velocity (MV), HR and rate of perceived exertion (RPE) were monitored at each trial. Additionally, partial MV was calculated in three phases: (1) start (first 400 m), (2) middle (400-9600 m) and (3) end (last 400 m). Results MV in CER condition was significantly higher than in PLA condition (11.8 ± 1.0 km·h-1 vs 11.4 ± 1.2 km·h-1; F = 6.200; P = 0.034; ŋp² = 0.408). HR and RPE values in CER condition were not different from PLA condition. Conclusions Our main finding was that the use of bioceramic clothes (CER) increased MV when compared to the PLA condition. Based on these results, bioceramic may be used as an ergogenic resource to increase performance.

OBJECTIVE: To test the hypotheses that 1) coronary artery disease patients with lower aerobic fitness exhibit a lower ventilatory efficiency and 2) coronary artery disease patients with lower initial aerobic fitness exhibit greater improvements in ventilatory efficiency with aerobic exercise training. METHOD: A total of 123 patients (61.0±0.7 years) with coronary artery disease were divided according to aerobic fitness status into 3 groups: group 1 (n = 34, peak VO2<17.5 ml/kg/min), group 2 (n = 67, peak VO2>17.5 and <24.5 ml/kg/min) and group 3 (n = 22, peak VO2>24.5 ml/kg/min). All patients performed a cardiorespiratory exercise test on a treadmill. Ventilatory efficiency was determined by the lowest VE/VCO2 ratio observed. The exercise training program comprised moderate-intensity aerobic exercise performed 3 times per week for 3 months. Clinicaltrials.gov: NCT02106533 RESULTS: Before intervention, group 1 exhibited both lower peak VO2 and lower ventilatory efficiency compared with the other 2 groups (p<0.05). After the exercise training program, group 1 exhibited greater improvements in aerobic fitness and ventilatory efficiency compared with the 2 other groups (group 1: ▵ = -2.5±0.5 units; group 2: ▵ = -0.8±0.3 units; and group 3: ▵ = -1.4±0.6 units, respectively; p<0.05). CONCLUSIONS: Coronary artery disease patients with lower aerobic fitness status exhibited lower ventilatory efficiency during a graded exercise test. In addition, after 3 months of aerobic exercise training, only the patients with initially lower levels of aerobic fitness exhibited greater improvements in ventilatory efficiency.

A criptococose é uma infecção que afeta humanos e animais sendo a etiologia atribuída às espécies Cryptococcus neoformans variedade neoformans, C. neoformans var. grubii e C. gattii. A doença é comum em cães e gatos, causando infecções respiratórias, neurológicas, cutâneas e oculares. Com o objetivo de entender melhor a epidemiologia da criptococose em animais, este trabalho descreve a ocorrência e a caracterização de espécies de Cryptococcus em pequenos animais doentes no Estado de Mato Grosso, Brasil. Amostras clínicas de quatro casos, dois em felinos e dois em caninos, foram submetidas à análise patológica, microbiológica e molecular. Microscopicamente, em três casos, nos cortes de tecidos corados pela hematoxilina notou-se desde ausência até acentuada inflamação granulomatosa composta por histiócitos, células multinucleadas e infiltração linfocitária. Em um caso, na análise citológica de "imprint" observou-se infiltrado inflamatório similar composto principalmente por células mononucleares e linfócitos. Em todos os casos havia variável quantidade de estruturas fúngicas intra e extracelulares compatíveis com Cryptococcus spp pela coloração do ácido períodico de Schiff (PAS). Todas as amostras foram positivas para a cultura em Sabouraud Dextrose Agar (SDA), e morfologicamente classificadas como Cryptococcus sp. Os isolados foram positivos no PCR para C. gattii, sendo confirmados pelo seqüenciamento. Os resultados caracterizaram a espécie envolvida na região, e contribuem para futuros estudos sobre a epidemiologia de C. gattii.

Cryptococcosis is an infection that affects humans and animals, the etiology is attributed to Cryptococcus neoformans variety neoformans, C. neoformans var. grubii and Cryptococcus gattii. The infection is common in dogs and cats, causing respiratory, neurological, cutaneous and ocular infections. Aiming to better understand the epidemiology of cryptococcosis in animals in the region, this paper describe the occurrence and characterization of the Cryptococcus species involved in this illness in pet animals at Mato Grosso State, Brazil. Clinical samples of four cases, two in cats and two dogs, were submitted for pathological, microbiological and molecular analysis. Microscopically, in three cases, tissue sections stained with hematoxylin and eosin had absence to severe granulomatous reaction composed by histiocytes, multinucleated cells and lymphocytes infiltration. In one case, citological imprint analysis showed similar inflammatory mainly mononuclear and lymphocyte cells infiltration. All cases had variable amounts of intracellular and extracellular fungal structures compatible with Cryptococcus sp. on Periodic Acid-Schiff (PAS) stain. All clinical samples were positive for culture on Sabouraud Dextrose Agar (SDA) and morphologically classified as Cryptococcus sp. The isolates were PCR positive for C. gatti, being confirmed by sequencing technique. The findings characterize the molecular species involved in animal infections in the region, and may contribute to future studies of the epidemiology of C. gattii.

The constant evolution of science and the growing demand for new technologies have led to new techniques in instrumentation that can improve detection, separation, resolution, and peak capacity. Comprehensive two-dimensional liquid chromatography (LC×LC) is presented as a powerful tool in complex sample analyses. During an analysis, a sample is subjected to two independent separation mechanisms that are combined, resulting in increased resolving power. For appropriate application of LC×LC, understanding the influence of parameters that require optimization is necessary. The main purpose of optimization is to predict the combination of stationary phases, separation conditions, and instrumental requirements to obtain the best separation performance. This review discusses theoretical, intrumental, and chemometric aspects of LC×LC and focuses on its applications in foods. It aims to provide a clear understanding of the aspects that can be used as strategies in the optimization of this analytical method.

Several species of Croton have been described with biological activities, mainly due to diterpenes, alkaloids and/or other secondary metabolites. These activities account for the traditional use of Croton species to treat certain diseases in South America, Asia and Western Africa. The crude methanol extracts obtained from leaves and steam bark of Croton dichrous Müll. Arg., C. erythroxyloides Baill., C. myrianthus Müll. Arg. and C. splendidus Mart. ex Colla were tested for antiproliferative activity against ten human cancer cell lines. Chemical analyses of all extracts were carried out by GC/MS and HPLC/MS/MS. The leaf extract obtained from C. erythroxyloides showed potent activity against PC-3 (prostate) and OVCAR-3 (ovary) cell lines. Lupeol is suggested to be involved in such activity. Tiliroside, an acyl-glycosilated flavonoid ubiquitous in all tested extracts, seems to play an important role in the observed moderate activity of most extracts against the leukemia K562 cell lineage.