Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures.

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L-1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.

Antiphospholipid Syndrome On Cloud (APSOnCloud): web-based monitoring of oral anticoagulation.

Antiphospholipid Syndrome (APS) is an autoimmune disease characterized by recurrent thromboses and fetal losses with the presence of antiphospholipid antibodies. The main treatment to prevent recurrent thrombotic events is oral anticoagulation with vitamin K antagonist (VKA), which requires frequent monitoring and dosage adjustments. Outpatient anticoagulation monitoring has its limitations, such as patients spending long hours between the testing procedure and waiting for the results to be adjusted. To optimize this adjustment and to improve APS patients-doctors relationship, we developed a website to help monitor APS patients, called Antiphospholipid Syndrome On Cloud or APSOnCloud. To test it, since March 2014 to March 2016, we registered 20 patients with APS that have inserted 132 international normalized ratio (INR) values. Sixty two percent were out of range and it took on average 7 hours for the doctor in charge to adjust these values. The mean time in therapeutic range was 58.1%. Our preliminary experience in monitoring VKA oral anticoagulation on APSOnCloud suggests that patients with APS might benefit from this web-based monitoring.

GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide.

Chromobacterium violaceum is a free-living bacillus with several genes that enables it survival under different harsh environments such as oxidative and temperature stresses. Here we performed a label-free quantitative proteomic study to unravel the molecular mechanisms that enable C. violaceum to survive oxidative stress. To achieve this, total proteins extracted from control and C. violaceum cultures exposed during two hours with 8 mM hydrogen peroxide were analyzed using GeLC-MS proteomics. Analysis revealed that under the stress condition, the bacterium expressed proteins that protected it from the damage caused by reactive oxygen condition and decreasing the abundance of proteins responsible for bacterial growth and catabolism. GeLC-MS proteomics analysis provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress ultimately aggregating knowledge of the response of this organism to environmental stress. This study identified approximately 1500 proteins, generating the largest proteomic coverage of C. violaceum so far. We also detected proteins with unknown function that we hypothesize to be part of new mechanisms related to oxidative stress defense. Finally, we identified the mechanism of clustered regularly interspaced short palindromic repeats (CRISPR), which has not yet been reported for this organism.

Lithium Induces Glycogen Accumulation in Salivary Glands of the Rat.

Lithium is administered for the treatment of mood and bipolar disorder. The aim of this study was to verify whether treatment with different concentrations of lithium may affect the glycogen metabolism in the salivary glands of the rats when compared with the liver. Mobilization of glycogen in salivary glands is important for the process of secretion. Two sets of experiments were carried out, that is, in the first, the rats received drinking water supplemented with LiCl (38,25 and 12 mM of LiCl for 15 days) and the second experiment was carried out by intraperitoneal injection of LiCl solution (12 mg/kg and 45 mg LiCl/kg body weight) for 3 days. The active form of glycogen phosphorylase was not affected by treatment with LiCl considering the two experiments. The active form of glycogen synthase presented higher activity in the submandibular glands of rats treated with 25 and 38 mM LiCl and in the liver, with 25 mM LiCl. Glycogen level was higher than that of control in the submandibular glands of rats receiving 38 and 12 mM LiCl, in the parotid of rats receiving 25 and 38 mM, and in the liver of rats receiving 12 mM LiCl. The absolute value of glycogen for the submandibular treated with 25 mM LiCl, and the liver treated with 38 mM LiCl, was higher than the control value, although not statistically significant for these tissues. No statistically significant difference was found in the submandibular and parotid salivary glands for protein concentration when comparing experimental and control groups. We concluded that LiCl administered to rats influences the metabolism of glycogen in salivary glands.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

Altered acetylcholine release in the hippocampus of dystrophin-deficient mice.

Mild cognitive impairments have been described in one-third of patients with Duchenne muscle dystrophy (DMD). DMD is characterized by progressive and irreversible muscle degeneration caused by mutations in the dystrophin gene and lack of the protein expression. Previously, we have reported altered concentrations of α7- and ß2-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal membranes of dystrophic (mdx) mice. This suggests that alterations in the central cholinergic synapses are associated with dystrophin deficiency. In this study, we examined the release of acetylcholine (ACh) and the level of the vesicular ACh transporter (VAChT) using synaptosomes isolated from brain regions that normally have a high density of dystrophin (cortex, hippocampus and cerebellum), in control and mdx mice at 4 and 12months of age. ACh release evoked by nicotinic stimulation or K(+) depolarization was measured as the tritium outflow from superfused synaptosomes preloaded with [(3)H]-choline. The results showed that the evoked tritium release was Ca(2+)-dependent and mostly formed by [(3)H]-ACh. ß2-containing nAChRs were involved in agonist-evoked [(3)H]-ACh release in control and mdx preparations. In hippocampal synaptosomes from 12-month-old mdx mice, nAChR-evoked [(3)H]-ACh release increased by 57% compared to age-matched controls. Moreover, there was a 98% increase in [(3)H]-ACh release compared to 4-month-old mdx mice. [(3)H]-ACh release evoked by K(+) depolarization was not altered, while the VAChT protein level was decreased (19%) compared to that of age-matched controls. In cortical and cerebellar preparations, there was no difference in nAChR-evoked [(3)H]-ACh release and VAChT levels between mdx and age-matched control groups. Our previous findings and the presynaptic alterations observed in the hippocampi of 12-month-old mdx mice indicate possible dysfunction of nicotinic cholinergic synapses associated with dystrophin deficiency. These changes may contribute to the cognitive and behavioral abnormalities described in dystrophic mice and patients with DMD.

Saberes e práticas populares de cuidado em saúde com o uso de plantas medicinais

Conclui-se, que o resgate desta prática traz consigo alternativas saudáveis, implantando o uso correto das plantas medicinais a fim de, fornecer subsídios e orientações para que a comunidade que busca a Assistência Básica de Saúde venha a usufruir dessa prática milenar que acompanha nossa história.

Saberes e práticas populares de cuidado em saúde com o uso de plantas medicinais / Knowledge and popular health practices with the use of medicinal plants

Conclui-se, que o resgate desta prática traz consigo alternativas saudáveis, implantando o uso correto das plantas medicinais a fim de, fornecer subsídios e orientações para que a comunidade que busca a Assistência Básica de Saúde venha a usufruir dessa prática milenar que acompanha nossa história.

[Postraumatic epidural arachnoid spinal cyst: case report]. / Quiste aracnoideo espinal epidural postraumático: presentación de un caso.

INTRODUCTION: Extradural arachnoid spinal cysts are unfrequent lesions that are associated with spinal trauma, surgery and less frequently with congenital anomalies. The clinical manifestations are similar to those seen with other compressive spinal cord lesions. Magnetic resonance techniques allow to diagnose correctly this pathology and to define its thopographic situation. The pathologic history of the patient is essencial to establish the ethiology. Surgery is the elective treatment in most cases. CLINICAL CASE: The patient is a 35 years old man who has a medical history of penetrating spinal trauma two years ago. In that instance he suffered an unilateral spinal cord section at D2-D3 level with the corresponding Brown Sequard syndrome. A small wound was detected at the skin dorsal level and it was closed without difficulties. At the beginning, he improved his motor right leg function with rehabilitation and vitamins. After two years of good recovery he came to our hospital suffering a neurological deterioration of six months of evolution. The physical examination revealed an spastic paraparesis. Magnetic resonance was performed demonstrating a cystic extradural collection compressing the spinal cord at D3-D4 level. Surgical decompressive treatment allowed to excise the cyst and it was possible to define a dural tear that was closed successfully. The outcome was good with restoration of the initial motor function that he had after the spinal trauma. CONCLUSIONS: Surgical management of postraumatic epidural arachnoid spinal cyst allows to detect the meningeal tear and to close it, which is highly effective on these kinds of lesions.

Analysis of the salivary proteome in gingivitis patients.

BACKGROUND AND OBJECTIVE: Gingivitis is a disease that is characterized by inflammation of the gingival tissue, which can progress to periodontitis and tooth loss. Although many studies have attempted to identify salivary proteins that are associated with the disease, this is the first study to use a proteomic approach to analyze and compare the proteomic profile of whole saliva from gingivitis patients and healthy controls. MATERIAL AND METHOD: To analyze the saliva proteome, two-dimensional gel electrophoresis and liquid chromatography were used, followed by mass spectrometry. RESULTS: The analyses showed that gingival inflammation was associated with increased amounts of blood proteins (serum albumin and hemoglobin), immunoglobulin peptides and keratins. In the control group, salivary cystatins, which were detected using capillary Liquid Chromatography on line to electrospray ionization Quadrupole Time-of-flight mass spectrometry, appeared to be more abundant. CONCLUSION: This approach provides novel insight into profiles of the salivary proteome during gingival inflammation, which may contribute to improvements in diagnosis.

Comparison of different diagnostic tests in dogs uninfected and naturally infected with visceral leishmaniasis

Uninfected dogs (n = 10) and those naturally infected with leishmaniasis (n = 10) were subjected to several diagnostic tests, namely: hemoculture, polymerase chain reaction (PCR) of hemoculture, indirect immunofluorescence (RIFI), cytological examination of lymph node aspirate, culture of lymph node aspirate and PCR of lymph node aspirate. RIFI - followed by PCR of lymph node aspirate culture - presented more positive results in infected dogs than in uninfected ones. In infected animals, RIFI was more effective than PCR of lymph node aspirate culture. There was no statistical difference in positivity between RIFI and hemoculture; lymph node aspirate culture/cytological examination of lymph node aspirate and PCR of hemoculture; and between PCR of lymph node aspirate culture and PCR of hemoculture. All infected and uninfected animals had positive and negative results in at least one test. In conclusion, the association of several tests improves the efficacy of canine visceral leishmaniasis diagnosis.(AU)

Comparison of different diagnostic tests in dogs uninfected and naturally infected with visceral leishmaniasis

Uninfected dogs (n = 10) and those naturally infected with leishmaniasis (n = 10) were subjected to several diagnostic tests, namely: hemoculture, polymerase chain reaction (PCR) of hemoculture, indirect immunofluorescence (RIFI), cytological examination of lymph node aspirate, culture of lymph node aspirate and PCR of lymph node aspirate. RIFI - followed by PCR of lymph node aspirate culture - presented more positive results in infected dogs than in uninfected ones. In infected animals, RIFI was more effective than PCR of lymph node aspirate culture. There was no statistical difference in positivity between RIFI and hemoculture; lymph node aspirate culture/cytological examination of lymph node aspirate and PCR of hemoculture; and between PCR of lymph node aspirate culture and PCR of hemoculture. All infected and uninfected animals had positive and negative results in at least one test. In conclusion, the association of several tests improves the efficacy of canine visceral leishmaniasis diagnosis.

Effects of Anacardium humile leaf extracts on the development of gastrointestinal nematode larvae of sheep.

The rapid development of nematode resistance to anthelmintics has limited the success of small ruminant gastrointestinal helminthiasis control in several countries, stimulating the search for alternatives. The aim of this work was to evaluate the in vitro anthelmintic activity of Anacardium humile (Anacardiaceae) on gastrointestinal nematodes of sheep. Aqueous and ethanolic extracts of A. humile leaves were evaluated by the larval development inhibition assay at concentrations of: 187.5, 150, 100, 50, and 30 mg ml(-1); and 100, 80, 60, 40, and 20 mg ml(-1), respectively. Phytochemical analysis demonstrated the presence of tannins, flavenoids, and alkaloids in the leaves. The LD(50) was 10.14 mg ml(-1) for the aqueous extract and 23.24 mg ml(-1) for the ethanolic extract, providing evidence for the potential of this plant in the endoparasite control.

Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis.

Leishmune, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group (p=0.0253), suggesting that the combination of additional chemotherapy with Leishmune-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p=0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune in the control and cure of CVL.

Ampelozyziphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria in the Amazon Region, hampers the development of Plasmodium berghei sporozoites.

Most medicinal plants used against malaria in endemic areas aim to treat the acute symptoms of the disease such as high temperature fevers with periodicity and chills. In some endemic areas of the Brazilian Amazon region one medicinal plant seems to be an exception: Ampelozyziphus amazonicus, locally named "Indian beer" or "Saracura-mira", used to prevent the disease when taken daily as a cold suspension of powdered dried roots. In previous work we found no activity of the plant extracts against malaria blood parasites in experimentally infected animals (mice and chickens) or in cultures of Plasmodium falciparum. However, in infections induced by sporozoites, chickens treated with plant extracts were partially protected against Plasmodium gallinaceum and showed reduced numbers of exoerythrocytic forms in the brain. We now present stronger evidence that the ethanolic extract of "Indian beer" roots hampers in vitro and in vivo development of Plasmodium berghei sporozoites, a rodent malaria parasite. Some mice treated with high doses of the plant extract did not become infected after sporozoite inoculation, whereas others had a delayed prepatent period and lower parasitemia. Our data validates the use of "Indian beer" as a remedy for malaria prophylaxis in the Amazon, where the plant exists and the disease represents an important problem which is difficult to control. Studies aiming to identify the active compounds responsible for the herein described causal prophylactic activity are needed and may lead to a new antimalarial prophylactic.