Growth regulators, culture media and antibiotics in the in vitro shoot regeneration from mature tissue of citrus cultivars

The objective of this study was to evaluate the effects of 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) combinations, basal media and beta-lactam antibiotics on in vitro organogenesis from mature stem segments of 'Pêra', 'Valência' and 'Bahia' sweet oranges and 'Cravo' rangpur lime. For induction of shoot regeneration, the segments of the four cultivars were placed on Murashige and Skoog (MS) medium containing the following BAP/NAA concentrations: 0.0/0.0; 0.25/0.0; 0.25/0.25; 0.5/0.0; 0.5/0.5; 1.0/0.0; 2.0/0.0; 2.0/0.25; 2.0/0.5; and 2.0/1.0 mg L-1. In order to test the influence of the culture media on shoot-bud induction, (MS), Murashige and Tucker (MT), and woody plant medium (WPM) formulations were evaluated, associated with the best combination of plant growth regulators obtained in the previous experiment. The influence of four beta-lactam antibiotics (timentin, cefotaxime sodium salt, meropenem trihydrate and augmentin) on shoot regeneration was determined. Better regeneration responses were achieved when internodal segments were cultured onto MS-based medium with 500 mg L-1 cefotaxime with the following BAP/NAA concentrations: 0.5 + 0.25 mg L-1 for 'Cravo', 1.0 + 0.25 mg L-1 for 'Valência' and 'Bahia', and 1.0 + 0.5 mg L-1 for 'Pêra'. Genotype, growth regulators, basal media and beta-lactam antibiotics affect the morphogenetic response in mature tissues of citrus.

Preliminary study of coconut water for graft tissues preservation in transplantation

OBJECTIVE: to verify the effectiveness of coconut water in preserving tissues for transplant. METHODS: Fifty male Wistar rats were randomly distributed in five groups, according to the following preservation solutions for tissue grafts: Group 1: Lactated Ringer; Group 2: Belzer solution; Group 3: mature coconut water; Group 4: green coconut water; Group 5: modified coconut water. In Group 5, the green coconut water has been modified like the Belzer solution. From each animal we harvasted the spleen, ovaries and skin of the back segment. These tissues were preserved for six hours in one of the solutions. Then, the grafts were reimplanted. The recovery of the function of the implanted tissues was assessed 90 days after surgery, by splenic scintigraphy and blood exame. The implanted tissues were collected for histopathological examination. RESULTS: The serum levels did not differ among groups, except for the animals in Group 5, which showed higher levels of IgG than Group 1, and differences in relation to FSH between groups 1 and 2 (p <0.001), 4 and 2 (p = 0.03) and 5 and 2 (p = 0.01). The splenic scintigraphy was not different between groups. The ovarian tissue was better preserved in mature coconut water (p <0.007). CONCLUSION: the coconut water-based solutions preserves spleen, ovary, and rat skin for six hours, maintaining their normal function.

Biochemical and morpho-anatomical analyses of strawberry vitroplants hyperhydric tissues affected by BA and gelling agents

In vitro propagation has become an effective practice for large-scale production of strawberry plants. The objective of this study was to evaluate the hyperhydricity and the multiplication capacity of two strawberry varieties (Fragaria x ananassa Duch. 'Dover' and 'Burkley') propagated in vitro. Plants maintained in MS medium supplemented with 1.0 mg L-1 BA were individualized and transferred to the same medium solidified with Agar (6.5 g L-1) or Phytagel® (2.5 g L-1) and BA at different concentrations (0; 0.5; 1.0; 2.0 and 3.0 mg L-1). Biochemical and anatomical analyses were carried out, as well as the analysis of the morphological hyperhydricity characteristics. The analysis of data showed: a) the increase in cytokinin concentration increased hyperhydricity frequency in both varieties; b) at concentrations up to 2.0 mg L-1 BA, the replacement of Agar by Phytagel® induced a higher formation of hyperhydric shoots; and c) the addition of BA induced oxidative stress, which is characterized by increased antioxidant activity and lipid peroxidation, as well as alterations at the cellular level, such as malformation of stomata and epidermal cells. In conclusion, the culture medium containing 0.5 mg L-1 BA solidified with Agar provided lower hyperhydricity percentages in association with higher rates of shoot proliferation in strawberry.

Diagnostic leaf to evaluate the nutritional status of Jatropha

ABSTRACT The analytical determination of nutrient levels in recently mature leaves in order to diagnose nutritional status is based on the fact that leaves are metabolically active and more sensitive to variation in nutrients of the soil. In most of cases, there is a direct well known between foliar content and the development and yield of the plant. However, for a more accurate interpretation, it is essential to establish the index leaf. There are few published studies about Jatropha with contrasting results. In order to establish the index leaf, in adult plants, the macronutrient levels were evaluated in samples collected in experimental plots, in which doses of nitrogen and phosphorus were applied, in two parts of the floral branches (in the top and in the middle thirds); and in three positions of leaves of the floral branch (between the 1st and 3rd, 6th and 8th, and 13th and 15th leaves below the inflorescence). The location of the leaf on the plant significantly affects nutrient contents. Nitrogen, phosphorus, potassium and sulfur tend to have higher concentration in young tissues. Calcium and magnesium showed higher levels in the basal leaves of floral branches. Samples collected in the top third of plants (between the 6th and 15th leaves of the floral branch) are more sensitive to variations of nitrogen and phosphorus fertilization. Therefore, we indicate the 6th to 15th leaves of the top third plants as index leaves estimate nutritional status of Jatropha.

Experimental life cycle of Lagochilascaris minor Leiper, 1909

The life cycle of Lagochilascaris minor was studied using material collected from human lesion and applying the experimental model: rodents (mice, hamsters), and carnivorae (cats, dogs). In mice given infective eggs, orally, hatch of the third stage larvae was noted in the gut wall, with migration to liver, lungs, skeletal musculature and subcutaneous tissue becoming, soon after, encysted. In cats infected with skinned carcasses of mice (60 to 235 days of infection) it was observed: hatch of third stage larvae from the nodules (cysts) in the stomach, migration through the oesophagus, pharynx, trachea, related tissues (rhino-oropharynx), and cervical lymphonodes developing to the mature stage in any of these sites on days 9-20 post inoculation (P.I.). There was no parasite development up to the mature stage in cats inoculated orally with infective eggs, which indicates that the life cycle of this parasite includes an obligatory intermediate host. In one of the cats (fed carcass of infected mice) necropsied on day 43 P.I., it was observed the occurence of the self-infective cycle of L. minor in the lung tissues and in the cervical region which was characterized by the finding of eggs in different stages of development, third stage larvae and mature worms. It's believed that some component of the carnivorae gastrointestinal tracts may preclude the development of third stage larvae from L. minor eggs what explains the interruption of the life cycle in animals fed infective eggs. It's also pointed out the role of the intermediate host in the first stages of the life cycle of this helminth.

Detection of parvovirus B19 infection in formalin-fixed and paraffin-embedded placenta and fetal tissues

Parvovirus B19 infection was first discovered in 1975 and it is implicated in fetal death from hydrops fetalis the world over. Diagnosis is usually made through histological identification of the intranuclear inclusion in placenta and fetal organs. However, these cells may be scarce or uncharacteristic, making definitive diagnosis difficult. We analyzed histologically placentas and fetal organs from 34 cases of non-immune hydrops fetalis, stained with Hematoxylin and Eosin (HE) and submitted to immunohistochemistry and polymerase chain reaction (PCR). Of 34 tissue samples, two (5.9%) presented typical intranuclear inclusion in circulating normoblasts seen in Hematoxylin and Eosin stained sections, confirmed by immunohistochemistry and PCR. However, PCR of fetal organs was negative in one case in which the placenta PCR was positive. We concluded that parvovirus B19 infection frequency is similar to the literature and that immunohistochemistry was the best detection method. It is highly specific and sensitive, preserves the morphology and reveals a larger number of positive cells than does HE with the advantage of showing cytoplasmic and nuclear positivity, making it more reliable. Although PCR is more specific and sensitive in fresh or ideally fixed material it is not so in formalin-fixed paraffin-embedded tissues, frequently the only one available in such cases.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs

Canine visceral leishmaniasis (CVL) is recognizable by characteristic signs of disease and is highly lethal. The infection, however, may be quite inapparent in some seropositive dogs, and this has raised the polemic question as to whether or not such animals can be a source of infection for Lutzomyia longipalpis, the vector of American visceral leishmaniasis (AVL). In this study we have examined 51 dogs with acute CVL from an AVL area in Pará State, northern Brazil, and compared the parasite density, amastigotes of Leishmania (L.) infantum chagasi, in the skin, lymph node and viscera of symptomatic with that of nine asymptomatic but seropositive dogs (IFAT-IgG). Post-mortem biopsy fragments of these tissues were processed by immunohistochemistry, using a polyclonal antibody against Leishmania sp. The X² and Mann Whitney tests were used to evaluate the means of infected macrophage density (p < 0.05). There was no difference (p > 0.05) in the skin (10.7/mm² x 15.5/mm²) and lymph node (6.3/mm² x 8.3/mm²), between asymptomatic and symptomatic dogs, respectively. It was higher (p < 0.05), however, in the viscera of symptomatic (5.3/mm²) than it was in asymptomatic (1.4/mm²) dogs. These results strongly suggest that asymptomatic or symptomatic L. (L.) i. chagasi-infected dogs can serve as a source of infection, principally considering the highest (p < 0.05) parasite density from skin (10.7/mm² x 15.5/mm²), the place where the vetor L. longipalpis takes its blood meal, compared with those from lymph node (6.3/mm² x 8.3/mm²) and viscera (1.4/mm²x 5.3/mm²).

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in Northern Brazil

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.

Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus

INTRODUCTION: Antimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined. METHODS: Rifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery. RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05). CONCLUSIONS: In our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

Titanium pigment in tissues of drug addicts: report of 5 necropsied cases

The aim of this report is to describe the anatomic-pathologic findings from necropsies of 5 drug addicts with titanium pigment in several organs after chronic intravenous injection of crushed propoxyphene hydrochloride tablets. Samples from liver, spleen, lungs, lymph nodes, and bone marrow were obtained, and after being grossly studied, they were submitted to evaluation using common light and polarized microscopy. In all 5 cases, a pigment with characteristics of titanium dioxide was found within tissue samples of the organs studied. Our findings suggest that research concerning titanium pigment within body tissues should be enhanced, considering the potential contribution of this morphologic data to forensic pathology.

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.