Biological and molecular characterization of two Anticarsia gemmatalis multiple nucleopolyhedrovirus clones exhibiting contrasting virulence.

Baculovirus natural populations are known to be genetically heterogeneous and such genotypic diversity could have implications in the performance of biocontrol agents. The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) has been widely used to control the velvetbean caterpillar, Anticarsia gemmatalis, in Brazil. In the present work, morphological and molecular analyses as well as the biological activity of AgMNPV genotypes derived from a Brazilian field isolate (AgMNPV-79) were carried out. The existence of genotypic variants in the population was confirmed by DNA restriction analysis. Although difference in virulence was observed among the variants, the most (Ag79-01) and the least (AgL-16) virulent clones do not show any morphological and cytopathological changes when compared to the most studied isolate (AgMNPV-2D). The complete genome analysis of the two viral clones showed the presence of single open reading frames (ORFs) of the pe-38 and he65 genes, which contrasts with the two split ORFs present in the genome of the AgMNPV-2D isolate. The viral clone AgL-16 has many variations in the ie-2 and pe-38 genes, which are transcription regulatory genes responsible for the regulation of viral early gene expression during insect cell infection. Furthermore, other genes showed alterations like the odv-e56, which have an essential role in the maturation and envelopment of the ODVs, and bro-a and bro-b genes which were fused to form a single ORF. For the Ag79-01, although the total number of single nucleotide variants (SNVs) was more prominent in the pe-38 gene, its genome showed very few modifications in comparison to the AgMNPV-2D genome.

Genome-wide diversity in temporal and regional populations of the betabaculovirus Erinnyis ello granulovirus (ErelGV).

BACKGROUND: Erinnyis ello granulovirus (ErelGV) is a betabaculovirus infecting caterpillars of the sphingid moth E. ello ello (cassava hornworm), an important pest of cassava crops (Manihot esculenta). In this study, the genome of seven field isolates of the virus ErelGV were deep sequenced and their inter- and intrapopulational sequence diversity were analyzed. RESULTS: No events of gene gain/loss or translocations were observed, and indels were mainly found within highly repetitive regions (direct repeats, drs). A naturally occurring isolate from Northern Brazil (Acre State, an Amazonian region) has shown to be the most diverse population, with a unique pattern of polymorphisms. Overall, non-synonymous substitutions were found all over the seven genomes, with no specific gathering of mutations on hotspot regions. Independently of their sizes, some ORFs have shown higher levels of non-synonymous changes than others. Non-core genes of known functions and structural genes were among the most diverse ones; and as expected, core genes were the least variable genes. We observed remarkable differences on diversity of paralogous genes, as in multiple copies of p10, fgf, and pep. Another important contrast on sequence diversity was found on genes encoding complex subunits and/or involved in the same biological processes, as late expression factors (lefs) and per os infectivity factors (pifs). Interestingly, several polymorphisms in coding regions lie on sequences encoding specific protein domains. CONCLUSIONS: By comparing and integrating information about inter- and intrapopulational diversity of viral isolates, we provide a detailed description on how evolution operates on field isolates of a betabaculovirus. Our results revealed that 35-41% of the SNPs of ErelGV lead to amino acid changes (non-synonymous substitutions). Some genes, especially non-core genes of unknown functions, tend to accumulate more mutations, while core genes evolve slowly and are more conserved. Additional studies would be necessary to understand the actual effects of such gene variations on viral infection and fitness.

Giant placental chorioangioma with favorable outcome: a case report and literature review of literature.

OBJECTIVE: To describe a case of prenatal diagnosis of a giant placental chorioangioma with favorable outcome. DESIGN: A case report. SETTING: Gynecology and Obstetrics Service, Federal University of Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. CASE REPORT: The placental chorioangioma is the most common benign tumor, but the type giant has a small prevalence, ranging from 1:16.000 to 1:50.000 pregnancies. We reported a case of a patient aged 18, pregnant for the first time, who performed a routine obstetric ultrasound was found to have polyhydramnios associated with placental vascular lesions suggestive of chorioangioma also was defined by fetal magnetic resonance imaging and confirmed by pathological examination.

A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining.

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

Successful co-infection of two different baculovirus species in the same cell line reveals a potential strategy for large in vitro production.

Baculoviruses have been applied for biocontrol of agricultural pests, such as velvetbean caterpillar (Anticarsia gemmatalis) and fall armyworm (Spodoptera frugiperda). Cell culture is an interesting approach for large-scale production of these viruses. Co-infection of a host cell with two distinct viruses can contribute to reduce costs due to saving cell culture media, bioreactor space and the resulting co-occluded polyhedra may help to reduce final biopesticide costs. The baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were chosen to test a model for in vitro co-infection in SF21 cells. Different proportions of SfMNPV/AgMNPV were evaluated along three in vitro passages by optical microscopy analysis of cells and real-time PCR (qPCR) of DNA obtained from budded viruses (BVs) and occlusion bodies (OBs). The kinetics of viral protein synthesis was carried out for analysis of the co-infection in first passage and bioassays with the resulting OBs were performed against A. gemmatalis and S. frugiperda larvae. The results demonstrated successful co-infection in these cells. The quantity of SfMNPV and AgMNPV in supernatants and sediments tends to be maintained stable during the three passages, although the amount of AgMNPV was higher than SfMPNV in most of the experiments. Analysis of the kinetics of radiolabed proteins showed that the cell protein synthesis was shut off and two distinct bands of about 30 kDa, regarded to be the polyhedrin of each virus, were strongly detected at 48 and 72 hp.i. Although the pathogenicity of the produced viruses was not completely satisfactory, the bioassays confirmed occurrence of co-infected larvae with disproportional amount of each virus.

Leaf trait variation during ontogeny in the endangered Brazilian rosewood tree.

Knowledge of plant responses to environmental heterogeneity during ontogeny is important to elucidate the changes that occur to promote resource capture in tropical forests. We tested the hypothesis that expression changes in leaf metamer traits of Brazilian rosewood (Dalbergia nigra), from seedlings to emergent canopy trees, occur as new microclimate environments are achieved. We also tested the hypothesis that increased light heterogeneity in the understorey leads to higher plasticity in leaf traits of seedlings and saplings than in sun-exposed metamers of emergent trees subject to stressful conditions. We compared leaf metamer traits of 53 individuals including seedlings, saplings and emergent trees. We also evaluated the light heterogeneity in vertical strata and the variations in leaf traits within individuals (among metamers of the same individual). These were associated with height of the individuals. Compared to understorey plants, emergent trees presented larger metamers, with lower specific leaf area (SLA), lower investment in leaf area per total dry mass of metamer (LARm ), lower specific petiole length (SPL) and lower specific internode length (SIL). Higher phenotypic variation within individuals was observed in seedlings, which decreased as the trees grew taller. The results suggest the integration of ontogenetic changes in leaf traits under new microclimate conditions as the plants reach different vertical strata in the forest. Additionally, our results support the hypothesis that increased light heterogeneity in the understorey shaped higher phenotypic variation within individuals in juveniles and that stressful conditions in sun-exposed leaf metamers of emergent trees led to increased phenotypic stability.

A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

Amifostine and glutathione prevent ifosfamide- and acrolein-induced hemorrhagic cystitis.

INTRODUCTION: Ifosfamide (IFS) is an antineoplastic alkylating agent whose major side effect is hemorrhagic cystitis (HC). This toxicity is attributed to the renal excretion of acrolein (ACR), a highly urotoxic IFS metabolite. Despite the clinical use of mesna to prevent HC, a significant percent ( approximately 33%) of patients present with at last one feature of HC, mainly hematuria. AIM: To investigate the use of two antioxidants-amifostine and glutathione-for the prevention of experimental IFS- and ACR-induced HC. MATERIALS AND METHODS: Male Swiss mice were treated intraperitoneal (i.p.) with saline (control), glutathione (125, 250 or 500 mg/kg) or amifostine (25, 50 or 100 mg/kg), and 30 min later they received a single i.p. injection of IFS at a dose of 400 mg/kg. To investigate the systemic effects of the antioxidants on ACR-induced HC, the animals were treated with saline, amifostine (50 mg/kg, i.p.) or glutathione (500 mg/kg, i.p.), and 30 min afterward with 75 mug ACR intravesically (i.ve.). In another set of experiments, the antioxidants were injected directly into the bladder, where the mice received a single i.ve injection of ACR (75 mug) plus amifostine (1.5 mg/kg) or glutathione (2 mg/kg). HC was measured 3 h after IFS or ACR injection according to bladder wet weight, macroscopic (edema and hemorrhage) and microscopic changes, i.e., edema, hemorrhage, cellular infiltration, fibrin deposition and urothelial desquamation. RESULTS: Pretreatments with amifostine or glutathione prevented IFS-induced HC in a dose-dependent manner. Furthermore, ACR-induced HC was also prevented by systemic (i.p.) or local (i.ve.) pretreatment with glutathione or amifostine. The greatest protective effect was seen with local amifostine treatment (2 mg/kg i.ve.) (P < 0.05). CONCLUSIONS: Glutathione and amifostine show a beneficial effect in experimental IFS- and ACR-induced HC. Thus, they should be investigated as an alternative treatment to prevent HC observed in patients undergoing IFS treatment.

Responses of Chironomidae (Diptera; Insecta) to the exclusion of shrimps and Ephemeroptera in a coastal forest stream, Rio de Janeiro, Brazil.

In a benthic community on a continuous flat granite substrate in a third-order coastal forest stream, the dominant chironomid (Cricotopus) increased in number when shrimps (Macrobrachium olfersi and Potimirim glabra) and baetid ephemeropterans were excluded by electricity. The response appeared to be mediated by an increase in periphyton and sediments, rather than a reduction of direct predation or interference. Chironomids, periphyton and sediments decreased significantly compared to the control when shrimps only were excluded. Baetid ephemeropteran appeared to be the most important determinants of periphyton and sediment mass; the density of chironomids appeared to follow the quantity of periphyton and sediments.

A model of hemorrhagic cystitis induced with acrolein in mice.

Acrolein is a urinary metabolite of cyclophosphamide and ifosfamide, which has been reported to be the causative agent of hemorrhagic cystitis induced by these compounds. A direct cytotoxic effect of acrolein, however, has not yet been demonstrated. In the present study, the effects of intravesical injection of acrolein and mesna, the classical acrolein chemical inhibitor, were evaluated. Male Swiss mice weighing 25 to 35 g (N = 6 per group) received saline or acrolein (25, 75, 225 microg) intravesically 3, 6, 12, and 24 h before sacrifice for evaluation of bladder wet weight, macroscopic and histopathological changes by Gray's criteria, and 3 and 24 h for assessment of increase in vascular permeability. In other animals, mesna was administered intravesically (2 mg) or systemically (80 mg/kg) 1 h before acrolein. Intravesical administration of acrolein induced a dose- and time-dependent increase in vascular permeability and bladder wet weight (within 3 h: 2.2- and 21-fold increases in bladder wet weight and Evans blue dye exuded, respectively, at doses of 75 microg/bladder), as confirmed by Gray's criteria. Pretreatment with mesna (2-mercaptoethanesulfonic acid), which interacts with acrolein resulting in an inactive compound, inhibited all changes induced by acrolein. Our results are the first demonstration that intravesical administration of acrolein induces hemorrhagic cystitis. This model of acrolein-induced hemorrhagic cystitis in mice may be an important tool for the evaluation of the mechanism by which acrolein induces bladder lesion, as well as for investigation of new uroprotective drugs.

A model validation and consensus building environment.

Over half of the failures in drug development are due to problems with the absorption, distribution, metabolism, excretion, and toxicity, or ADME/Tox properties of a candidate compound. The utilization of in silico tools to predict ADME/Tox and physicochemical properties holds great potential for reducing the attrition rate in drug research and development, as this technology can prioritize candidate compounds in the pharmaceutical R&D pipeline. However, a major concern surrounding the use of in silico ADME/Tox technology is the reliability of the property predictions. Bio-Rad Laboratories, Inc. has created a computational environment that addresses these concerns. This environment is referred to as KnowItAll. Within this platform are encoded a number of ADME/Tox predictors, the ability to validate these predictors with/without in-house data and models, as well as build a 'consensus' model that may be a much better model than any of the individual predictive model. The KnowItAll system can handle two types of predictions: real number and categorical classification.

Is the combination of pyrazinamide plus rifampicin safe for treating latent tuberculosis infection in persons not infected by the human immunodeficiency virus?

SETTING: Nine public health care centres in four Spanish cities. OBJECTIVE: To evaluate the efficacy and safety of 2 months of rifampicin (R) plus pyrazinamide (Z) therapy (2RZ) compared with a 6-month course of isoniazid therapy (6H) for treating latent tuberculosis infection (LTBI). DESIGN: Multicentered, randomised, comparative and prospective trial conducted in HIV-seronegative contacts of infectious pulmonary TB cases. RESULTS: Of 352 individuals, 199 received 6H and 153 2RZ; 73% of contacts receiving 6H and 71% receiving 2RZ completed treatment (P = 0.73). Treatment interruption due to hepatotoxicity (ALT/AST > 5 times upper limit of normal) was observed in 10% of contacts in the 2RZ group and in 2.5% of the 6H group (P = 0.007). This higher than expected rate of hepatotoxicity in the 2RZ arm led to premature termination of the study. Severe or fatal liver injury was not detected. Liver function tests normalised after discontinuation of treatment. We conclude that the use of RZ should only be considered when other regimens are unsuitable and intensive monitoring of liver function is feasible.

Bleeding reduction after topical application of tranexamic acid together with Betadine solution in total knee arthroplasty. A randomised controlled study.

INTRODUCTION: Topical application of tranexamic acid to the knee joint before closure in total knee arthroplasty reduces postoperative bleeding without increase in complication. However, it is unknown the effectiveness of topic TXA performed with other topical medications, like povidone-iodine solution. MATERIALS AND METHODS: One hundred and twenty-five patients were randomized to receive 100mL of povidone-iodine solution (control: group A) or 1.5 (group B) and 3.0 g (group C) of topical TXA in povidone-iodine solution applied into the knee before closure in total knee arthroplasty. RESULTS: The patients in the TXA groups had higher mean postoperative hemoglobin levels (P=0.01 and P=0.03 in groups B and C, respectively) and a reduced postoperative blood loss in the TXA groups (P=0.07 and P=0.09 in groups B and C, respectively). No significant complications were observed. DISCUSSION: In this study, topical application of tranexamic acid after total knee arthroplasty together with povidone-iodine solution results in higher postoperative hemoglobin levels and lower blood loss compared with those in the control group without other complications. LEVEL OF EVIDENCE: I - I: high-powered prospective randomized trial.

The Bradyrhizobium japonicum noeD gene: a negatively acting, genotype-specific nodulation gene for soybean.

Bradyrhizobium japonicum strain USDA 110 is restricted for nodulation by soybean genotype PI 417566. We previously reported the identification of a USDA 110 Tn5 mutant, strain D4.2-5, that had the ability to overcome nodulation restriction conditioned by PI 417566 (S. M. Lohrke, J. H. Orf, E. Martínez-Romero, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:2378-2383, 1995). In this study, we report the cloning and characterization of the negatively acting DNA region mutated in strain D4.2-5 that is involved in the genotype-specific nodulation of soybean. The Tn5 integration site was localized to a 5.2-kb EcoRI fragment isolated from wild-type USDA 110 genomic DNA. Saturation Tn5 mutagenesis of this 5.2-kb region and DNA homogenitization studies indicated that a 0.9-kb DNA region was involved in the genotype-specific nodulation of PI 417566. A single open reading frame (ORF) of 474 nucleotides, encoding a predicted protein of 158 amino acids, was identified within this region by DNA sequencing. This ORF was named noeD. Computer comparisons with available data bases revealed no significant similarities between the noeD DNA or predicted amino acid sequence and any known genes or their products. However, comparisons done with the region upstream of noeD revealed a high degree of similarity (about 76% similarity and 62% identity) to the N-terminal regions of the Rhizobium leguminosarum bv. viciae and R. meliloti nodM genes, which have been postulated to encode a glucosamine synthase. Southern hybridization analysis indicated that noeD is not closely linked to the main or auxiliary nodulation gene clusters in B. japonicum and that both nodulation-restricted and -unrestricted B. japonicum serogroup 110 strains contain a noeD homolog. High-performance liquid chromatography and fast atom bombardment-mass spectrometry analyses of the lipo-chitin oligosaccharide (LCO) nodulation signals produced by an noeD mutant showed a higher level of acetylation than that found with wild-type USDA 110. These results suggest that specific LCO signal molecules may be one of the factors influencing nodulation specificity in this symbiotic system.

Ethanol induces hyper and hypoglycemia in both fasted and nonfasted rats dependent on the ambient temperature.

An experiment was undertaken to characterize the influence of ambient temperature on ethanol-induced glycemic alterations in rats. Animals under two different feeding conditions (nonfasted or 48-hr fasted) were IP injected with 4.0 g/kg of ethanol. Blood glucose and body temperature were measured before, 90 and 180 min after drug administration. The rats were tested under ambient temperatures of 16, 21 and 32 degrees C. Fed animals with a mean pre-drug glycemia of near 105 mg/100 ml presented a variation of blood glucose ranging from 50 mg/100 ml at 16 degrees C to 140 mg/100 ml at 32 degrees C. The glycemia from fasted rats, with a starting value of 70 mg/100 ml ranged from 20 to 115 mg/100 ml at 16 and 32 degrees C, respectively. It was concluded that the administration of ethanol can render nonfasted as well as fasted rats hypo or hyperglycemic, depending upon the environmental temperature.

Does hypothermia play a relevant role in the glycemic alterations induced by ethanol?

The potential of ethanol-induced hypothermia on the glycemic alterations induced by this drug were evaluated. In Experiment 1 ambient temperature was manipulated. After 4.0 g/kg of ethanol blood glucose levels and body temperature were assessed in fed or 48 hr starved rats at either 21 degrees C or 28 degrees C room temperature. Hyper or hypoglycemia was observed depending on both the feeding condition and the environmental temperature. In Experiment 2, this hypothesis was tested by determining if rats tolerant to the thermic effects of ethanol would show a decreased glycemic response. The results support this assumption.

Blood glucose and body temperature alterations induced by ethanol in rats submitted to different levels of food deprivation.

The effects of 1.0, 3.0 and 5.0 g/kg of ethanol on blood glucose levels and body temperature were examined in rats submitted to either acute food deprivation (24 or 48 hr), chronic starvation, or to both chronic plus acute food deprivation. The results show that: (a) 3.0 and 5.0 g/kg produced either an increase or a decrease of glucose levels depending on the state of fasting; (b) rats not deprived of food presented hyperglycemia while being hypothermic; (c) a marked hypothermia was present when no substantial alterations in glycemia were observed; and (d) in cases where hypoglycemia and hypothermia occurred, the fall in body temperature paralleled or preceded the decrease in glucose levels.

Hypoglycemia and hypothermia induced by ethanol: antagonism by indomethacin.

The influence of pretreatment with 5.0 or 10.0 mg/kg of indomethacin, a prostaglandin synthetase inhibitor, on the alterations in body temperature produced by 3.0 and 4.0 g/kg of ethanol, was studied in food-deprived and free-feeding rats. A partial antagonism of ethanol's hypothermic effect resulted from indomethacin pretreatments and this effect was found to be ethanol dose-dependent. This result could account for the conflicting reports in the literature on the effectiveness of indomethacin in antagonizing ethanol-induced hypothermia. Indomethacin (5.0 mg/kg) also antagonized ethanol-induced hypoglycemia in 48 hr starved rats. The relationship between two effects of ethanol, hypothermia and hypoglycemia, is discussed.

The excitatory effect of ethanol: absence in rats, no tolerance and increased sensitivity in mice.

Three questions related to ethanol's stimulating effect (ESE) were studied. The first referred to the reported absence of tolerance to ESE in mice. It was determined whether tolerance would develop if the period of ethanol treatment were extended significantly beyond those normally found in the literature. No evidence of tolerance to ESE was found over a 5-month period of treatment. The second issue related to the possibility that mice not only do not develop tolerance but actually become more responsive to ESE after chronic exposure. A dose of ethanol that acutely did not produce a significant activating effect did induce a marked excitation after the animals were chronically treated with ethanol. Finally, the issue was addressed of whether the absence of ESE in some strains of rats could in part be due to a masking effect by the depressant component of this drug. To test this possibility rats were treated with ethanol for a 4-month period. Tolerance to the depressant effect was observed but no ESE was detected.

Lack of effect of the benzodiazepine receptor antagonist Ro 15-1788 on ethanol-induced intoxication in mice.

The effects of 3, 6 and 48 mg/kg of the benzodiazepine receptor antagonist Ro 15-1788 on the ethanol-induced depressant action were evaluated in mice. These results support and extend previous findings in experimental animals and show that Ro 15-1788 in doses devoid of intrinsic effects, does not antagonize the motor impairment, hypnotic effect or lethality induced by ethanol.