Vector within-host feeding preference mediates transmission of a heterogeneously distributed pathogen

2. We documented the within-host distribution of two vector species that differ in transmission efficiency, the leafhoppers Draeculacephala minerva and Graphocephala atropunctata, and which are free to move throughout entirely caged alfalfa plants. The more efficient vector D. minerva fed preferentially at the base of the plant near the soil surface, whereas the less efficient G. atropunctata preferred overwhelming the top of the plant. 3. Next we documented X. fastidiosa heterogeneity in mechanically inoculated plants. Infection rates were up to 50% higher and mean bacterial population densities were 100-fold higher near the plant base than at the top or in the taproot. 4. Finally, we estimated transmission efficiency of the two leafhoppers when they were confined at either the base or top of inoculated alfalfa plants. Both vectors were inefficient when confined at the top of infected plants and were 20-60% more efficient when confined at the plant base. 5. These results show that vector transmission efficiency is determined by the interaction between leafhopper within-plant feeding behaviour and pathogen within-plant distribution. Fine-scale vector and pathogen overlap is likely to be a requirement generally for efficient transmission of vector-borne pathogens.

Spatial portability of numerical models of leaf wetness duration based on empirical approaches

Leaf wetness duration (LWD) models based on empirical approaches offer practical advantages over physically based models in agricultural applications, but their spatial portability is questionable because they may be biased to the climatic conditions under which they were developed. In our study, spatial portability of three LWD models with empirical characteristics - a RH threshold model, a decision tree model with wind speed correction, and a fuzzy logic model - was evaluated using weather data collected in Brazil, Canada, Costa Rica, Italy and the USA. The fuzzy logic model was more accurate than the other models in estimating LWD measured by painted leaf wetness sensors. The fraction of correct estimates for the fuzzy logic model was greater (0.87) than for the other models (0.85-0.86) across 28 sites where painted sensors were installed, and the degree of agreement k statistic between the model and painted sensors was greater for the fuzzy logic model (0.71) than that for the other models (0.64-0.66). Values of the k statistic for the fuzzy logic model were also less variable across sites than those of the other models. When model estimates were compared with measurements from unpainted leaf wetness sensors, the fuzzy logic model had less mean absolute error (2.5 h day(-1)) than other models (2.6-2.7 h day(-1)) after the model was calibrated for the unpainted sensors. The results suggest that the fuzzy logic model has greater spatial portability than the other models evaluated and merits further validation in comparison with physical models under a wider range of climate conditions. (C) 2010 Elsevier B.V. All rights reserved.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

Surgical Correction of Aesthetically Deformed Eyebrows Using Local Transposition Flaps

Eyebrow positions differ in many ways. They vary in shape, thickness, length, and distance between the eyebrows, making the face more or less harmonious. When a large distance exists between the eyebrows and the medial brow is slanting downward, the glabellar area is larger, giving the face an awkward appearance. To correct this deformity, the authors propose using two Z-plasties to allow transposition of flaps in the region of the medial brow. The Z-shaped flap is outlined at the medial third of the brow, in the glabellar region, with the eyebrow centered in the lower portion of the ""Z"" and the hairless skin in the upper portion. The flaps then are transposed and sutured. Transposition of the flaps, lifting the brow flap to the glabellar region, results in horizontal positioning of the medial and central third of the eyebrow. The proposed transpositioning of ""Z"" flaps in this region corrects this type of deformity of the medial and central portions of the eyebrows, with an aesthetically satisfactory result.

Myotomy of the Levator Labii Superioris Muscle and Lip Repositioning: A Combined Approach for the Correction of Gummy Smile

Background: Treatment of excessive gingival display usually involves procedures such as Le Fort impaction or maxillary gingivectomies. The authors propose an alternative technique that reduces the muscular function of the elevator of the upper lip muscle and repositioning of the upper lip. Methods: Fourteen female patients with excessive gingival exposure were operated on between February of 2008 and March of 2009. They were filmed before and at least 6 months after the procedure. They were asked to perform their fullest smile, and the maximum gingival exposures were measured and analyzed using ImageJ software. Patients were operated on under local anesthesia. Their gingival mucosa was freed from the maxilla using a periosteum elevator. Skin and subcutaneous tissue were dissected bluntly from the underlying musculature of the upper lip. A frenuloplasty was performed to lengthen the upper lip. Both levator labii superioris muscles were dissected and divided. Results: The postoperative course was uneventful in all of the patients. The mean gingival exposure before surgery was 5.22 +/- 1.48 mm; 6 months after surgery, it was 1.91 +/- 1.50 mm. The mean gingival exposure reduction was 3.31 +/- 1.05 mm (p < 0.001), ranging from 1.59 to 4.83 mm. Conclusion: This study shows that the proposed technique was efficient in reducing the amount of exposed gum during smile in all patients in this series. (Plast. Reconstr. Surg. 126: 1014, 2010.)

Surgical correction of Tessier number 0 cleft

The no. 0-14 cleft involves the midline of the face and cranium. It may include both a true and a false median cleft lip, with or without associated hypotelorism or hypertelorism. The no. 0 cleft is the most common of the craniofacial clefts. The objective of this study was to review the functional outcome and aesthetic results of the different techniques applied for each case. We have conducted a retrospective analysis of our series consisting of 32 cases of Tessier no. 0 cleft, in the period between 1997 and 2007. The patients were divided into 2 groups: those with the true median cleft and those with the false median cleft. The clinical findings, lip malformation, alveolar cleft, nasal appearance, septal involvement, associated deformities, and surgical procedures, were all reviewed. Holoprosencephaly was present in 9 cases, with a false median cleft upper lip and an absence of the premaxilla, septum, and columella (only 1 patient underwent lip and columella reconstruction at 2 years of age). Nine patients had an incomplete median cleft lip. Seven of these cases had associated median alveolar cleft, and 1 had an intranasal tumor, associated with lipoma of corpus callosum, characteristic of the Pai syndrome. Six cases of a bifid nose were seen, 2 of which were associated with an alveolar median cleft and hypertelorism. An isolated median alveolar cleft was present in 7 cases, 2 of them associated with a no. 30 cleft. This article presents a large series of Tessier no. 0 cleft, describing the differences between the false and the true median cleft. The surgical procedures may vary in relation to the type of involvement.

Endostatin- and interleukin-2-expressing retroviral bicistronic vector for gene therapy of metastatic renal cell carcinoma

Background Metastatic renal cell carcinoma (mRCC) is one of the most treatment-resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)-2 using an orthotopic metastatic RCC mouse model. Methods Balb/C-bearing Renca cells were treated with NIH/3T3-LendIRES-IL-2-SN cells. In the survival studies, mice were monitored daily until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and the number of metastatic nodules, nodule area, tumor vessels and proliferation of tumor-infiltrating Renca cells were determined. Results Inoculation of NIH/3T3-LendIRES-IL-2-SN cells resulted in an increase in ES and IL-2 levels in the treated group (p < 0.05). There was a significant decrease in lung wet weight, lung nodule area and tumor vessels in the treated group compared to the control group (p < 0.001). The proliferation of Renca cells in the bicistronic-treated group was significantly reduced compared to the control group (p < 0.05). Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice submitted to bicistronic therapy (log-rank test, p = 0.0016). Bicistronic therapy caused an increase in the infiltration of CD4, CD4 interferon (IFN)gamma-producing, CD8, CD8 IFN gamma-producing and natural killer (CD49b) cells. Conclusions Retroviral bicistronic gene transfer led to the secretion of functional ES and IL-2 that was sufficiently active to: (i) inhibit tumor angiogenesis and tumor cell proliferation and (ii) increase the infiltration of immune cells (C) Copyright. 2011 John Wiley & Sons, Ltd.

An fMRI Normative Database for Connectivity Networks Using One-Class Support Vector Machines

The application of functional magnetic resonance imaging (fMRI) in neuroscience studies has increased enormously in the last decade. Although primarily used to map brain regions activated by specific stimuli, many studies have shown that fMRI can also be useful in identifying interactions between brain regions (functional and effective connectivity). Despite the widespread use of fMRI as a research tool, clinical applications of brain connectivity as studied by fMRI are not well established. One possible explanation is the lack of normal pattern, and intersubject variability-two variables that are still largely uncharacterized in most patient populations of interest. In the current study, we combine the identification of functional connectivity networks extracted by using Spearman partial correlation with the use of a one-class support vector machine in order construct a normative database. An application of this approach is illustrated using an fMRI dataset of 43 healthy Subjects performing a visual working memory task. In addition, the relationships between the results obtained and behavioral data are explored. Hum Brain Mapp 30:1068-1076, 2009. (C) 2008 Wiley-Liss. Inc.

The impact of functional connectivity changes on support vector machines mapping of fMRI data

Functional magnetic resonance imaging (fMRI) is currently one of the most widely used methods for studying human brain function in vivo. Although many different approaches to fMRI analysis are available, the most widely used methods employ so called ""mass-univariate"" modeling of responses in a voxel-by-voxel fashion to construct activation maps. However, it is well known that many brain processes involve networks of interacting regions and for this reason multivariate analyses might seem to be attractive alternatives to univariate approaches. The current paper focuses on one multivariate application of statistical learning theory: the statistical discrimination maps (SDM) based on support vector machine, and seeks to establish some possible interpretations when the results differ from univariate `approaches. In fact, when there are changes not only on the activation level of two conditions but also on functional connectivity, SDM seems more informative. We addressed this question using both simulations and applications to real data. We have shown that the combined use of univariate approaches and SDM yields significant new insights into brain activations not available using univariate methods alone. In the application to a visual working memory fMRI data, we demonstrated that the interaction among brain regions play a role in SDM`s power to detect discriminative voxels. (C) 2008 Elsevier B.V. All rights reserved.

A hypothesis for explaining single outbreaks (like the Black Death in European cities) of vector-borne infections

We propose a mechanism by which single outbreaks of vector-borne infections can happen even when the value of the basic reproduction number, R(o), of the infection is below one. With this hypothesis we have shown that dynamical models simulations demonstrate that the arrival of a relatively small (with respect to the host population) number of infected vectors can trigger a short-lived epidemic but with a huge number of cases. These episodes are characterized by a sudden outbreak in a previously virgin area that last from weeks to a few months, and then disappear without leaving vestiges. The hypothesis proposed in this paper to explain those single outbreaks of vector-borne infections, even when total basic reproduction number, Ro, is less than one (which explain the fact that those infections fail to establish themselves at endemic levels), is that the vector-to-host component of Ro is greater than one and that a sufficient amount of infected vectors are imported to the vulnerable area, triggering the outbreak. We tested the hypothesis by performing numerical simulations that reproduce the observed outbreaks of chikungunya in Italy in 2007 and the plague in Florence in 1348. The theory proposed provides an explanation for isolated outbreaks of vector-borne infections, ways to calculate the size of those outbreaks from the number of infected vectors arriving in the affected areas. Given the ever-increasing worldwide transportation network, providing a high degree of mobility from endemic to virgin areas, the proposed mechanism may have important implications for public health planning. (C) 2009 Elsevier Ltd. All rights reserved.

Age-Related Metabolic Profiles in Cognitively Healthy Elders: Results from a Voxel-Based [(18)F]Fluorodeoxyglucose-Positron-Emission Tomography Study with Partial Volume Effects Correction

BACKGROUND AND PURPOSE: Functional brain variability has been scarcely investigated in cognitively healthy elderly subjects, and it is currently debated whether previous findings of regional metabolic variability are artifacts associated with brain atrophy. The primary purpose of this study was to test whether there is regional cerebral age-related hypometabolism specifically in later stages of life. MATERIALS AND METHODS: MR imaging and FDG-PET data were acquired from 55 cognitively healthy elderly subjects, and voxel-based linear correlations between age and GM volume or regional cerebral metabolism were conducted by using SPM5 in images with and without correction for PVE. To investigate sex-specific differences in the pattern of brain aging, we repeated the above voxelwise calculations after dividing our sample by sex. RESULTS: Our analysis revealed 2 large clusters of age-related metabolic decrease in the overall sample, 1 in the left orbitofrontal cortex and the other in the right temporolimbic region, encompassing the hippocampus, the parahippocampal gyrus, and the amygdala. The division of our sample by sex revealed significant sex-specific age-related metabolic decrease in the left temporolimbic region of men and in the left dorsolateral frontal cortex of women. When we applied atrophy correction to our PET data, none of the above-mentioned correlations remained significant. CONCLUSIONS: Our findings suggest that age-related functional brain variability in cognitively healthy elderly individuals is largely secondary to the degree of regional brain atrophy, and the findings provide support to the notion that appropriate PVE correction is a key tool in neuroimaging investigations.

Pleural fluid: Are temperature and storage time critical preanalytical error factors in biochemical analyses?

Background: Biochemical analysis of fluid is the primary laboratory approach hi pleural effusion diagnosis. Standardization of the steps between collection and laboratorial analyses are fundamental to maintain the quality of the results. We evaluated the influence of temperature and storage time on sample stability. Methods: Pleural fluid from 30 patients was submitted to analyses of proteins, albumin, lactic dehydrogenase (LDH), cholesterol, triglycerides, and glucose. Aliquots were stored at 21 degrees, 4 degrees, and-20 degrees C, and concentrations were determined after 1, 2, 3, 4, 7, and 14 days. LDH isoenzymes were quantified in 7 random samples. Results: Due to the instability of isoenzymes 4 and 5, a decrease in LDH was observed in the first 24 h in samples maintained at -20 degrees C and after 2 days when maintained at 4 degrees C. Aside from glucose, all parameters were stable for up to at least day 4 when stored at room temperature or 4 degrees C. Conclusions: Temperature and storage time are potential preanalytical errors in pleural fluid analyses, mainly if we consider the instability of glucose and LDH. The ideal procedure is to execute all the tests immediately after collection. However, most of the tests can be done in refrigerated sample;, excepting LDH analysis. (C) 2010 Elsevier B.V. All rights reserved.

Neonatal BCG Immunization Followed by DNAhsp65 Boosters: Highly Immunogenic but not Protective Against Tuberculosis - a Paradoxical Effect of the Vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Ultrasonographic Evaluation of Achilles Tendon Repair After Percutaneous Sectioning for the Correction of Congenital Clubfoot Residual Equinus

Background: Most cases of congenital clubfoot treated with the Ponseti technique require percutaneous Achilles tenotomy to correct the residual equinus. Clinical evidence suggests that complete healing occurs between the cut tendon stumps, but there have not yet been any detailed studies investigating this reparative process. This study was performed to assess Achilles tendon repair after percutaneous section to correct the residual equinus of clubfoot treated with the Ponseti method. Method: A prospective study analyzed 37 tenotomies in 26 patients with congenital clubfoot treated with the Ponseti technique, with a minimum follow-up of 1 year after the section. The tenotomy was performed percutaneously with a large-bore needle bevel with patient sedation and local anesthesia. Ultrasonographic scanning was performed after section to ascertain that the tenotomy had been completed and to measure the stump separation. In the follow-up period, the reparative process was followed ultrasonographically and assessed at 3 weeks, 6 months, and 1 year posttenotomy. Results: The ultrasonography performed immediately after the procedure showed that in some cases, residual strands between the tendon ends persisted, and these were completely sectioned under ultrasound control. A mean retraction of 5.65 mm +/- 2.26 mm (range, 2.3 to 11.0 mm) between tendon stumps after section was observed. Unusual bleeding occurred in one case and was controlled by digital pressure, with no interference with the final treatment. After 3 weeks, ultrasonography showed tendon repair with the tendon gap filled with irregular hypoechoic tissue, and also with transmission of muscle motion to the heel. Six months after tenotomy, there was structural filling with a fibrillar aspect, mild or moderate hypoechogenicity, and tendon scar thickening when compared with a normal tendon. One year after tenotomy, ultrasound showed a fibrillar structure and echogenicity at the repair site that was similar to a normal tendon, but with persistent tendon scarring thickness. Conclusions: There is a fast reparative process after Achilles tendon percutaneous section that reestablishes continuity between stumps. The reparative tissue evolved to tendon tissue with a normal ultrasonographic appearance except for mild thickening, suggesting a predominantly intrinsic repair mechanism.