Newcastle disease virus (NDV) was isolated from an outbreak in layer chickens in the Dominican Republic in 2008. Infections with this isolate led to a 100% apparent case fatality rate in birds. Complete genome sequencing revealed that the isolate does not belong to any of the previously described NDV genotypes. Similarly, large differences were observed in the amino acid sequence of the fusion and hemagglutinin-neuraminidase proteins in comparison with all known NDV genotypes, suggesting the existence of an unknown reservoir for NDV. The work presented here represents the first complete genome sequence of NDV in the Dominican Republic.

Many hypotheses have been proposed to explain high species diversity in Amazonia, but few generalizations have emerged. In part, this has arisen from the scarcity of rigorous tests for mechanisms promoting speciation, and from major uncertainties about palaeogeographic events and their spatial and temporal associations with diversification. Here, we investigate the environmental history of Amazonia using a phylogenetic and biogeographic analysis of trumpeters (Aves: Psophia), which are represented by species in each of the vertebrate areas of endemism. Their relationships reveal an unforeseen ‘complete’ time-slice of Amazonian diversification over the past 3.0 Myr. We employ this temporally calibrated phylogeny to test competing palaeogeographic hypotheses. Our results are consistent with the establishment of the current Amazonian drainage system at approximately 3.0–2.0 Ma and predict the temporal pattern of major river formation over Plio-Pleistocene times. We propose a palaeobiogeographic model for the last 3.0 Myr of Amazonian history that has implications for understanding patterns of endemism, the temporal history of Amazonian diversification and mechanisms promoting speciation. The history of Psophia, in combination with new geological evidence, provides the strongest direct evidence supporting a role for river dynamics in Amazonian diversification, and the absence of such a role for glacial climate cycles and refugia.

Five Newcastle disease virus strains isolated from geese were classified into a new genotype, designated genotype XII. The complete genome sequences of two strains indicated that these viruses were distinct from viruses of genotype VII. More investigations need to be conducted for us to understand the origin of these new strains.

T2*-weighted gradient-echo MRI images at high field (≥ 7 Tesla) have shown rich image contrast within and between brain regions. The source for these contrast variations has been primarily attributed to tissue magnetic susceptibility differences. In this study, the contribution of myelin to both T2* and frequency contrasts is investigated using a mouse model of demyelination based on a cuprizone diet. The demyelinated brains showed significantly increased T2* in white matter and a substantial reduction in gray-white matter frequency contrast, suggesting that myelin is a primary source for these contrasts. Comparison of in-vivo and in-vitro data showed that, although tissue T2* values were reduced by formalin fixation, gray-white matter frequency contrast was relatively unaffected and fixation had a negligible effect on cuprizone-induced changes in T2* and frequency contrasts.

SUMMARY

An article by Hook et al. (2012) provides insight into the mechanisms confining membrane protrusions to the front of migrating neutrophils. The authors rule out a role for diffusion of inhibitory signals and show that membrane tension is a necessary and sufficient means to restrict signals that lead to protrusions.

Background

Phenolic compounds combine antioxidant and hypocholesterolemic activities and, consequently, are expected to prevent or minimize cardiometabolic risk.

Methods

To evaluate the effect of an aqueous extract (AQ) and non-esterified phenolic fraction (NEPF) from rosemary on oxidative stress in diet-induced hypercholesterolemia, 48 male 4-week old Wistar rats were divided into 6 groups: 1 chow diet group (C) and 5 hypercholesterolemic diet groups, with 1 receiving water (HC), 2 receiving AQ at concentrations of 7 and 140 mg/kg body weight (AQ70 and AQ140, respectively), and 2 receiving NEPF at concentrations of 7 and 14 mg/kg body weight (NEPF7 and NEPF14, respectively) by gavage for 4 weeks.

Results

In vitro, both AQ and NEPF had remarkable antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH●) assay, which was similar to BHT. In vivo, the group that received AQ at 70 mg/kg body weight had lower serum total cholesterol (−39.8%), non-HDL-c (−44.4%) and thiobarbituric acid reactive substance (TBARS) levels (−37.7%) compared with the HC group. NEPF (7 and 14 mg/kg) reduced the tissue TBARS levels and increased the activity of tissular antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). Neither AQ nor NEPF was able to ameliorate the alterations in the hypercholesterolemic diet-induced fatty acid composition in the liver.

Conclusions

These data suggest that phenolic compounds from rosemary ameliorate the antioxidant defense in different tissues and attenuate oxidative stress in diet-induced hypercholesterolemic rats, whereas the serum lipid profile was improved only in rats that received the aqueous extract.

Abstract

Introduction

We sought to evaluate the ability of various metabolic syndrome definitions in predicting primary cardiovascular disease (CVD) outcomes in a vast multiethnic U.S. cohort.

Methods

This study included 6,814 self-identified men and women aged 45–84 years enrolled in the Multi-Ethnic Study of Atherosclerosis (MESA) study. Gender-stratified analyses were performed to calculate hazard ratios of CVD, stroke, and mortality associated with various metabolic syndrome definitions and their individual constructs.

Results

The hazard ratios [95% confidence interval (CI)] for all-cause CVD in men were 2.90 (2.18–3.85), 2.64 (1.98–3.51), 2.16 (1.62–2.88), 2.56 (1.91–3.44), 1.82 (1.35–2.46), and 2.92 (2.15–3.95) for the National Cholesterol Education Program (NCEP), American Heart Association (AHA), World Health Organization (WHO), International Diabetes Federation (IDF), European Group for the Study of Insulin Resistance (EGIR), and the newly defined consensus criteria. Hazard ratios in women were 2.11 (1.41–3.15), 2.17 (1.45–3.27), 2.04 (1.37–3.06), 1.91 (1.27–2.88), 1.85 (1.23–2.79), and 2.08 (1.37–3.14), respectively. Metabolic syndrome was strongly associated with stroke risk only in males. In men, all constitutive metabolic syndrome components were continuously and strongly associated with CVD. In women, high-density lipoprotein and triglycerides did not appear to be associated with short term CVD risk.

Conclusion

We found the newly defined consensus criteria for metabolic syndrome to be similarly predictive of cardiovascular events when compared to existing definitions. Significant gender differences exist in the association between metabolic syndrome, its individual components, and CVD.

Human Herpesvirus 6 (HHV-6) is a ubiquitous virus with an estimated seroprevalence of 95% in the adult population. HHV-6 is associated with several neurologic disorders, including multiple sclerosis, an inflammatory demyelinating disease affecting the CNS. Animal models of HHV-6 infection would help clarify its role in human disease but have been slow to develop because rodents lack CD46, the receptor for cellular entry. Therefore, we investigated the effects of HHV-6 infections in a non-human primate, the common marmoset Callithrix jacchus. We inoculated a total of 12 marmosets with HHV-6A and HHV-6B intravenously and HHV-6A intranasally. Animals were monitored for 25 weeks post-inoculation clinically, immunologically and by MRI. Marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms and generated virus-specific antibody responses, while those inoculated intravenously with HHV-6B were asymptomatic and generated comparatively lower antibody responses. Viral DNA was detected at a low frequency in paraffin-embedded CNS tissue of a subset of marmosets inoculated with HHV-6A and HHV-6B intravenously. When different routes of HHV-6A inoculation were compared, intravenous inoculation resulted in virus-specific antibody responses and infrequent detection of viral DNA in the periphery, while intranasal inoculation resulted in negligible virus-specific antibody responses and frequent detection of viral DNA in the periphery. Moreover, marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms, while marmosets inoculated with HHV-6A intranasally were asymptomatic. We demonstrate that a marmoset model of HHV-6 infection can serve to further define the contribution of this ubiquitous virus to human neurologic disorders.

Author Summary

The human herpesviruses HHV-6A and HHV-6B are widely distributed in the human population, but also specifically associated with several central nervous system (CNS) diseases. We investigated HHV-6A and HHV-6B infections in the common marmoset, a non-human primate naturally susceptible to infection, unlike rodents. We inoculated marmosets with HHV-6A and HHV-6B intravenously, and with HHV-6A intranasally, to represent a more physiologic route of infection. Following intravenous HHV-6A inoculation, marmosets exhibited clinical symptoms with evidence of spinal cord pathology. Animals inoculated intravenously with HHV-6B were asymptomatic and without detectable CNS pathology. Both groups developed robust anti-viral antibody responses, and we detected viral DNA infrequently in the periphery. By contrast, marmosets inoculated intranasally with HHV-6A were asymptomatic, failed to generate anti-viral antibodies, and we frequently detected viral DNA in the periphery. Interestingly, HHV-6 DNA was detected in brain and spinal cord sections of several intravenously inoculated animals, demonstrating that HHV-6 can gain access to and persist in the CNS. These observations help to define the contributions of ubiquitous herpesviruses to neurologic disease development in a non-human primate. As little is known about the acquisition and host response to HHV-6A, this model may clarify how this virus may trigger or potentiate disease.

Background

Identification of delirium in emergency departments (ED) is often underestimated; within EDs, studies on delirium assessment and relation with patient outcome in Intermediate Care Units (IMCU) appear missing in European hospital settings. Here we aimed to determine delirium prevalence in an EDIMCU (Hospital de Braga, Braga, Portugal) and assessed routine biochemical parameters that might be delirium indicators.

Methods

The study was prospective and observational. Sedation level was assessed via the Richmond Agitation-Sedation Scale and delirium status by the Confusion Assessment Method for the ICU. Information collected included age and gender, admission type, Charlson Comorbidity Index combined condition score (Charlson score), systemic inflammatory response syndrome criteria (SIRS), biochemical parameters (blood concentration of urea nitrogen, creatinine, hemoglobin, sodium and potassium, arterial blood gases, and other parameters as needed depending on clinical diagnosis) and EDIMCU length of stay (LOS). Statistical analyses were performed as appropriate to determine if baseline features differed between the ‘Delirium’ and ‘No Delirium’ groups. Multivariate logistic regression was performed to assess the effect of delirium on the 1-month outcome.

Results

Inclusion and exclusion criteria were met in 283 patients; 238 were evaluated at 1-month for outcome follow-up after EDIMCU discharge (“good” recovery without complications requiring hospitalization or institutionalization; “poor” institutionalization in permanent care-units/assisted-living or death). Delirium was diagnosed in 20.1% patients and was significantly associated with longer EDIMCU LOS. At admission, Delirium patients were significantly older and had significantly higher blood urea, creatinine and osmolarity levels and significantly lower hemoglobin levels, when compared with No Delirium patients. Delirium was an independent predictor of increased EDIMCU LOS (odds ratio 3.65, 95% CI 1.97-6.75) and poor outcome at 1-month after discharge (odds ratio 3.51, CI 1.84-6.70), adjusted for age, gender, admission type, presence of SIRS criteria, Charlson score and osmolarity at admission.

Conclusions

In an EDIMCU setting, delirium was associated with longer LOS and poor outcome at1-month post-discharge. Altogether, findings support the need for delirium screening and management in emergency settings.

An assessment of the concentrations of thirteen different therapeutic pharmaceutical compounds was conducted on water samples obtained from different wastewater treatment plants (WWTPs) using solid phase extraction and high- and ultra-high-performance liquid chromatography with mass spectrometry detection (HPLC-MS/MS and UHPLC-MS/MS), was carried out.

The target compounds included ketoprofen and naproxen (anti-inflammatories), bezafibrate (lipid-regulating), carbamazepine (anticonvulsant), metamizole (analgesic), atenolol (β-blocker), paraxanthine (stimulant), fluoxetine (antidepressant), and levofloxacin, norfloxacin, ciprofloxacin, enrofloxacin and sarafloxacin (fluoroquinolone antibiotics).

The relative standard deviations obtained in method were below 11%, while the detection and quantification limits were in the range of 0.3 – 97.4 ng·L-1 and 1.1 – 324.7 ng·L-1, respectively. The water samples were collected from two different WWTPs located on the island of Gran Canaria in Spain over a period of one year. The first WWTP (denoted as WWTP1) used conventional activated sludge for the treatment of wastewater, while the other plant (WWTP2) employed a membrane bioreactor system for wastewater treatment.

Most of the pharmaceutical compounds detected in this study during the sampling periods were found to have concentrations ranging between 0.02 and 34.81 μg·L-1.

The spatiotemporal characteristics of the hemodynamic response to increased neural activity were investigated at the level of individual intracortical vessels using BOLD-fMRI in a well-established rodent model of somatosensory stimulation at 11.7T. Functional maps of the rat barrel cortex were obtained at 150×150×500µm spatial resolution every 200ms. The high spatial resolution allowed separation of active voxels into those containing intracortical macro vessels, mainly vein/venules (referred to as macrovasculature), and those enriched with arteries/capillaries and small venules (referred to as microvasculature) since the macro vessel can be readily mapped due to the fast T2* decay of blood at 11.7T. The earliest BOLD response was observed within layers IV–V by 0.8s following stimulation and encompassed mainly the voxels containing the microvasculature and some confined macrovasculature voxels. By 1.2s, the BOLD signal propagated to the macrovasculature voxels where the peak BOLD signal was 2–3 times higher than that of the microvasculature voxels. The BOLD response propagated in individual venules/veins far from neuronal sources at later times. This was also observed in layers IV–V of the barrel cortex after specific stimulation of separated whisker rows. These results directly visualized that the earliest hemodynamic changes to increased neural activity occur mainly in the microvasculature and spread towards the macrovasculature. However, at peak response, the BOLD signal is dominated by penetrating venules even at layer IV–V of the cortex.

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice (susceptible to dental fluorosis, n = 18) and 129P3/J mice (resistant, n = 18) were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies.

Identification of thymocyte regulators is a central issue in T cell biology. Interestingly, growing evidence indicates that common key molecules control neuronal and immune cell functions. The neurotrophic factor receptor RET mediates critical functions in foetal hematopoietic subsets, thus raising the possibility that RET-related molecules may also control T cell development. We show that Ret, Gfra1 and Gfra2 are abundantly expressed by foetal and adult immature DN thymocytes. Despite the developmentally regulated expression of these genes, analysis of foetal thymi from Gfra1, Gfra2 or Ret deficient embryos revealed that these molecules are dispensable for foetal T cell development. Furthermore, analysis of RET gain of function and Ret conditional knockout mice showed that RET is also unnecessary for adult thymopoiesis. Finally, competitive thymic reconstitution assays indicated that Ret deficient thymocytes maintained their differentiation fitness even in stringent developmental conditions. Thus, our data demonstrate that RET/GFRα signals are dispensable for thymic T cell development in vivo, indicating that pharmacological targeting of RET signalling in tumours is not likely to result in T cell production failure.

OBJECTIVE:

Large vessel occlusion in acute ischemic stroke is associated with low recanalization rates under intravenous thrombolysis. We evaluated the safety and efficacy of the Solitaire AB stent in treating acute ischemic stroke.

METHODS:

Patients presenting with acute ischemic stroke were prospectively evaluated. The neurological outcomes were assessed using the National Institutes of Health Stroke Scale and the modified Rankin Scale. Time was recorded from the symptom onset to the recanalization and procedure time. Recanalization was assessed using the thrombolysis in cerebral infarction score.

RESULTS:

Twenty-one patients were evaluated. The mean patient age was 65, and the National Institutes of Health Stroke Scale scores ranged from 7 to 28 (average 17±6.36) at presentation. The vessel occlusions occurred in the middle cerebral artery (61.9%), distal internal carotid artery (14.3%), tandem carotid occlusion (14.3%), and basilar artery (9.5%). Primary thrombectomy, rescue treatment and a bridging approach represented 66.6%, 28.6%, and 4.8% of the performed procedures, respectively. The mean time from symptom onset to recanalization was 356.5±107.8 minutes (range, 80-586 minutes). The mean procedure time was 60.4±58.8 minutes (range, 14-240 minutes). The overall recanalization rate (thrombolysis in cerebral infarction scores of 3 or 2b) was 90.4%, and the symptomatic intracranial hemorrhage rate was 14.2%. The National Institutes of Health Stroke Scale scores at discharge ranged from 0 to 25 (average 6.9±7). At three months, 61.9% of the patients had a modified Rankin Scale score of 0 to 2, with an overall mortality rate of 9.5%.

CONCLUSIONS:

Intra-arterial thrombectomy with the Solitaire AB device appears to be safe and effective. Large randomized trials are necessary to confirm the benefits of this approach in acute ischemic stroke.

Introduction:

Somatostatin analogs (SSAs) are used as part of standard treatment for advanced neuroendocrine tumors (NETs). The mechanisms behind the antiproliferative action of SSAs remain largely unknown, but a connection with the mammalian target of rapamycin (mTOR) signaling pathway has been suggested. Our purpose was to evaluate the activation status of the AKT/mTOR pathway in advanced metastatic NETs and identify biomarkers of response to SSA therapy.

Patients and methods:

Expression of phosphatase and tensin homolog (PTEN), phosphorylated (p)-AKT(Ser473), and p-S6(Ser240/244) was evaluated using immunohistochemistry in archival paraffin samples from 23 patients. Expression levels were correlated with clinicopathological parameters and progression-free survival under treatment with SSAs.

Results:

A positive association between p-AKT and p-S6 expression was identified (P = 0.01) and higher expression of both markers was observed in pancreatic NETs. AKT/mTOR activation was observed without the loss of PTEN expression. Tumors showing AKT/mTOR signaling activation progressed faster when treated with SSAs: higher expression of p-AKT or p-S6 predicted a median progression-free survival of 1 month vs 26.5 months for lower expression (P = 0.02).

Conclusion:

Constitutive activation of the AKT/mTOR pathway was associated with shorter time-to-progression in patients undergoing treatment with SSAs. Larger case series are needed to validate whether p-AKT(Ser473) and p-S6(Ser240/244) can be used as prognostic markers of response to therapy with SSAs.

Background

There is significant heterogeneity in reported sensitivities and specificities of diagnostic serological assays for Chagas disease, as might be expected from studies that vary widely according to setting, research design, antigens employed, and reference standard. The purpose of this study is to summarize the reported accuracy of serological assays and to identify sources of heterogeneity including quality of research design. To avoid associated spectrum bias, our analysis was limited to cohort studies.

Methods

We completed a search of PubMed, a bibliographic review of potentially relevant articles, and a review of articles identified by a study author involved in this area of research. Studies were limited to prospective cohort studies of adults published since 1985. Measures of diagnostic accuracy were pooled using a Der Simonian Laird Random Effects Model. A subgroup analysis and meta regression were employed to identify sources of heterogeneity. The QUADAS tool was used to assess quality of included studies and Begg's funnel plot was used to assess publication bias.

Results

Eighteen studies and 61 assays were included in the final analysis. Significant heterogeneity was found in all pre-determined subgroups. Overall sensitivity was 90% (95% CI: 89%–91%) and overall specificity was 98% (95% CI: 98%–98%).

Conclusion

Sensitivity and specificity of serological assays for the diagnosis of Chagas disease appear less accurate than previously thought. Suggestions to improve the accuracy of reporting include the enrollment of patients in a prospective manner, double blinding, and providing an explicit method of addressing subjects that have an indeterminate diagnosis by either the reference standard or index test.

Author Summary

Chagas disease, an infectious disease endemic to Latin America, is caused by the protozoan parasite Trypanosoma cruzi. T. cruzi can be transmitted through blood transfusions, organ transplants, or from mother to fetus, although it is most commonly transmitted through insect vectors. Infections can remain silent for many years before manifesting as potentially fatal damage to the cardiac and/or digestive system. Diagnosis of Chagas disease during its chronic asymptomatic phase is crucial to preventing future infections with T. cruzi and is often performed using serological tests that detect antibodies in the blood. Because there is currently no gold standard for serological diagnostic tests, multiple forms of serologic testing are often used in conjunction. The purpose of this study was to compare reports on the accuracy of serological tests. After limiting studies by certain criteria, the authors found a lower estimate of accuracy than has previously been reported in the literature and suggest quality improvements that can be made to standardize future reports.

Background

Low-density lipoprotein cholesterol-lowering therapy is an important aspect of primary prevention of cardiovascular disease (CVD). Statins are the most widely used drug therapy for achieving low-density lipoprotein goals based on an individual's 10-year risk. However, substantial risk of CVD events still exists even when a person is on statins. We sought to explore the predictors of future CVD events in individuals on statins with no pre-existing CVD.

Methods

The analysis was done on subjects who were on statins (n = 919) at baseline in the Multi-Ethnic Study of Atherosclerosis limited access dataset from the National Heart, Lung and Blood Institute. The primary outcome variable was all-cause CVD events (n = 67). Multivariate regression Cox proportional hazard analysis was done to identify potential independent predictors of all-cause CVD.

Results

Our cohort consisted of 47% males, with a mean age of 66 ± 9 years. Sixty-seven participants (7.3%) experienced CVD events during a mean follow-up of 4.4 years. A higher coronary artery calcium score, homocysteine levels, waist circumference and a lower large arterial elasticity index were identified as independent predictors of CVD events.

Conclusion

Homocysteine, waist circumference, coronary artery calcification and the large artery elasticity index appear to be the major independent predictors of CVD events in individuals on statins with no pre-existing CVD. In addition to emphasizing weight loss, alternative approaches beyond lipid reduction may need to be explored to better characterize and attenuate the residual risk in subjects on statin therapy for primary prevention.

Cotia virus (COTV) SPAn232 was isolated in 1961 from sentinel mice at Cotia field station, São Paulo, Brazil. Attempts to classify COTV within a recognized genus of the Poxviridae have generated contradictory findings. Studies by different researchers suggested some similarity to myxoma virus and swinepox virus, whereas another investigation characterized COTV SPAn232 as a vaccinia virus strain. Because of the lack of consensus, we have conducted an independent biological and molecular characterization of COTV. Virus growth curves reached maximum yields at approximately 24 to 48 h and were accompanied by virus DNA replication and a characteristic early/late pattern of viral protein synthesis. Interestingly, COTV did not induce detectable cytopathic effects in BSC-40 cells until 4 days postinfection and generated viral plaques only after 8 days. We determined the complete genomic sequence of COTV by using a combination of the next-generation DNA sequencing technologies 454 and Illumina. A unique contiguous sequence of 185,139 bp containing 185 genes, including the 90 genes conserved in all chordopoxviruses, was obtained. COTV has an interesting panel of open reading frames (ORFs) related to the evasion of host defense, including two novel genes encoding C-C chemokine-like proteins, each present in duplicate copies. Phylogenetic analysis revealed the highest amino acid identity scores with Cervidpoxvirus, Capripoxvirus, Suipoxvirus, Leporipoxvirus, and Yatapoxvirus. However, COTV grouped as an independent branch within this clade, which clearly excluded its classification as an Orthopoxvirus. Therefore, our data suggest that COTV could represent a new poxvirus genus.

The spatial and temporal control of chromosome duplication and segregation is crucial for proper cell division. While this process is well studied in eukaryotic and some prokaryotic organisms, relatively little is known about it in prokaryotic polyploids such as Synechococcus elongatus PCC 7942, which is known to possess one to eight copies of its single chromosome. Using a fluorescent repressor-operator system, S. elongatus chromosomes and chromosome replication forks were tagged and visualized. We found that chromosomal duplication is asynchronous and that the total number of chromosomes is correlated with cell length. Thus, replication is independent of cell cycle and coupled to cell growth. Replication events occur in a spatially random fashion. However, once assembled, replisomes move in a constrained manner. On the other hand, we found that segregation displays a striking spatial organization in some cells. Chromosomes transiently align along the major axis of the cell and timing of alignment was correlated to cell division. This mechanism likely contributes to the non-random segregation of chromosome copies to daughter cells.

Background

Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease.

Methodology/Principal Findings

Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages.

Conclusions/Significance

Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.

Author Summary

Cutaneous leishmaniasis is a widespread tropical disease caused by different species of Leishmania protozoa that are transmitted by infected sandflies. Clinical presentations are extremely diverse and dependent on a variety of parasite and host factors that are poorly understood. Leishmania (V.) braziliensis infection may result in a devastating disease manifestation characterized by the development of destructive lesions in the oral, nasal, and pharyngeal mucosal. Ecto-nucleotidases are enzymes that are involved in the hydrolysis of extracellular nucleotides. These enzymes have been shown to correlate with virulence of Leishmania parasites. In this work, we evaluated the ecto-nucleotidase activity of promastigotes from the twenty three different L. braziliensis isolates. We demonstrated that isolates obtained from mucosal lesions present higher levels of ecto-nucleotidase activity than those from cutaneous lesions. In addition, we show that in the murine model of cutaneous leishmaniasis, promastigote forms of parasite with higher activity induce a delayed/decreased immune response that may correlate with spreading of the parasites throughout the body. Thus, we propose that the level of ecto-nucleotidase activity of promastigotes may be a marker for the development of severe clinical forms of cutaneous leishmaniasis and also a possible target for future therapeutic intervention.

Introduction

Cerebral palsy (CP) is a posture and movement disorder and different therapeutic modalities, such as the use of braces, have sought to favor selective motor control and muscle coordination in such patients. The aim of the proposed study is to determine the effect of the combination of posture-control insoles and ankle-foot orthoses (AFOs) improving functional limitation in children with CP.

Methods/Design

The sample will be composed of 24 children with CP between four and 12 years of age. After the signing of the statement of informed consent, the children will be randomly allocated to two groups: a control group using AFOs alone and an experimental group using both posture-control insoles and AFOs. Evaluations will be performed on five occasions: without any accessory (insoles or AFOs), immediately after, one month after, six months after and one year after AFOs or insole and AFOs use. The evaluation will involve the analysis of gait, static and functional balance, mobility and hypertonia. The three-dimensional assessment of gait will involve the eight-camera SMART-D SMART-D 140® system (BTS Engineering), two Kistler force plates (model 9286BA) and an eight-channel, wireless FREEEMG® electromyography (BTS Engineering). Static balance will be assessed using a Kistler force plate (model 9286BA). Clinical functional balance and mobility will be assessed using the Berg Balance Scale, Timed Up-and-Go Test and Six-Minute Walk Test. The posture-control insoles will be made of ethylene vinyl acetate, with thermal molding for fixation. The fixed orthoses will be made of polypropylene and attached to the ankle region (AFO). The results will be analyzed statistically, with the level significance set to 5% (p < 0.05).

Trial Registration

Trial Registration Number: RBR6d342s (http://www.ensaiosclinicos.gov.br/news/)

Background

HIV-1 subtype B is the most prevalent in developed countries and, consequently, it has been extensively studied. On the other hand, subtype C is the most prevalent worldwide and therefore is a reasonable target for future studies. Here we evaluate the acquisition of resistance and the viability of HIV-1 subtype B and C RT clones from different isolates that were subjected to in vitro selection pressure with zidovudine (ZDV) and lamivudine (3TC).

Methods/Principal Findings

MT4 cells were infected with chimeric virus pseudotyped with RT from subtype B and C clones, which were previously subjected to serial passage with increasing concentrations of ZDV and 3TC. The samples collected after each passage were analyzed for the presence of resistance mutations and VL. No differences were found between subtypes B and C in viral load and resistance mutations when these viruses were selected with 3TC. However, the route of mutations and the time to rebound of subtype B and C virus were different when subjected to ZDV treatment. In order to confirm the role of the mutations detected, other clones were generated and subjected to in vitro selection. RT subtype B virus isolates tended to acquire different ZDV resistance mutations (Q151M and D67N or T215Y, D67D/N and F214L) compared to subtype C (D67N, K70R, T215I or T215F).

Conclusions/Significance

This study suggests that different subtypes have a tendency to react differently to antiretroviral drug selection in vitro. Consequently, the acquisition of resistance in patients undergoing antiretroviral therapy can be dependent on the subtypes composing the viral population.

Here we report the biological and molecular characterization of a virulent genotype VII Newcastle disease virus (NDV) circulating in Venezuela and the assessment of the vaccination efficacy under field conditions compared to controlled rearing conditions. Biological pathotyping showed a mean embryo dead time of 50 h and an intracerebral pathogenicity index of 1.86. Sequence-based phylogenetic analysis demonstrated that the virus belongs to genotype VII in class II (a genotype often found in Asia and Africa), representing the first report of the presence of this genotype in the continent of South America. A vaccine-challenge trial in commercial broilers reared in fields or in a experimental setting included dual (live/killed) priming of 1-day-old chicks plus two live NDV and infectious bursal disease virus (IBDV) field vaccinations at days 7 and 17, followed by a very stringent genotype VII NDV challenge at day 28. Serology for NDV and IBDV, bursal integrity, and protection against NDV lethal challenge were assessed. At 28 days, field vaccinates showed significantly lower NDV (1,356 versus 2,384) and higher IBD (7,295 versus 1,489) enzyme-linked immunosorbent assay (ELISA) antibody titers than the experimentally reared birds. A lower bursal size and bursa-body weight ratio (P < 0.05) and higher bursa lesion score were also detected in the field set. Only 57.1% of field vaccinates survived the lethal challenge, differing (P < 0.05) from 90.5% survival in the experimental farm. Overall, results confirmed the presence of the genotype VII viruses in South America and suggest that field-associated factors such as immunosuppression compromise the efficacy of the vaccination protocols implemented.

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that “stressful conditions” will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

Author Summary

Leishmania parasites are the causative agent of a spectrum of diseases characterized by severe lesions in skin or life threatening visceral infections. In the parasite life cycle, a range of morphological transitions can be found such as amastigotes (hosted in humans and others mammals) and promastigotes (located in the sand fly vector). The disease begins when the infective non-dividing promastigote metacyclic form is transmitted to mammalian hosts by the bite of an infected sand fly. The conditions for the development of these metacyclic promastigotes are still not fully understood. Here we tested the hypothesis that the presence or absence of purine will determine whether the parasite will proliferate or differentiate into the metacyclic form. Our experiments indicate that the presence of purines in the culture medium of Leishmania parasites interfere with the development of metacyclic promastigotes. Our results also show that although decreased proliferation and metacyclic differentiation occur simultaneously these two phenomena are independently regulated. Finally we show, for the first time in a natural vector, that metacyclogenesis is inhibited in vivo by adenosine, suggesting a new level of cell differentiation control in these protozoan parasites which is driven by purine sensing.