Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments

Polymerase chain reaction (PCR) is widely applied in clinical and environmental microbiology. Primer design is key to the development of successful assays and is often performed manually by using multiple nucleic acid alignments. Few public software tools exist that allow comprehensive design of degenerate primers for large groups of related targets based on complex multiple sequence alignments. Here we present a method for designing such primers based on tree building followed by application of a set covering algorithm, and demonstrate its utility in compiling Multiplex PCR primer panels for detection and differentiation of viral pathogens

Automated Classification of Airborne Laser Scanning Point Clouds

Making sense of the physical world has always been at the core of mapping. Up until recently, this has always dependent on using the human eye. Using airborne lasers, it has become possible to quickly "see" more of the world in many more dimensions. The resulting enormous point clouds serve as data sources for applications far beyond the original mapping purposes ranging from flooding protection and forestry to threat mitigation. In order to process these large quantities of data, novel methods are required. In this contribution, we develop models to automatically classify ground cover and soil types. Using the logic of machine learning, we critically review the advantages of supervised and unsupervised methods. Focusing on decision trees, we improve accuracy by including beam vector components and using a genetic algorithm. We find that our approach delivers consistently high quality classifications, surpassing classical methods

Nonparametric methods for the analysis of single-color pathogen microarrays

<p>Abstract</p> <p>Background</p> <p>The analysis of oligonucleotide microarray data in pathogen surveillance and discovery is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied.</p> <p>Results</p> <p>Positive predictive value and false positive rates were examined to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney <it>U</it>, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, the chi-square proved most useful.</p> <p>Conclusions</p> <p>The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy.</p

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.The authors thank Valeria Bussetti for expert technical assistance. This study was supported by the National Institutes of Health (awards 5R21AI067281, AI057158, 5R21AI067281 and AI088647), the United States Department of Defense and an intramural grant from Iowa State University. AEF is supported by a grant from the Wellcome Trust (award 106207).This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00042

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico.

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.The authors thank Valeria Bussetti for expert technical assistance. This study was supported by the National Institutes of Health (awards 5R21AI067281, AI057158, 5R21AI067281 and AI088647), the United States Department of Defense and an intramural grant from Iowa State University. AEF is supported by a grant from the Wellcome Trust (award 106207).This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00042

Microbial biopesticides for integrated crop management : an assessment of environmental and regulatory sustainability

Herbivorous insects and mites, plant diseases and weeds are major impediments to the production of food crops and are increasingly difficult to control with conventional chemicals. This paper focuses on microbial control agents with an emphasis on augmentation. There are marked differences in the availability of products in different countries which can be explained in terms of differences in their regulatory systems. Regulatory failure arises from the application of an inappropriate synthetic pesticides model. An understanding of regulatory innovation is necessary to overcome these problems. Two attempts at remedying regulatory failure in the UK and the Netherlands are assessed. Scientific advances can feed directly into the regulatory process and foster regulatory innovation

Induction of Inflammation by West Nile virus Capsid through the Caspase-9 Apoptotic Pathway

West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid (Cp) by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade

Clinical Characteristics, Management, and Outcomes of Patients Diagnosed With Acute Pulmonary Embolism in the Emergency Department

Objectives In a large U.S. sample, this study measured the presentation features, testing, treatment strategies, and outcomes of patients diagnosed with pulmonary embolism (PE) in the emergency department (ED). Background No data have quantified the demographics, clinical features, management, and outcomes of outpatients diagnosed with PE in the ED in a large, multicenter U.S. study. Methods Patients of any hemodynamic status were enrolled from the ED after confirmed acute PE or with a high clinical suspicion prompting anticoagulation before imaging for PE. Exclusions were inability to provide informed consent (where required) or unavailability for follow-up. Results A total of 1,880 patients with confirmed acute PE were enrolled from 22 U.S. EDs. Diagnosis of PE was based upon positive results of computerized tomographic pulmonary angiogram in most cases (n = 1,654 [88%]). Patients represented both sexes equally, and racial and ethnic composition paralleled the overall U.S. ED population. Most (79%) patients with PE were employed, and one-third were older than age 65 years. The mortality rate directly attributed to PE was 20 in 1,880 (1%; 95% confidence interval [CI]: 0% to 1.6%). Mortality from hemorrhage was 0.2%, and the all-cause 30-day mortality rate was 5.4% (95% CI: 4.4% to 6.6%). Only 3 of 20 patients with major PE that ultimately proved fatal had systemic anticoagulation initiated before diagnostic confirmation, and another 3 of these 20 received a fibrinolytic agent. Conclusions Patients diagnosed with acute PE in U.S. EDs have high functional status, and their mortality rate is low. These registry data suggest that appropriate initial medical management of ED patients with severe PE with anticoagulation is poorly standardized and indicate a need for research to determine the appropriate threshold for empiric treatment when PE is suspected before diagnostic confirmation

Panmicrobial Oligonucleotide Array for Diagnosis of Infectious Diseases

To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004–2005

Addressing Grand Challenges in Earth Observation Science: The Earth Observation Data Centre for Water Resources Monitoring

Earth observation is entering a new era where the increasing availability of free and open global satellite data sets combined with the computing power offered by modern information technologies opens up the possibility to process high-resolution data sets at global scale and short repeat intervals in a fully automatic fashion. This will not only boost the availability of higher level earth observation data in purely quantitative terms, but can also be expected to trigger a step change in the quality and usability of earth observation data. However, the technical, scientific, and organisational challenges that need to be overcome to arrive at this point are significant. First of all, Petabyte-scale data centres are needed for storing and processing complete satellite data records. Second, innovative processing chains that allow fully automatic processing of the satellite data from the raw sensor records to higher-level geophysical products need to be developed. Last but not least, new models of cooperation between public and private actors need to be found in order to live up to the first two challenges. This paper offers a discussion of how the Earth Observation Data Centre for Water Resources Monitoring (EODC) – a catalyser for an open and international cooperation of public and private organisations – will address these three grand challenges with the aim to foster the use of earth observation for monitoring of global water resources