Automatic local resolution-based sharpening of cryo-EM maps.

MOTIVATION: Recent technological advances and computational developments have allowed the reconstruction of Cryo-Electron Microscopy (cryo-EM) maps at near-atomic resolution. On a typical workflow and once the cryo-EM map has been calculated, a sharpening process is usually performed to enhance map visualization, a step that has proven very important in the key task of structural modeling. However, sharpening approaches, in general, neglects the local quality of the map, which is clearly suboptimal. RESULTS: Here, a new method for local sharpening of cryo-EM density maps is proposed. The algorithm, named LocalDeblur, is based on a local resolution-guided Wiener restoration approach of the original map. The method is fully automatic and, from the user point of view, virtually parameter-free, without requiring either a starting model or introducing any additional structure factor correction or boosting. Results clearly show a significant impact on map interpretability, greatly helping modeling. In particular, this local sharpening approach is especially suitable for maps that present a broad resolution range, as is often the case for membrane proteins or macromolecules with high flexibility, all of them otherwise very suitable and interesting specimens for cryo-EM. To our knowledge, and leaving out the use of local filters, it represents the first application of local resolution in cryo-EM sharpening. AVAILABILITY AND IMPLEMENTATION: The source code (LocalDeblur) can be found at https://github.com/I2PC/xmipp and can be run using Scipion (http://scipion.cnb.csic.es) (release numbers greater than or equal 1.2.1). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Validation of electron microscopy initial models via small angle X-ray scattering curves.

MOTIVATION: Cryo electron microscopy (EM) is currently one of the main tools to reveal the structural information of biological macromolecules. The re-construction of three-dimensional (3D) maps is typically carried out following an iterative process that requires an initial estimation of the 3D map to be refined in subsequent steps. Therefore, its determination is key in the quality of the final results, and there are cases in which it is still an open issue in single particle analysis (SPA). Small angle X-ray scattering (SAXS) is a well-known technique applied to structural biology. It is useful from small nanostructures up to macromolecular ensembles for its ability to obtain low resolution information of the biological sample measuring its X-ray scattering curve. These curves, together with further analysis, are able to yield information on the sizes, shapes and structures of the analyzed particles. RESULTS: In this paper, we show how the low resolution structural information revealed by SAXS is very useful for the validation of EM initial 3D models in SPA, helping the following refinement process to obtain more accurate 3D structures. For this purpose, we approximate the initial map by pseudo-atoms and predict the SAXS curve expected for this pseudo-atomic structure. The match between the predicted and experimental SAXS curves is considered as a good sign of the correctness of the EM initial map. AVAILABILITY AND IMPLEMENTATION: The algorithm is freely available as part of the Scipion 1.2 software at http://scipion.i2pc.es/.

Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice.

Dengue viruses (DENV) are transmitted to humans by the bite of Aedes aegypti or Aedes albopictus mosquitoes, with millions of infections annually in over 100 countries. The diseases they produce, which occur exclusively in humans, are dengue fever (DF) and dengue hemorrhagic fever (DHF). We previously developed a humanized mouse model of DF in which mice transplanted with human hematopoietic stem cells produced signs of DENV disease after injection with low-passage, wild-type isolates. Using these mice, but now allowing infected A. aegypti to transmit dengue virus during feeding, we observed signs of more severe disease (higher and more sustained viremia, erythema, and thrombocytopenia). Infected mice mounted innate (gamma interferon [IFN-γ] and soluble interleukin 2 receptor alpha [sIL-2Rα]) and adaptive (anti-DENV antibodies) immune responses that failed to clear viremia until day 56, while a mosquito bite alone induced strong immunomodulators (tumor necrosis factor alpha [TNF-α], IL-4, and IL-10) and thrombocytopenia. This is the first animal model that allows an evaluation of human immunity to DENV infection after mosquito inoculation.

RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro.

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

Humanized mice show clinical signs of dengue fever according to infecting virus genotype.

We demonstrated that the infection of humanized NOD-scid IL2r gamma(null) mice with different strains (representing the four genotypes) of dengue virus serotype 2 (DEN-2) can induce the development of human-like disease, including fever, viremia, erythema, and thrombocytopenia. Newborn mice were irradiated and received transplants by intrahepatic inoculation of human cord blood-derived hematopoietic progenitor cells (CD34(+)). After 6 weeks, mouse peripheral blood was tested by flow cytometry to determine levels of human lymphocytes (CD45(+) cells); rates of reconstitution ranged from 16 to 80% (median, 52%). Infection (with approximately 10(6) PFU, the equivalent of a mosquito bite) of these humanized mice with eight low-passage-number strains produced a high viremia extending to days 12 to 18 postinfection. We observed a significant decrease in platelets at day 10 in most of the mice and an increase in body temperature (fever) and erythema (rash) in comparison with humanized mice inoculated with cell culture medium only. Comparison of Southeast (SE) Asian and other genotype viruses (American, Indian, and West African) in this model showed significant differences in magnitude and duration of viremia and rash, with the SE Asian viruses always being highest. Indian genotype viruses produced lower viremias and less thrombocytopenia than the others, and West African (sylvatic) viruses produced the shortest periods of viremia and the lowest rash measurements. These results correlate with virulence and transmission differences described previously for primary human target cells and whole mosquitoes and may correlate with epidemiologic observations around the world. These characteristics make this mouse model ideal for the study of dengue pathogenesis and the evaluation of vaccine attenuation and antivirals.

DeepRes: a new deep-learning- and aspect-based local resolution method for electron-microscopy maps.

In this article, a method is presented to estimate a new local quality measure for 3D cryoEM maps that adopts the form of a 'local resolution' type of information. The algorithm (DeepRes) is based on deep-learning 3D feature detection. DeepRes is fully automatic and parameter-free, and avoids the issues of most current methods, such as their insensitivity to enhancements owing to B-factor sharpening (unless the 3D mask is changed), among others, which is an issue that has been virtually neglected in the cryoEM field until now. In this way, DeepRes can be applied to any map, detecting subtle changes in local quality after applying enhancement processes such as isotropic filters or substantially more complex procedures, such as model-based local sharpening, non-model-based methods or denoising, that may be very difficult to follow using current methods. It performs as a human observer expects. The comparison with traditional local resolution indicators is also addressed.

Identification of blood meal source and infection with Trypanosoma cruzi of Chagas disease vectors using a multiplex cytochrome b polymerase chain reaction assay.

Long-term control of triatomine bugs in Chagas endemic regions will depend on a full understanding of vector-parasite-host interactions. Herein we describe a cytochrome b multiplex polymerase chain reaction (PCR)-based strategy for blood meal source identification in bug foregut contents. This technique discriminates human from animal blood, and has been tested in five Triatoma species from México. Host identification has been validated for human, four rodent species, two bat species, dog, rabbit, sheep, and opossum. In addition, Trypanosoma cruzi can be identified simultaneously using S34/S67-specific kinetoplast DNA primers. Both host and parasite identification were possible as long as 10 weeks after bug feeding, and in samples stored up to 6 years. The blood meal identification procedure described here represents a powerful tool for large-scale studies identifying the biological, ecological, and environmental variables associated with Chagas disease transmission.

Appropriateness of lumbar spine magnetic resonance imaging in Spain.

OBJECTIVES: To determine the minimum percentage of lumbar spine magnetic resonance imaging (LSMRI) which are inappropriately prescribed in routine practice. METHODS: LSMRI performed prospectively on 602 patients in 12 Radiology Services across 6 regions in Spain, were classified as "appropriate", "uncertain" or "inappropriate" based on the indication criteria established by the National Institute for Clinical Excellence, the American College of Physicians and Radiology, and current evidence-based clinical guidelines. Studies on patients reporting at least one "red flag" were classified as "appropriate". A logistic regression model was developed to identify factors associated with a higher likelihood of inappropriate LSMRI, including gender, reporting of referred pain, health care setting (private/public), and specialty of prescribing physician. Before performing the LSMRI, the radiologists also assessed the appropriateness of the prescription. RESULTS: Eighty-eight percent of LSMRI were appropriate, 1.3% uncertain and 10.6% inappropriate. The agreement of radiologists' assessment with this classification was substantial (k=0.62). The odds that LSMRI prescriptions were inappropriate were higher for patients without referred pain [OR (CI 95%): 13.75 (6.72; 28.16)], seen in private practice [2.25 (1.20; 4.22)], by orthopedic surgeons, neurosurgeons or primary care physicians [2.50 (1.15; 5.56)]. CONCLUSION: Efficiency of LSMRI could be improved in routine practice, without worsening clinical outcomes.

Dengue virus tropism in humanized mice recapitulates human dengue fever.

Animal models of dengue virus disease have been very difficult to develop because of the virus' specificity for infection and replication in certain human cells. We developed a model of dengue fever in immunodeficient mice transplanted with human stem cells from umbilical cord blood. These mice show measurable signs of dengue disease as in humans (fever, viremia, erythema and thrombocytopenia), and after infection with the most virulent strain of dengue serotype 2, humanized mice showed infection in human cells in bone marrow, spleen and blood. Cytokines and chemokines were secreted by these human cells into the mouse bloodstream. We demonstrated that the pathology of dengue virus infection in these mice follows that reported in human patients, making this the first valid and relevant model for studying dengue fever pathogenesis in humans.

Stress fractures of the metacarpal bones in adolescent tennis players: a case series.

BACKGROUND: There are 12 reported cases of metacarpal stress fractures in athletes, with only 4 of them involving the second metacarpal. PURPOSE: The authors describe stress fracture of the second metacarpal bone in teenaged tennis players and the relationship with sport intensity and type of grip used. They also demonstrate that magnetic resonance imaging is the diagnostic study of choice to differentiate this entity from the most common cause of pain in this region of the hand in tennis players-the carpal boss. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Seven adolescent tennis players (mean age, 16.5 years; 6 female, 1 male) with dorsal hand pain produced by playing tennis were examined by radiographs and initial magnetic resonance imaging. In 2 cases, bone scintigraphy was performed. In the first 2 cases, the presumptive diagnosis was a carpal boss, but with this experience, the diagnostic evaluation of the last 5 cases was oriented toward a stress reaction at this level. Radiologic follow-up was performed. The authors also evaluated the grip type used by each tennis player. RESULTS: Clinical evaluation and imaging studies resulted in a diagnosis of stress injury of the second metatarsal in 6 of 7 cases, with the seventh case involving the third metacarpal. Initial imaging was positive in 3 cases, revealing an increased signal in the marrow without hairline crack and cortical thickening of the shaft or simply an increased signal in the marrow. In all cases, there was a history of recent increase in the sport training load. Six of the 7 tennis players were using a semi-Western or Western grip. CONCLUSION: Stress fractures of the second metacarpal are characteristic of adolescent tennis players and are associated with an increased intensity of tennis play and may be associated with use of the semi-Western or Western grip. Magnetic resonance imaging is the most useful tool for obtaining a definitive diagnosis.

Induction of protective antibodies against dengue virus by tetravalent DNA immunization of mice with domain III of the envelope protein.

Dengue fever is a growing public health concern around the world and despite vaccine development efforts, there are currently no effective dengue vaccines. In the present study we report the induction of protective antibodies against dengue virus by DNA immunization with domain III (DIII) region of the envelope protein (E) in a mouse model. The DIII region of all four dengue virus serotypes were cloned separately into pcDNA 3 plasmid. Protein expression was tested in COS-7 cells. Each plasmid, or a tetravalent combination, were used to immunize BALB/c mice by intramuscular route. Presence of specific antibodies was evaluated by ELISA, and neutralizing antibodies were tested using a cytopathogenic effect (CPE) inhibition assay in BHK-21 cells, as well as in newborn mice challenged intracranially with dengue 2 virus. Mice immunized with individual DIII constructs or the tetravalent formulation developed antibodies against each corresponding dengue serotype. Antibody titers by ELISA were similar for all serotypes and no significant differences were observed when boosters were administered, although antibody responses were dose-dependent. CPE inhibition assays using Den-2 virus showed neutralization titers of 1:10 in mice immunized with individual DIII plasmid or those immunized with the tetravalent formulations. 43% of newborn mice challenged with Den-2 in combination with sera from mice immunized with Den-2 DIII plasmid were protected, whereas sera from mice immunized with the tetravalent formulation conferred 87% protection. Our results suggest that DIII can be used as a tetravalent DNA formulation to induce neutralizing and protective antibodies against dengue virus.

Phylogenetic analysis of the envelope protein (domain III) of dengue 4 viruses.

OBJECTIVE: To evaluate the genetic variability of domain III of envelope (E) protein and to estimate phylogenetic relationships of dengue 4 (Den-4) viruses isolated in Mexico and from other endemic areas of the world. MATERIAL AND METHODS: A phylogenetic study of domain III of envelope (E) protein of Den-4 viruses was conducted in 1998 using virus strains from Mexico and other parts of the world, isolated in different years. Specific primers were used to amplify by RT-PCR the domain III and to obtain nucleotide sequence. Based on nucleotide and deduced aminoacid sequence, genetic variability was estimated and a phylogenetic tree was generated. To make an easy genetic analysis of domain III region, a Restriction Fragment Length Polymorphism (RFLP) assay was performed, using six restriction enzymes. RESULTS: Study results demonstrate that nucleotide and aminoacid sequence analysis of domain III are similar to those reported from the complete E protein gene. Based on the RFLP analysis of domain III using the restriction enzymes NIa III, Dde I and Cfo I, Den-4 viruses included in this study were clustered into genotypes 1 and 2 previously reported. CONCLUSIONS: Study results suggest that domain III may be used as a genetic marker for phylogenetic and molecular epidemiology studies of dengue viruses. The English version of this paper is available too at: http://www.insp.mx/salud/index.html.

The distal semimembranosus complex: normal MR anatomy, variants, biomechanics and pathology.

OBJECTIVE: To describe the normal MR anatomy and variations of the distal semimembranosus tendinous arms and the posterior oblique ligament as seen in the three orthogonal planes, to review the biomechanics of this complex and to illustrate pathologic examples. RESULTS AND CONCLUSION: The distal semimembranosus tendon divides into five tendinous arms named the anterior, direct, capsular, inferior and the oblique popliteal ligament. These arms intertwine with the branches of the posterior oblique ligament in the posterior medial aspect of the knee, providing stability. This tendon-ligamentous complex also acts synergistically with the popliteus muscle and actively pulls the posterior horn of the medial meniscus during knee flexion. Pathologic conditions involving this complex include complete and partial tears, insertional tendinosis, avulsion fractures and bursitis.

Desarrollo de agentes inmunizantes contra el dengue / Development of immunizing agents against dengue

El complejo de los cuatro flavivirus del dengue es transmitido principalmente por el mosquito Aedes aegypti. Se han atribuido epidemias a la actividad de A. albopictus, A. polynesiensis y a varias especies del complejo A. scutellaris. Los factores de riesgo que determinan la probabilidad de enfermar o morir por dengue est n relacionados tanto con el huésped (características genéticas, estado inmunitario, forma de vida y condiciones de salud, saneamiento b sico de la vivienda y abastecimiento de agua potable) como con el virus (variabilidad genética de cepas entre y dentro de los serotipos, diferente capacidad patogénica y distribución geogr fica). A pesar de la falta de conocimiento sobre la inmunobiopatología del dengue, se han hecho importantes avances para conseguir una respuesta inmunitaria protectora con virus atenuados y con antígenos obtenidos por medio de tecnologías recombinantes. Desde los años 40, se ha intentado desarrollar vacunas contra el dengue. La inmunidad que se adquiere por infección natural es específica para cada serotipo y se han documentado infecciones por tres serotipos diferentes en la misma persona, por lo que probablemente sea necesaria una vacuna tetravalente. En voluntarios se han probado vacunas contra los cuatro serotipos que han sido inmunogénicas y seguras. Aunque las vacunas con virus atenuados son prometedoras, son necesarios nuevos estudios sobre su eficacia y seguridad. Actualmente est n en curso estudios para producir vacunas contra el virus del dengue mediante tecnologías de ADN recombinante y otras técnicas de biología molecular, utilizando como antígenos proteínas estructurales (principalmente la glicoproteína E) y no estructurales. Con el mismo propósito se han usado varios vectores de expresión como Escherichia coli, baculovirus, virus de la vacuna y virus de la fiebre amarilla. Lamentablemente, no se han obtenido resultados satisfactorios en el hombre. La necesidad de desarrollar vacunas eficaces contra el dengue tiene especial importancia si se toma en cuenta la magnitud del problema de la transmisión de los cuatro serotipos en el mundo. La inmunización efectiva contra el dengue contribuir a su prevención y la relación costo-beneficio ser positiva. El hecho de que el dengue endémico afecte a niños de corta edad hace necesaria su inmunización, aprovechando la oportunidad que ofrece el Programa Ampliado de Inmunizacion

The four serotypes of dengue flaviviruses are transmitted mainly by the Aedes aegypti mosquito, and some epidemics have been attributed to Ae. albopictus, Ae. polynesiensis, and various species of the Ae. scutellaris complex. The risk factors involved in dengue mortality and morbidity are related to the human host (genetic characteristics of infected persons; lifestyles, immune status, and health conditions of people; basic sanitation of dwellings; and water supply) and to the virus (genetic variability between and among serotypes, different pathogenicities, and geographic distribution). Notwithstanding the lack of knowledge of the immunopathobiology of dengue fever, important advances have been made in terms of a protective immune response, using attenuated dengue viruses or antigens produced by means of recombinant technologies. Efforts have been made since the 1940s to develop dengue vaccines. Immunity acquired from natural infection is specific for each serotype, and as many as three different serotype infections have been reported in one individual. For this reason, a tetravalent vaccine may likely be needed. Candidate vaccines against the four serotypes have been tested in volunteers and have proven to be immunogenic and safe. Although attenuated live virus vaccines are promising, more study is needed regarding their effectiveness and safety. Currently, several studies are ongoing to develop dengue vaccines using antigens from structural proteins (particularly E glycoprotein) and nonstructural proteins, with recombinant DNA technology and other biomolecular technologies. With the same goal, various expression vectors are being used, including Escherichia coli, baculovirus, vaccinia virus, and yellow fever virus. Unfortunately, no satisfactory results have been obtained in humans. The need for effective dengue vaccines is great, given the serious worldwide problem of the transmission of the four serotypes. Effective immunization against dengue would contribute to its prevention, with a positive costbenefit relationship. Endemic dengue affects young children, and they should be immunized through the Expanded Program on Immunization.

Phylogenetic analysis of the envelope protein (domain lll) of dengue 4 viruses

Objective. To evaluate the genetic variability of domain III of envelope (E) protein and to estimate phylogenetic relationships of dengue 4 (Den-4) viruses isolated in Mexico and from other endemic areas of the world. Material and Methods. A phylogenetic study of domain III of envelope (E) protein of Den-4 viruses was conducted in 1998 using virus strains from Mexico and other parts of the world, isolated in different years. Specific primers were used to amplify by RT-PCR the domain III and to obtain nucleotide sequence. Based on nucleotide and deduced aminoacid sequence, genetic variability was estimated and a phylogenetic tree was generated. To make an easy genetic analysis of domain III region, a Restriction Fragment Length Polymorphism (RFLP) assay was performed, using six restriction enzymes. Results. Study results demonstrate that nucleotide and aminoacid sequence analysis of domain III are similar to those reported from the complete E protein gene. Based on the RFLP analysis of domain III using the restriction enzymes Nla III, Dde I and Cfo I, Den-4 viruses included in this study were clustered into genotypes 1 and 2 previously reported. Conclusions. Study results suggest that domain III may be used as a genetic marker for phylogenetic and molecular epidemiology studies of dengue viruses.

Objetivo. Evaluar la variabilidad genética del dominio III de la proteína de envoltura (E) y estimar la relación filogenética de los virus dengue 4 (Den-4) aislados en México y en otras regiones endémicas del mundo. Material y métodos. En el presente trabajo reportamos un estudio filogenético del dominio III de la proteína de envoltura (E) que se realizó en 1998 con virus Den-4 aislados en distintos años en México y en otras partes del mundo. Se usaron oligonucleótidos específicos para amplificar por RT-PCR la región del dominio III y para obtener la secuencia de nucleótidos. Mediante el análisis de la secuencia de nucleótidos y de la secuencia deducida de aminoácidos se estimó la variabilidad genética y se generó un árbol filogenético. Para facilitar el análisis genético del dominio III se usó la técnica basada en el polimorfismo de fragmentos generados con enzimas de restricción (PFER) utilizando seis enzimas de restricción. Resultados. Los datos demuestran que la información del análisis de la secuencia de nucleótidos y de aminoácidos de la región del dominio III es similar a la del gene completo de la proteína E. El análisis de PFER con las enzimas de restricción Nla III, Dde I y Cfo I, mostró que los virus Den-4 incluidos en este estudio se agruparon en los genotipos 1 y 2 reportados previamente. Conclusiones. Los resultados sugieren que el dominio III se puede utilizar como un marcador para estudios filogenéticos y de epidemiología molecular del virus Den-4.

Prevalence of antibodies to the repeat epitope of the circumsporozoite protein of Plasmodium vivax in San Luis Potosi, Mexico

The prevalence of antibodies against the repeat epitope of the circumsporozoite protein (cs) of the standard (PV210) and variant (PVK247) strain of Plasmodium vivax was determined by ELISA in 1170 sera from individual residents of seven localities of the Region Huasteca of San Luis Potosi, Mexico. The capture antigens were the synthetic peptides DDAAD and (ANGAGNQPG)4 that correspond to the repeats of the PV210 and PVK247 cs proteins, respectively. Of the analyzed serum samples, 34.1 percent (400/1170) were positive with one or both of these antigens. Of the sera, 18.2 percent (214/1170) reacted with the DDAADF peptide and 6.6 percent (78/1170) were positive with the variant synthetic peptide. Additionally, 9.2 percent (108/1170) of the samples reacted with both peptides. A sample of 10 percent of positive sera for the variant cs repeat (18/78) was tested with the cs repeat peptide of P. malariae/P. brasilianum (NAAG); almost all of them (16/18, 89 percent) being positive. These results confirm that the transmission of the variant strain of P. vivax is a common Phenomenon in endemic regions in Latin America, as well as in other tropical regions of the world. These findings may have implications for the development of a P. vivax vaccine since that based on the standard cs repeat only would not be universally protective