Heavy metal resistance in the Yanomami and Tunapuco microbiome

BACKGROUND The Amazon Region hosts invaluable and unique biodiversity as well as mineral resources. Consequently, large illegal and artisanal gold mining areas exist in indigenous territories. Mercury has been used in gold mining, and some has been released into the environment and atmosphere, primarily affecting indigenous people such as the Yanomami. In addition, other heavy metals have been associated with gold mining and other metal-dispersing activities in the region. OBJECTIVE Investigate the gut microbiome of two semi-isolated groups from the Amazon, focusing on metal resistance. METHODS Metagenomic data from the Yanomami and Tunapuco gut microbiome were assembled into contigs, and their putative proteins were searched against a database of metal resistance proteins. FINDINGS Proteins associated with mercury resistance were exclusive in the Yanomami, while proteins associated with silver resistance were exclusive in the Tunapuco. Both groups share 77 non-redundant metal resistance (MR) proteins, mostly associated with multi-MR and operons with potential resistance to arsenic, nickel, zinc, copper, copper/silver, and cobalt/nickel. Although both groups harbour operons related to copper resistance, only the Tunapuco group had the pco operon. CONCLUSION The Yanomami and Tunapuco gut microbiome shows that these people have been exposed directly or indirectly to distinct scenarios concerning heavy metals.

Comprehensive Evaluation and Application of Experimental Sources of Variation in Gut Microbiome Sequencing Studies / 中国生物化学与分子生物学报

Gut microbiome sequencing studies have great potential to translate microbial analysis outcomes into human health research. Sequencing strategies of 16S amplicon and whole-metagenome shotgun (WMS) are two main methods in microbiome research with respective advantages. However, how sample heterogeneity, sequencers and library preparation protocols affect the sequencing reproducibility of gut microbiome needs further investigation. This study aims to provide a reference for the selection of sequencing technologies by comparing differences in microbial composition from different sampling sites. The results of three widely adopted sequencers showed that the technical repetition correlation (r= 0. 94) was high in WMS method, while the biological repetition correlation (r = 0. 69) was low. Bray-Curtis distance identified that dissimilarity from biological replicates was larger than that of technical replicates (P<0. 001). In addition, dissimilarity and specific taxonomic profiles were observed between 16S and WMS datasets. Our results imply that homogenization is a necessary step before sample DNA extraction. The sequencers contributed less to taxonomic variation than the library preparation protocols. We developed an empirical Bayes approach that " borrowed information" in calculations and analyzed batch effect parameters using standardized data and prior distributions of (non-) parameters, which may improve population comparability between 16S and WMS and provide a basis for further application to fusion analysis of published 16S and microbial datasets.

Lesión del nervio ciático mayor por proyectil de escopeta / Lesion of the major sciatic nerve due to a shotgun projectile

Se describe el caso clínico de un paciente de 26 años de edad, atendido en la consulta de Ortopedia y Traumatología del Centro de Diagnóstico Integral Salvador Allende de Caracas, en la República Bolivariana de Venezuela, quien desde hacía 6 meses había recibido un disparo de escopeta en la cara posterior de ambos muslos. Esta vez acudió a consulta con dolor intenso en el miembro inferior izquierdo, sobre todo por debajo de la rodilla, acompañado de parestesias en la zona de inervación de ambas ramas de bifurcación del nervio ciático. Se pudo comprobar que el joven presentaba múltiples impactos de proyectiles en la cara posterior de ambos muslos y marcha claudicante. Los estudios radiográficos confirmaron la presencia de 8 proyectiles, 2 en el muslo derecho y 6 en el izquierdo. En la vista lateral se observó que uno de ellos se encontraba a una profundidad que se correspondía con la región anatómica del nervio ciático izquierdo. Para la intervención quirúrgica se utilizó anestesia local, sedación y un intensificador de imágenes. Con la cooperación del paciente se pudo extraer el proyectil alojado en el epineuro del ciático sin complicaciones. A los 3 meses habían desaparecido las molestias y pudo reincorporarse a sus actividades habituales.

The case report of a 26 years patient is described. He was assisted in the Orthopedics and Traumatology Service of Salvador Allende Integral Diagnostic Center from Caracas, in the Bolivian Republic of Venezuela who had received a shotgun shot in the posterior face of both thighs 6 months ago. This time he went to visit the doctor with intense pain in the left inferior member, mainly below the knee, accompanied by paresthesias in the innervation area of both bifurcation branches of the sciatic nerve. It could be demonstrated that the young man presented multiple projectile impacts in the posterior face of both thighs and hesitating gait. The radiographic studies confirmed the presence of 8 projectiles, two in the right thigh and 6 in the left one. In the lateral view it was observed that one of them was at a depth that matched with the anatomical region of the left sciatic nerve. Local anesthesia, sedation and an images intensifier were used for the operation. The projectile located in the epineurium of the sciatic nerve could be removed with the patient cooperation without complications. After 3 months the discomfort had disappeared and he could return to his usual activities.

Analysis of peptides and proteins from Asini Corii Colla using nano LC-Q-Exactive-MS/MS / 中国中药杂志

This paper aims to systematically analyze the peptides and proteins from Asini Corii Colla(ACC) through shotgun proteomics. After high-pH reversed-phase fractionation, the proteins and peptides in the hydrolysate of ACC were further separated by nano LC-Q-Exactive-MS/MS under the following conditions: Thermo Scientific EASY column(100 μm×2 cm, 5 μm, C_(18)) as precolumn, Thermo Scientific EASY column(75 μm×100 mm, 3 μm, C_(18)) for solid phase extraction, gradient elution with 0.1% formic acid in water(mobile phase A) and 84% acetonitrile in water containing 0.1% formic acid(mobile phase B), and MS in positive ion mode. Based on Uniprot_Equus caballus, MS data, and literature, 2 291 peptides were identified from ACC by MaxQuant, with 255 Maillard reactions(AML, CML, CEL)-modified peptides identified for the first time. Through alignment, the peptides were found to belong to 678 equine proteins. In conclusion, the combination of nano LC-Q-Exactive-MS/MS and shotgun proteomics achieved rapid and accurate identification of the proteins and peptides in ACC, which provides the key information and new insights for further investigation of chemicals and effective substances in ACC.

Shotgun metagenome sequencing of Chinese gut microbiota: a review / 生物工程学报

The research on the relationship between gut microbiota and human health continues to be a hot topic in the field of life science. Culture independent 16S rRNA gene high-throughput sequencing is the current main research method. However, with the reduction of sequencing cost and the maturity of data analysis methods, shotgun metagenome sequencing is gradually becoming an important method for the study of gut microbiome due to its advantages of obtaining more information. With the support from the human microbiome project, 30 805 metagenome samples were sequenced in the United States. By searching NCBI PubMed and SRA databases, it was found that 72 studies collecting about 10 000 Chinese intestinal samples were used for metagenome sequencing. To date, only 56 studies were published, including 16 related to metabolic diseases, 16 related to infectious and immune diseases, and 12 related to cardiovascular and cerebrovascular diseases. The samples were mainly collected in Beijing, Guangzhou, Shanghai and other cosmopolitan cities, where great differences exist in sequencing platforms and methods. The outcome of most studies are based on correlation analysis, which has little practical value in guiding the diagnosis and treatment of clinical diseases. Standardizing sampling methods, sequencing platform and data analysis process, and carrying out multi center parallel research will contribute to data integration and comparative analysis. Moreover, insights into the functional verification and molecular mechanism by using the combination of transcriptomics, proteomics and culturomics will enable the gut microbiota research to better serve the clinical diagnosis and treatment.

Analysis of components of proteins from Echinococcus granulosus cyst fluid / 中国血吸虫病防治杂志

Objective To analyze the components of proteins from Echinococcus granulosus cyst fluid using the shotgun method, and to identify the active components with potential regulatory effects for immune dysregulation diseases. Methods The E. granulosus cyst fluid was collected aseptically from the hepatic cysts of patients with cystic echinococcosis, and characterized by liquid chromatography (LC) tandem mass spectrometry (MS/MS) following digestion with trypsin. The protein data were searched using the software MaxQuant version 1.6.1.0 and the cellular components, molecular functions, and biological processes of the identified proteins were analyzed using the Gene Ontology (GO) method. Results The E. granulosus cyst fluid separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) had a relative molecular mass of 25 to 70 kDa. LS-MS/MS analysis identified 37 proteins, including 32 known proteins and 5 unknown proteins. At least 4 proteins were preliminarily found to exhibit potential regulatory effects for immune dysregulation diseases, including antigen B, glutathione-S-transferase (GST), thioredoxin peroxidase (TPX) and malate dehydrogenase (MDH). GO enrichment analysis showed that the identified proteins had 149 molecular functions and were involved in 341 biological processes. Conclusions E. granulosus cyst fluid has a variety of protein components, and four known proteins are preliminarily identified to be associated with immune dysregulation diseases.

Analysis of Glass Fracture Pattern on Soda Lime and Tempered Glass Caused by Shotgun Bullet Impact

@#Introduction: Broken glass exhibits unique fracture patterns depend upon the nature of the impact. The fracture patterns provide information like point and angle of impact, direction of force and sequence of firing. Recent studies have shown that the use of shotgun in Malaysia is increasing, yet, the relationship existing among the fracture pattern and the projectile impact factors are not well documented. The objective was to analyse the fracture characteristics on different glass types of variable thickness and distance made by shotgun ammunition. Methods: Soda lime and tempered glass panel with dimension of 12’x 12’ with 3 or 4 mm thickness were shot from various distances of 4, 6 and 8 m from the muzzle end of the shotgun. Samples were analysed under fixed parameters and observations were recorded. Results: It is found that the bullet hole diameter of 4 mm tempered glass were larger compared to 4 mm soda lime glass ranged from 14.33 to 24.17 cm as distance increased. Tempered glass surface also exhibited dicing fragments unlike soda lime glass where only radial fracture patterns are evident. This can be attributed to high inherent strength and ductility that makes the tempered glass remarkably resistant to external force. Conclusion: The findings from this study can lead to distinguish the type of glass through examination of fracture patterns, whether it is soda lime silica or tempered glass. The type of glass and the source of impact can be determined using the fragments, no reconstruction necessary.

Enfoque metagenómico para la caracterización del microbioma de aves corral. Revisión / Metagenomic approaches for characterization of poultry microbiome. A review

RESUMEN El pollo y el huevo son una fuente importante de proteína para el ser humano a nivel mundial. La producción de estos alimentos se ha intensificado durante los últimos años y se prevé que se produzca alrededor de 1 50 millones de toneladas de carne de pollo en 2020 (OCDE / FAO, 2018). Sin embargo, uno de los mayores problemas ligados a los procesos de producción avícola lo constituyen las enfermedades infecciosas ocasionadas por microorganismos patógenos. Entre los más relevantes se encuentran microorganismos como Salmonella ssp, Campylobacter spp, y Escherichia coli. Por lo tanto, es importante comprender los mecanismos implicados en la colonización de microorganismos patógenos que afectan a las aves de corral y sus interacciones con la microbiota gastrointestinal las cuales son clave en la mejora de la absorción de nutrientes y el fortalecimiento del sistema inmune, que influye en el crecimiento, el bienestar y la salud de las aves de corral. Sin embargo, hay poca información relacionada con la microbiota gastrointestinal de pollos parrilleros y gallinas productoras de huevo. Hasta hace poco, la caracterización se limitaba a los microorganismos que podían recuperarse a través de cultivos tradicionales. Por lo anterior, en el último tiempo se ha intensificado el uso de técnicas moleculares, entre las que se destaca la metagenómica, la cual ofrece una alternativa para una mejor comprensión de las interacciones bacterianas, la identificación de genes de resistencia a los antibióticos, identificación de elementos genéticos móviles, y el diseño de estrategias para intervenciones más efectivas con el objetivo de romper la cadena de transmisión de microorganismos patógenos durante el ciclo de producción avícola. En esta revisión, se describen los principales enfoques metagenómicos para el estudio de microbiomas de aves de corral, las técnicas de secuenciación y herramientas bioinformáticas usadas para su caracterización.

ABSTRACT Chicken and eggs are an important source of protein for humans worldwide. Production of these foods has been intensified in recent years and around 150 million tonnes of chicken meat is expected to be produced by 2020 (OECD / FAO, 2018). However, one of the biggest problems linked to poultry production processes are the infectious diseases caused by pathogenic microorganisms. Among the most relevant are found microorganisms such as Salmonella ssp, Campylobacter spp, and Escherichia coli. Therefore, it is important to understand the mechanisms involved in the colonization of pathogenic microorganisms that can affect poultry and their interactions with the gastrointestinal microbiota, which are key in improving nutrient absorption and strengthening the immune system, which it influences the growth, welfare and health of the chicken. However, there is little information related to the gastrointestinal microbiota of chicken. Until recently, the characterization was limited to microorganisms that could be recovered through culture traditional. Therefore, in the last time, it has been intensified use of molecular techniques, among those is remarked metagenomics, which offers an alternative for a better understanding of bacterial interactions, the identification of antibiotic resistance genes, identification of mobile genetic elements, and the design of strategies for more effective interventions with the aim of breaking the chain of transmission of pathogenic microorganisms during the poultry production cycle. In this review, the main metagenomics approaches are describe aimed to study microbiomes from poultry, sequencing techniques and bioinformatics tools used for its characterization.

The genetic diversity of wild and cultivated Manila clam (Ruditapes philippinarum) revealed by 29 novel microsatellite markers

Background: Microsatellite loci often used as a genetic tool for estimating genetic diversity population variation in a wide variety of different species. The application of microsatellite markers in genetics and breeding includes investigating the genetic differentiation of wild and cultured populations, assessing and determining the genetic relationship of different populations. The aim of this work is to develop several microsatellite markers via highthroughput sequencing and characterize these markers in commercially important bivalve Ruditapes philippinarum. Results: Among the two populations of R. philippinarum studied, 110 alleles were detected. The number of alleles at the cultured population ranged from 3 to 17 (mean NA = 6.897) and wild population ranged from 2 to 15 (mean NA = 6.793). The observed and expected heterozygosities of cultured population ranged from 0.182 to 0.964, and from 0.286 to 0.900, with an average of 0.647 and 0.692, respectively. The observed and expected heterozygosities of wild population ranged from 0.138 to 1.000, and from 0.439 to 0.906, with an average of 0.674 and 0.693, respectively. The polymorphism information content ranged from 0.341 to 0.910 with an average of 0.687. Sixteen and thirteen microsatellite loci deviated significantly from Hardy­Weinberg equilibrium after correction for multiple tests in cultured and wild population, respectively. Conclusions: Twenty-nine novel microsatellite loci were developed using Illumina paired-end shotgun sequencing and characterized in two population of R. philippinarum.

Genome of Leptospira borgpetersenii strain 4E, a highly virulent isolate obtained from Mus musculus in southern Brazil

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.

Study of ocular surface macro genome in dry eye patients / 眼科新进展

Objective To investigate the difference in ocular surface microbiota between dry eye patients and healthy subjects,and discuss the role of microbiota in dry eye.Methods Twenty cases of dry eye patients and 90 cases of healthy subjects were collected in the PLA General Hospital and Zhongshan Ophthalmic Center.The samples of conjunctiva impression cytology were collected from all subjects,and then metagenomic shotgun sequencing was performed following the DNA extraction.The differences in alpha diversity and metabolic pathways of the ocular surface microbiota between dry eye patients and healthy subjects were evaluated.Results There was no significant difference in alpha diversity of the microbial community between dry eye patients and healthy subjects (P =0.13).However,an increase of 15 species and a decrease of 10 species were detected on the ocular surface of dry eye patients.The enriched antibiotic resistance genes in dry eye patients were more than healthy subjects.Conclusion Although the alpha diversity of the microbial community on ocular surface between dry eye patients and healthy subjects are not distinguishable,a significant difference could be found in relative abundance and metabolic pathways,suggest that these specific microbiome may be related to the pathogenesis and disease progression of dry eye.

Identificação de adesinas de Leptospira interrogans por shotgun phage display / Identification of Leptospira interrogans adhesins by shotgun phage display

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente. Em ensaio de Western Blot os soros foram específicos no reconhecimento das proteínas recombinantes e as proteínas nativas foram verificadas em extratos de sorovares patogênicos de L. interrogans. Em ensaios de adesão, as proteínas recombinantes aderiram às células A31, LLC-PK1 e Vero e especificamente à laminina. Em ensaios de interferência em células usando laminina houve um aumento da adesão das proteínas recombinantes, o que pode ser explicado pela ligação da laminina às células e uma maior ligação das LICs estudadas. Em ensaio de localização celular usando imunofluorescência e microscopia eletrônica, foi observado que ambas as proteínas se encontram na superfície da L. interrogans. No experimento de desafio animal, a LIC12976 e a LIC13418 não se mostraram protetoras. Este trabalho contribuiu para a identificação das novas adesinas LIC13418 e LIC12976 que podem participar da virulência de leptospiras patogênicas envolvendo a primeira etapa da infecção na interação patógeno-hospedeiro

In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts. In binding assays, recombinant proteins bind to A31, LLC-PK1 and Vero cells and specifically to laminin. In interference cell assay using laminin there was an increase of recombinant protein bindings, which can be explained by the laminin binding to cells and further binding of the recombinant LICs. In cellular localization assay using immunofluorescence and electron microscopy, it was observed that both are surface proteins of L. interrogans. In the animal challenge, the LIC12976 and LIC13418 were not protective. As a whole, this work contributed to the identification of LIC12976 and LIC13418 as new adhesins and they can participate in the virulence of pathogenic Leptospira in the first stage of host pathogen interaction.

Blunt Splenic Injury by Gunshot / 대한구급학회지

Trauma is frequently not purely penetrating or purely blunt. Such mixed trauma can result from the mechanism of injury. Recently, we encountered a patient who accidentally shot himself with a shotgun. He had a 15 x 8-cm-sized penetrating injury on left flank that did not penetrate into the peritoneal cavity and a blunt splenic injury with hemoperitoneum. Surgical and interventional treatments were performed for each injury. We present this case with a review of the related literature.

Peripheral arterial embolism of pellets after shotgun injury to the abdomen: A case report.

Dealing with shotgun injury to the abdomen it is important to be aware of the possibility of missile emboli and their potential clinical effects because it usually causes vascular trauma but intravascular missile embolism is relatively rare. Vascular trauma following shotgun injuries may involve laceration of the vessel wall, pseudoaneurysm, arteriovenous fistula or missile embolism. A pellet embolus should be suspected in all cases where gunshot entry wound is present with or without an exit wound. We recently encountered a case of a close-range shotgun injury to the abdomen with subsequent embolisation of pellets to bifurcation of popliteal artery both lower limbs. However, pellet embolus is asymptomatic, there is still debate over best management because conservative management avoids surgical risks and operative removal prevents the possibility of embolus related life threatening complications. This case shows that it is necessary to do whole body imaging in all cases of shotgun injury whether exit wound present or not.

Identificação de adesinas de Leptospira interrogans por shotgun phage display / Identification of Leptospira interrogans adhesins by shotgun phage display

Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente...

In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts...

Recent progress in the methods of genome sequencing

Genome sequencing is a very important tool for the development of genetic diagnosis, drugs of gene engineering, pharmacogenetics, etc. As the HGP comes into people's ears, there is an emerging need for the genome sequencing. During the recent years, there are two different traditional strategies available for this target: shotgun sequencing and hierarchical sequencing. Besides these, many efforts are pursuing new ideas to facilitate fast and cost-effective genome sequencing, including 454 GS system, polony sequencing, single molecular array, nanopore sequencing, with each having different unique characteristics, but remains to be fully developed.

Sequenciação do genoma foi um instrumento muito importante para o desenvolvimento do diagnóstico genético, a droga da engenharia genética, farmacogenética, etc. Como o HGP entrar em ouvidos do povo, há uma necessidade emergente para a sequenciação do genoma. Durante os últimos anos, há duas diferentes estratégias tradicionais disponíveis para este objectivo: seqüenciamento shotgun hierárquico e sequenciação. Além desses, muitos esforços estão a prosseguir novas idéias para facilitar a rápida e eficaz em termos de custos sequenciação do genoma, incluindo 454 GS sistema, polony seqüenciamento, único molecular array, nanopore seqüenciamento, com cada um dos quais com diferentes características únicas, e que resta para ser mais desenvolvido.

Analysis of proteins secreted by bone marrow stromal cells using shotgun mass spectrometry / 解剖学报

Objective Using shotgun mass spectrometry to detect proteins probably contained in bone marrow stromal cells(BMSCs) conditioned medium. MethodsMixed with BMSCs conditioned medium was divided into two parts which is(>5kD and <5kD) by means of ultrafiltration. The two parts were used to culture neural stem cells(NSCs) separately, and the proportions of neurons, astrocytes and oligodendrocytes in the offsprings of NSCs were calculated, then the effective part that could regulate the differentiation of NSCs was detected by Shotgun mass spectrometry. Results The BMSCs conditioned medium which is >5kD could promote the NSCs differentiate into more neurons and oligodendrocytes. The SDS-PAGE of this part showed that the most proteins were above 14kD, then the protein bands were enzymed. In total, 456 proteins were identified by Shotgun mass spectrometry after all the protein bands were enzymed, there were 154 similar proteins, 17 hypothetical proteins and 56 unknown proteins. And in the rest of 229 proteins, most of them were cytoskeletal proteins, secreted proteins, signal transduction proteins, enzymes, transporter and so on. Conclusion Many proteins secreted by BMSCs could regulate the differentiation of NSCs so as to prove the protein components probably existed in the BMSCs conditioned medium.

A Shotgun Injury with Billiard Ball Ricochet Effect: A Case Report / 대한법의학회지

Because of the strict gun control system in Korea, deaths from gunfire injury cannot be frequently seen in the legal autopsies. Injuries or deaths among the registered hunters using shotguns, though not common, have been reported via mass media. We experienced a 52-year-old male's shotgun death in which the manner of death was mostly assumed to be a suicide. The shotgun injury presented the billiard ball ricochet effect, in which a large wound was formed on the inner side of the left thoracic cavity by spraying out of pellets while a small entry wound by bunched pellets was on the left anterior chest wall.

CitEST libraries

In order to obtain a better understanding of what is citrus, 33 cDNA libraries were constructed from different citrus species and genera. Total RNA was extracted from fruits, leaves, flowers, bark, seeds and roots, and subjected or not to different biotic and abiotic stresses (pathogens and drought) and at several developmental stages. To identify putative promoter sequences, as well as molecular markers that could be useful for breeding programs, one shotgun library was prepared from sweet orange (Citrus sinensis var. Olimpia). In addition, EST libraries were also constructed for a citrus pathogen, the oomycete Phythophthora parasitica in either virulent or avirulent form. A total of 286,559 cDNA clones from citrus were sequenced from their 5’ end, generating 242,790 valid reads of citrus. A total of 9,504 sequences were produced in the shotgun library and the valid reads were assembled using CAP3. In this procedure, we obtained 1,131 contigs and 4,083 singletons. A total of 19,200 cDNA clones from P. parasitica were sequenced, resulting in 16,400 valid reads. The number of ESTs generated in this project is, to our knowledge, the largest citrus sequence database in the world.

Two Shotgun Wounds / 대한법의학회지

Shotguns were developed as an alternative to single-missile rifle or handgun in order to enable gunner to spray a wide area with shot and thus kill birds and small animals too difficult to hit with single-missile weapons. As the popularity of shotguns grew, ammunition was developed for large game. The use of shotguns on humans has been markedly increasing in various manner of death. In forensic aspect, the shotgun wounds have characteristic findings due to their own characteristics and medical examiners may determine the range, distance, direction and kinds of gun. When the muzzle of the shotgun is placed tightly against the surface of the abdomen or thorax, the consequent wound of entrance will be single in number and circular in shape, and will have a diameter approximately equal to that of the bore of the weapon. However, when the wound is made over a site with underlying bone, especially in the scalp, the wound may have a different appearance, showing star shaped entrance due to tears from the sides of the wound and accompanying soot and burn effect. When the muzzle is held short to mid-range, considerable variation occurs in the appearance of the wound. We report two cases of shotgun wounds which showed typical findings of contact shots and distant shots.