Extracellular Heat Shock Protein (Hsp)70 and Hsp90α Assist in Matrix Metalloproteinase-2 Activation and Breast Cancer Cell Migration and Invasion

Breast cancer is second only to lung cancer in cancer-related deaths in women, and the majority of these deaths are caused by metastases. Obtaining a better understanding of migration and invasion, two early steps in metastasis, is critical for the development of treatments that inhibit breast cancer metastasis. In a functional proteomic screen for proteins required for invasion, extracellular heat shock protein 90 alpha (Hsp90α) was identified and shown to activate matrix metalloproteinase 2 (MMP-2). The mechanism of MMP-2 activation by Hsp90α is unknown. Intracellular Hsp90α commonly functions with a complex of co-chaperones, leading to our hypothesis that Hsp90α functions similarly outside of the cell. In this study, we show that a complex of co-chaperones outside of breast cancer cells assists Hsp90α mediated activation of MMP-2. We demonstrate that the co-chaperones Hsp70, Hop, Hsp40, and p23 are present outside of breast cancer cells and co-immunoprecipitate with Hsp90α in vitro and in breast cancer conditioned media. These co-chaperones also increase the association of Hsp90α and MMP-2 in vitro. This co-chaperone complex enhances Hsp90α-mediated activation of MMP-2 in vitro, while inhibition of Hsp70 in conditioned media reduces this activation and decreases cancer cell migration and invasion. Together, these findings support a model in which MMP-2 activation by an extracellular co-chaperone complex mediated by Hsp90α increases breast cancer cell migration and invasion. Our studies provide insight into a novel pathway for MMP-2 activation and suggest Hsp70 as an additional extracellular target for anti-metastatic drug development

Monitoring the Long-Term Molecular Epidemiology of the Pneumococcus and Detection of Potential ‘Vaccine Escape’ Strains

While the pneumococcal protein conjugate vaccines reduce the incidence in invasive pneumococcal disease (IPD), serotype replacement remains a major concern. Thus, serotype-independent protection with vaccines targeting virulence genes, such as PspA, have been pursued. PspA is comprised of diverse clades that arose through recombination. Therefore, multi-locus sequence typing (MLST)-defined clones could conceivably include strains from multiple PspA clades. As a result, a method is needed which can both monitor the long-term epidemiology of the pneumococcus among a large number of isolates, and analyze vaccine-candidate genes, such as pspA, for mutations and recombination events that could result in 'vaccine escape' strains.We developed a resequencing array consisting of five conserved and six variable genes to characterize 72 pneumococcal strains. The phylogenetic analysis of the 11 concatenated genes was performed with the MrBayes program, the single nucleotide polymorphism (SNP) analysis with the DNA Sequence Polymorphism program (DnaSP), and the recombination event analysis with the recombination detection package (RDP).The phylogenetic analysis correlated with MLST, and identified clonal strains with unique PspA clades. The DnaSP analysis correlated with the serotype-specific diversity detected using MLST. Serotypes associated with more than one ST complex had a larger degree of sequence polymorphism than a serotype associated with one ST complex. The RDP analysis confirmed the high frequency of recombination events in the pspA gene.The phylogenetic tree correlated with MLST, and detected multiple PspA clades among clonal strains. The genetic diversity of the strains and the frequency of recombination events in the mosaic gene, pspA were accurately assessed using the DnaSP and RDP programs, respectively. These data provide proof-of-concept that resequencing arrays could play an important role within research and clinical laboratories in both monitoring the molecular epidemiology of the pneumococcus and detecting 'vaccine escape' strains among vaccine-candidate genes

Population Genetics of Streptococcus dysgalactiae Subspecies equisimilis Reveals Widely Dispersed Clones and Extensive Recombination

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an emerging global pathogen that can colonize and infect humans. Although most SDSE isolates possess the Lancefield group G carbohydrate, a significant minority have the group C carbohydrate. Isolates are further sub-typed on the basis of differences within the emm gene. To gain a better understanding of their molecular epidemiology and evolutionary relationships, multilocus sequence typing (MLST) analysis was performed on SDSE isolates collected from Australia, Europe and North America.The 178 SDSE isolates, representing 37 emm types, segregate into 80 distinct sequence types (STs) that form 17 clonal complexes (CCs). Eight STs recovered from all three continents account for >50% of the isolates. Thus, a small number of STs are highly prevalent and have a wide geographic distribution. Both ST and CC strongly correlate with group carbohydrate. In contrast, eleven STs were associated with >1 emm type, suggestive of recombinational replacements involving the emm gene; furthermore, 35% of the emm types are associated with genetically distant STs. Data also reveal a history of extensive inter- and intra-species recombination involving the housekeeping genes used for MLST. Sequence analysis of single locus variants identified through goeBURST indicates that genetic change mediated by recombination occurred approximately 4.4 times more frequently than by point mutation.A few genetic lineages with an intercontinental distribution dominate among SDSE causing infections in humans. The distinction between group C and G isolates reflects recent evolution, and no long-term genetic isolation between them was found. Lateral gene transfer and recombination involving housekeeping genes and the emm gene are important mechanisms driving genetic variability in the SDSE population

Validation of the content of the prevention protocol for early sepsis caused by Streptococcus agalactiaein newborns

AbstractObjective: to validate the content of the prevention protocol for early sepsis caused by Streptococcus agalactiaein newborns.Method: a transversal, descriptive and methodological study, with a quantitative approach. The sample was composed of 15 judges, 8 obstetricians and 7 pediatricians. The validation occurred through the assessment of the content of the protocol by the judges that received the instrument for data collection - checklist - which contained 7 items that represent the requisites to be met by the protocol. The validation of the content was achieved by applying the Content Validity Index.Result: in the judging process, all the items that represented requirements considered by the protocol obtained concordance within the established level (Content Validity Index > 0.75). Of 7 items, 6 have obtained full concordance (Content Validity Index 1.0) and the feasibility item obtained a Content Validity Index of 0.93. The global assessment of the instruments obtained a Content Validity Index of 0.99.Conclusion: the validation of content that was done was an efficient tool for the adjustment of the protocol, according to the judgment of experienced professionals, which demonstrates the importance of conducting a previous validation of the instruments. It is expected that this study will serve as an incentive for the adoption of universal tracking by other institutions through validated protocols

Quasispecies Theory and the Behavior of RNA Viruses

A large number of medically important viruses, including HIV, hepatitis C virus, and influenza, have RNA genomes. These viruses replicate with extremely high mutation rates and exhibit significant genetic diversity. This diversity allows a viral population to rapidly adapt to dynamic environments and evolve resistance to vaccines and antiviral drugs. For the last 30 years, quasispecies theory has provided a population-based framework for understanding RNA viral evolution. A quasispecies is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. Many predictions of quasispecies theory run counter to traditional views of microbial behavior and evolution and have profound implications for our understanding of viral disease. Here, we discuss basic principles of quasispecies theory and describe its relevance for our understanding of viral fitness, virulence, and antiviral therapeutic strategy

Analysis of whole genome sequencing for the Escherichia coli O157:H7 typing phages

Background: Shiga toxin producing Escherichia coli O157 can cause severe bloody diarrhea and haemolytic uraemic syndrome. Phage typing of E. coli O157 facilitates public health surveillance and outbreak investigations, certain phage types are more likely to occupy specific niches and are associated with specific age groups and disease severity. The aim of this study was to analyse the genome sequences of 16 (fourteen T4 and two T7) E. coli O157 typing phages and to determine the genes responsible for the subtle differences in phage type profiles. Results: The typing phages were sequenced using paired-end Illumina sequencing at The Genome Analysis Centre and the Animal Health and Veterinary Laboratories Agency and bioinformatics programs including Velvet, Brig and Easyfig were used to analyse them. A two-way Euclidian cluster analysis highlighted the associations between groups of phage types and typing phages. The analysis showed that the T7 typing phages (9 and 10) differed by only three genes and that the T4 typing phages formed three distinct groups of similar genomic sequences: Group 1 (1, 8, 11, 12 and 15, 16), Group 2 (3, 6, 7 and 13) and Group 3 (2, 4, 5 and 14). The E. coli O157 phage typing scheme exhibited a significantly modular network linked to the genetic similarity of each group showing that these groups are specialised to infect a subset of phage types. Conclusion: Sequencing the typing phage has enabled us to identify the variable genes within each group and to determine how this corresponds to changes in phage type.Public Health EnglandNational Institute for Health Research scientific research development fundBiotechnology and Biological Sciences Research Council (BBSRC

Stress-path effects on the grading of an artificial material with crushable grains

Granular materials forming part of natural slopes, embankments, foundations, pavement structures, and rail track structures are subjected to both static and dynamic loads. As a result of this particle breakage may occur. This work shows the results of an experimental investigation into the mechanical behaviour of an artificial granular material, consisting of crushed expanded clay pellets, commercially known under the brand name LECA (Light Expanded Clay Aggregate). The maximum particle size of the material tested was 2 mm; the material was reconstituted to obtain grading curves with the same mean diameter D50, and different coefficients of uniformity, U (= 3.5, 7, 14, 28) or the same coefficient of uniformity, U, and different D50 (= 0.5, 1 mm). The constant volume friction angle and the minimum and maximum densities corresponding to each grading were determined before testing in one dimensional and triaxial compression at different stress levels. The changes of grading of the material after testing were described using two parameters defined as the ratio of the mean diameter and coefficient of uniformity of the current particle size distribution and initial distribution, which were both assumed to be consistent with self-similar grading with varying fractal dimensionLes matériaux granulaires qui font partie de la pente naturelle, digues, fondations, structures de chaussées et des structures de voie ferrée se sont soumis à la fois aux charges statiques et dynamiques. À la suite de cette rupture des particules de charges peut se produire. Ce travail présente les résultats d'une étude expérimentale sur le comportement mécanique d'un matériau granulaire artificielle, composée de boulettes d'argile expansée concassées, plus communément connus sous le nom de marque LECA, comme un acronyme pour granulats d'argile expansé léger. La taille maximale des particules du matériau testé est de 2 mm, le matériau a été reconstitué à obtenir des courbes de gradation avec le même diamètre moyen D50, et différents coefficients d'uniformité, U (= 3.5, 7, 14, 28) ou le même coefficient d'uniformité , U et D50 différent (= 0,5, 1 mm). L'angle de volume constant de friction et les densités minimale et maximale correspondant à chaque classement ont été déterminés avant l'essai dans une compression triaxiale dimensions et à différents niveaux de contrainte. Les changements de classement du matériau après les essais ont été décrites à l'aide d'un seul paramètre en fonction du rapport des aires sous la granulométrie et la répartition limitant, qui ont tous deux été supposé être compatible avec auto-similaire classement avec plus ou moins la dimension fractal

Stress-path effects on the grading of an artificial material with crushable grains

Granular materials forming natural slopes, embankments, foundations, pavement structures, and rail track structures are subjected to static and dynamic loads, which may cause particle breakage to occur. Results are presented of an experimental investigation into the mechanical response of an artificial granular material, consisting of crushed expanded clay pellets, commercially known under the brand name LECA (Light Expanded Clay Aggregate) to various stress path tests. The material was reconstituted, with a maximum particle size of 2 mm, to obtain grading curves with the same mean diameter d50, and different coefficients of uniformity, U (= 3.5, 7, 14, 28) or the same U and a different d50 (= 0.5, 1 mm). The constant volume friction angle and the minimum and maximum densities corresponding to each grading were determined before stress path testing in one dimensional and triaxial compression at different stress levels. Changes in the LECA grading after the stress path tests were described using two parameters defined respectively as the mean diameter and coefficient of uniformity of the final distribution over the value of the initial distributions, both of which were assumed to be consistent with self-similar grading with varying fractal dimension