Transcriptomic Changes of Piscirickettsia salmonis During Intracellular Growth in a Salmon Macrophage-Like Cell Line.

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. P. salmonis infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of P. salmonis during host infection, we established an in vitro model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of P. salmonis genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of P. salmonis LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during P. salmonis intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during P. salmonis infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular P. salmonis alternates between a replicative phase and a stationary phase in which the stringent response is activated.

Colgajos Microvascularizados en Reconstrucción Maxilofacial: Avances de la Microcirugía / Microvascularized flaps in Maxillofacial Reconstruction: Advances in Microsurgery

RESUMEN: La reconstrucción de la cabeza y el cuello contempla avances importantes a lo largo de los años. Los colgajos microvasculares se han convertido en la primera opción de tratamiento en grandes defectos del territorio maxilofacial, mientras tanto, la tecnología con el uso de microscopía y luego las imágenes como CT, angiografía por tomografía computarizada, dispositivo ultrasónico, RNM o Doppler contribuyen a lograr una predictibilidad excepcional de estos colgajos microvasculares. Por lo general, la técnica de anastomosis consiste en una sutura de 9-0 en 360°, pero existen autores que han descrito diversos métodos que no son de sutura con un rendimiento aceptable. Existe un buen número de diferentes colgajos microvasculares, cuatro de ellos son los más comunes en la reconstrucción maxilofacial: fíbula, ilíaco, antebrazo radial, escápula. Además el colgajo anterolateral, muy útil en defectos de piel y tejidos blandos. La evolución de los colgajos microvasculares implica los colgajos quiméricos, muy útiles en defectos grandes. El objetivo de este artículo es describir y exponer el desarrollo de la microcirugía y las diversas opciones de colgajos microvasculares en la reconstrucción maxilofacial.

ABSTRACT: Head and neck reconstruction have shown important advances over the years. Microvasculars flaps transfer has become the first treatment option in large defects of the maxillofacial area. Meanwhile technology through the use of microscopy and the subsequent use of images such as CT, CT angiography, RNM or Doppler ultrasonic device, and additional new techniques have contributed to an exceptional predictability of these microvascular flaps. Typically, the anastomosis technique consists in 9-0 suture in 360°, but since the vascular flaps exist, authors have described diverse non-suture methods with acceptable performance. There are a number of different microvasculars flaps, four of them are the most common in maxillofacial reconstruction: fibula, iliac, radial forearm, scapula. In addition the anterolateral tight flap, very useful in skin and soft tissues defects. The microvascular flaps evolution involves the chimeric flaps that are useful in large defects. The aim of this article is to describe and expose microsurgery development and the diverse microvascular flap options in maxillofacial reconstruction.

Epidemics which never came: yellow fever (1883) and bubonic plague (1902-1903) in Baja California / Las epidemias que nunca llegaron: fiebre amarilla (1883) y peste bubónica (1902-1903) en Baja California.

This paper seeks to clarify the epidemic panorama that was generated in Baja California in the late nineteenth and early twentieth 20th century's, specifically that occurred in 1883 and 1902, years in which it is claimed occurred epidemics of yellow fever and bubonic plague respectively. However, as demonstrated in our study they never occurred due to social-demographic conditions in the area.

Este artículo busca aclarar el panorama epidémico que se generó en Baja California a finales del siglo XIX y principios del XX, específicamente el que se dio en 1883 y 1902, años en los que se afirma que ocurrieron epidemias de fiebre amarilla y peste bubónica, respectivamente. Sin embargo, como se demuestra en nuestro estudio, nunca ocurrieron debido a las condiciones sociodemográficas de la zona.

Target genes of Dpp/BMP signaling pathway revealed by transcriptome profiling in the early D.melanogaster embryo.

In the early Drosophila melanogaster embryo, the gene regulatory network controlled by Dpp signaling is involved in the subdivision of dorsal ectoderm into the presumptive dorsal epidermis and amnioserosa. In this work, we aimed to identify new Dpp downstream targets involved in dorsal ectoderm patterning. We used oligonucleotide D. melanogaster microarrays to identify the set of genes that are differential expressed between wild type embryos and embryos that overexpress Dpp (nos-Gal4>UAS-dpp) during early stages of embryo development. By using this approach, we identified 358 genes whose relative abundance significantly increased in response to Dpp overexpression. Among them, we found the entire set of known Dpp target genes that function in dorsal ectoderm patterning (zen, doc, hnt, pnr, ush, tup, and others) in addition to several up-regulated genes of unknown functions. Spatial expression pattern of up-regulated genes in response to Dpp overexpression as well as their opposing transcriptional responses to Dpp loss- and gain-of-function indicated that they are new candidate target genes of Dpp signaling pathway. We further analyse one of the candidate genes, CG13653, which is expressed at the dorsal-most cells of the embryo during a restricted period of time. CG13653 orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa. We characterized the enhancer region of CG13653 and revealed that CG13653 is directly regulated by Dpp signaling pathway.

Spatial and temporal distribution of Patched-related protein in the Drosophila embryo.

Patched-related (Ptr) encodes a protein with 12 potential transmembrane domains and a sterol-sensing domain that is closely related in predicted topology and domain organization to Patched, the canonical receptor of the Hedgehog pathway. Here we describe the production of an antibody specific for Drosophila Ptr and analyse its spatial and temporal distribution in the embryo. We find that at early developmental stages Ptr is predominantly localized at cell periphery but later on it becomes strongly and almost exclusively expressed in hemocytes. Interestingly Ptr null mutant embryos died without hatching. Our findings suggest that Ptr plays an essential function in Drosophila development, perhaps as a new receptor of embryonic hemocytes.

Complete genome sequence of Piscirickettsia salmonis LF-89 (ATCC VR-1361) a major pathogen of farmed salmonid fish.

Piscirickettsia salmonis, the causative agent of salmonid rickettsial septicemia (SRS), is a significant threat to the healthy and sustainable production of salmonid farming industry. This Gram-negative bacterium, originally isolated from a coho salmon in Southern Chile, produces a systemic infection characterized by colonization of several fish organs. P. salmonis is able to infect, survive, and replicate inside salmonid macrophages however little is known about its mechanisms of pathogenesis. Here, we present the whole genome sequence and annotation of the P. salmonis reference strain LF-89 (ATCC VR-1361). The genome contains one circular chromosome of 3,184,851 bp and three plasmids, pPSLF89-1 (180,124 bp), pPSLF89-2 (33,516 bp) and pPSLF89-3 (51,573 bp). A total of 2850 protein-coding genes, 56 tRNAs and six copies of 5S-16S-23S rRNA.

Transcriptional response of Atlantic salmon families to Piscirickettsia salmonis infection highlights the relevance of the iron-deprivation defence system.

BACKGROUND: Piscirickettsiosis or Salmonid Rickettsial Septicaemia (SRS) is a bacterial disease that has a major economic impact on the Chilean salmon farming industry. Despite the fact that Piscirickettsia salmonis has been recognized as a major fish pathogen for over 20 years, the molecular strategies underlying the fish response to infection and the bacterial mechanisms of pathogenesis are poorly understood. We analysed and compared the head kidney transcriptional response of Atlantic salmon (Salmo salar) families with different levels of susceptibility to P. salmonis infection in order to reveal mechanisms that might confer infection resistance. RESULTS: We ranked forty full-sibling Atlantic salmon families according to accumulated mortality after a challenge with P. salmonis and selected the families with the lowest and highest cumulative mortalities for microarray gene expression analysis. A comparison of the response to P. salmonis infection between low and high susceptibility groups identified biological processes presumably involved in natural resistance to the pathogen. In particular, expression changes of genes linked to cellular iron depletion, as well as low iron content and bacterial load in the head kidney of fish from low susceptibility families, suggest that iron-deprivation is an innate immunity defence mechanism against P. salmonis. To complement these results, we predicted a set of iron acquisition genes from the P. salmonis genome. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed that most of these genes form part of the Fur regulon of P. salmonis. CONCLUSIONS: This study revealed, for the first time, differences in the transcriptional response to P. salmonis infection among Atlantic salmon families with varied levels of susceptibility to the infection. These differences correlated with changes in the abundance of transcripts encoding proteins directly and indirectly involved in the immune response; changes that highlighted the role of nutritional immunity through iron deprivation in host defence mechanisms against P. salmonis. Additionally, we found that P. salmonis has several mechanisms for iron acquisition, suggesting that this bacterium can obtain iron from different sources, including ferric iron through capturing endogenous and exogenous siderophores and ferrous iron. Our results contribute to determining the underlying resistance mechanisms of Atlantic salmon to P. salmonis infection and to identifying future treatment strategies.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-ß superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.

Functionalities of free and open electronic health record systems.

OBJECTIVES: The aim of this study was to examine open-source electronic health record (EHR) software to determine their level of functionalities according to the International Organization for Standardization (ISO) standards. METHODS: ISO standards were used as a guideline to determine and describe the reference architecture and functionalities of a standard electronic health record system as well the environmental context for which the software has been built. Twelve open-source EHR systems were selected and evaluated according to two-dimensional criteria based on ISO/TS 18308:2004 functional requirements and ISO/TR 20514:2005 context of the EHR system. RESULTS: Open EHR software programs mostly fulfill structural, procedural, evolutional, and medicolegal requirements at the minimal and full functionality levels. Communication, privacy, and security requirements are accomplished in less than 23 percent of the cases, mainly at minimal functional level. Ethical, cultural, and consumer requirements still need to be fulfilled by free and open-source EHR applications. CONCLUSIONS: Most analyzed systems had several functional limitations. Nevertheless, especially for clinicians and decision makers in developing countries, open-source EHR systems are an option. The limited functionalities are likely to become requirements for further releases of open-source EHR systems.

Biometrics for electronic health records.

Securing electronic health records, in scenarios in which the provision of care services is share among multiple actors, could become a complex and costly activity. Correct identification of patients and physician, protection of privacy and confidentiality, assignment of access permissions for healthcare providers and resolutions of conflicts rise as main points of concern in the development of interconnected health information networks. Biometric technologies have been proposed as a possible technological solution for these issues due to its ability to provide a mechanism for unique verification of an individual identity. This paper presents an analysis of the benefit as well as disadvantages offered by biometric technology. A comparison between this technology and more traditional identification methods is used to determine the key benefits and flaws of the use biometric in health information systems. The comparison as been made considering the viability of the technologies for medical environments, global security needs, the contemplation of a share care environment and the costs involved in the implementation and maintenance of such technologies. This paper also discusses alternative uses for biometrics technologies in health care environments. The outcome of this analysis lays in the fact that even when biometric technologies offer several advantages over traditional method of identification, they are still in the early stages of providing a suitable solution for a health care environment.

Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis.

BACKGROUND: Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis. RESULTS: Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction. CONCLUSION: Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.See related minireview by Vichas and Zallen: http://www.jbiol.com/content/8/8/76.

The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts.

Porcine circovirus type 2 (PCV2)-associated diseases are considered to be the biggest problem for the worldwide swine industry. The PCV2 capsid protein (Cap) is an important antigen for development of vaccines. At present, most anti-PCV2 vaccines are produced as injectable formulations. Although effective, these vaccines have certain drawbacks, including stress with concomitant immunosuppresion, and involve laborious and time-consuming procedures. In this study, Saccharomyces cerevisiae was used as a vehicle to deliver PCV2 antigen in a preliminary attempt to develop an oral vaccine, and its immunogenic potential in mice was tested after oral gavage-mediated delivery. The cap gene with a yeast-optimized codon usage sequence (opt-cap) was chemically synthesized and cloned into Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2, under the control of the Gal1 promoter. Intracellular expression of the Cap protein was confirmed by Western blot analysis and its antigenic properties were compared with those of baculovirus/insect cell-produced Cap protein derived from the native PCV2 cap gene. It was further demonstrated by electron micrography that the yeast-derived PCV2 Cap protein self-assembles into virus-like particles (VLPs) that are morphologically and antigenically similar to insect cell-derived VLPs. Feeding raw yeast extract containing Cap protein to mice elicited both serum- and fecal-specific antibodies against the antigen. These results show that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles, and that oral yeast-mediated antigen delivery is an alternative strategy to efficiently induce anti-PCV2 antibodies in a mouse model, which is worthy of further investigation in swine.

Regulatory network for cell shape changes during Drosophila ventral furrow formation.

Rapid and sequential cell shape changes take place during the formation of the ventral furrow (VF) at the beginning of Drosophila gastrulation. At the cellular level, this morphogenetic event demands close coordination of the proteins involved in actin cytoskeletal reorganization. In order to construct a regulatory network that describes these cell shape changes, we have used published genetic and molecular data for 18 genes encoding transcriptional regulators and signaling pathway components. Based on the dynamic behavior of this network we explored the hypothesis that the combination of three recognizable phenotypes describing wild type or mutant cell types, during VF invagination, correspond to different activation states of a specific set of these gene products, which are point attractors of the regulatory network. From our results, we recognize missing components in the regulatory network and suggest alternative pathways in the regulation of cell shape changes during VF formation.

Identification of genes expressed during Drosophila melanogaster gastrulation by using subtractive hybridization.

A subtractive hybridization approach was used to identify genes that are expressed at the beginning of gastrulation. We used tester DNA complimentary to RNA (cDNA) prepared from stages 6-7 embryos (gastrula) and excess driver cDNA from stages 2-4 embryos (syncytial blastoderm) to generate a gastrula-subtracted cDNA library. A reverse Northern blot procedure used to analyze 105 subtracted clones showed that 65% had a level of expression at least 2.5-fold higher in stages 6-7 versus stages 2-4 embryos. We determined the nucleotide sequence of these clones and identified 49 individual sequences, including 33 previously uncharacterized genes. We verified the level of expression of 24 genes during Drosophila melanogaster embryogenesis using a semiquantitative polymerase chain reaction (PCR) approach. As expected, all of the selected clones showed their highest level of expression after stages 2-4 of embryogenesis, including several that displayed peaks of expression during gastrulation. Three genes that were expressed at their highest levels in stages 6-7 were further analyzed by 5'-rapid amplification of cDNA ends (RACE) analysis, Northern blot assays and in situ hybridization. Our results indicate that these genes exhibited temporal and spatially restricted patterns of expression in developing embryos, and moreover, their transcripts were detected in cells that undergo morphological changes during the gastrulation stage. Characterizing the role of these genes will be important to increase our understanding of the molecular mechanisms that regulate cellular activities during D. melanogaster gastrulation.

A Model System for Studying the Penetration of Microorganisms into Meat.

This study assessed the effect of line pressure used in spraying meat surfaces on removal of inoculated bacteria and on penetration of Blue Lake, an insoluble dye, into sprayed meat. Pressures used in this analysis were 690, 2070, 4140, and 6200 kPa. The highest pressure, 6200 kPa, removed significantly less aerobic bacteria than the other three lower pressures. No significant differences in removal of Enterobacteriaceae were noted due to pressure. Nozzle type was also a variable in the study on penetration. Type of nozzle had a significant effect on the penetration of the Blue Lake into the meat at a pressure of 6200 kPa. An equation describing penetration of the Blue Lake into the meat is given.