Transcriptional responses are oriented towards different components of the rearing environment in two Drosophila sibling species.

BACKGROUND: The chance to compare patterns of differential gene expression in related ecologically distinct species can be particularly fruitful to investigate the genetics of adaptation and phenotypic plasticity. In this regard, a powerful technique such as RNA-Seq applied to ecologically amenable taxa allows to address issues that are not possible in classic model species. Here, we study gene expression profiles and larval performance of the cactophilic siblings Drosophila buzzatii and D. koepferae reared in media that approximate natural conditions and evaluate both chemical and nutritional components of the diet. These closely related species are complementary in terms of host-plant use since the primary host of one is the secondary of the other. D. koepferae is mainly a columnar cactus dweller while D. buzzatii prefers Opuntia hosts. RESULTS: Our comparative study shows that D. buzzatii and D. koepferae have different transcriptional strategies to face the challenges posed by their natural resources. The former has greater transcriptional plasticity, and its response is mainly modulated by alkaloids of its secondary host, while the latter has a more canalized genetic response, and its transcriptional plasticity is associated with the cactus species. CONCLUSIONS: Our study unveils a complex pleiotropic genetic landscape in both species, with functional links that relate detox responses and redox mechanisms with developmental and neurobiological processes. These results contribute to deepen our understanding of the role of host plant shifts and natural stress driving ecological specialization.

Veterinary-focused assessment with sonography for trauma-airway, breathing, circulation, disability and exposure: a prospective observational study in 64 canine trauma patients.

OBJECTIVE: To describe the technique and findings of the 'veterinary focused assessment with sonography for trauma-airway, breathing, circulation, disability and exposure' protocol in dogs suffering from trauma. MATERIALS AND METHODS: Prospective observational study on a new point-of-care ultrasound protocol on 64 dogs suffering from trauma and comparison of findings with radiology. RESULTS: Comparison of the results of this new ultrasound protocol for trauma patients with radiography findings for pneumothorax, pleural effusion, alveolar-interstitial syndrome and abdominal effusion revealed positive agreement of 89, 83, 100 and 87% and negative agreement of 76, 83, 76 and 92%, respectively. Novel findings of the 'veterinary focused assessment with sonography for trauma-airway, breathing, circulation, disability and exposure' exam, which were not previously reported for dogs undergoing focused assessment with sonography for trauma, included alveolar-interstitial syndrome (suggestive of pulmonary contusions), diaphragmatic hernia, retroperitoneal effusion and tracheal injury. Our new technique may also help identify increased intracranial pressure via changes in optic nerve sheath diameter and haemodynamic instability through the evaluation of the caudal vena cava and cardiac function. CLINICAL SIGNIFICANCE: The described ultrasound examination protocol can be rapidly performed on dogs suffering from trauma during resuscitation and it may detect injuries previously undetectable using other veterinary point-of-care ultrasound protocols.

Molecular typing of Escherichia coli O157:H7 isolates from Swedish cattle and human cases: population dynamics and virulence.

While all verotoxin-producing Escherichia coli O157:H7 bacteria are considered potential pathogens, their genetic subtypes appear to differ in their levels of virulence. The aim of this study was to compare the distribution of subtypes of E. coli O157:H7 in the cattle reservoir and in human cases with and without severe complications in order to gain clues about the relationship between subtype and relative virulence. A lineage-specific polymorphism assay (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a novel real-time PCR assay to identify clade 8 were applied to a large and representative set of isolates from cattle from 1996 to 2009 (n = 381) and human cases from 2008 to 2011 (n = 197) in Sweden. Draft genome sequences were produced for four selected isolates. The E. coli O157:H7 isolates in Swedish cattle generally belonged to four groups with the LSPA-6 profiles 211111 (clade 8/non-clade 8), 213111, and 223323. The subtype composition of the cattle isolates changed dramatically during the study period with the introduction and rapid spread of the low-virulence 223323 subtype. The human cases presumed to have been infected within the country predominantly carried isolates with the profiles 211111 (clade 8) and 213111. Cases progressing to hemolytic-uremic syndrome (HUS) were mostly caused by clade 8, with MLVA profiles consistent with Swedish cattle as the source. In contrast, infections contracted abroad were caused by diverse subtypes, some of which were associated with a particular region. The work presented here confirms the high risk posed by the clade 8 variant of E. coli O157:H7. It also highlights the dynamic nature of the E. coli O157:H7 subtype composition in animal reservoirs and the importance of this composition for the human burden of disease.

Genome Sequence of Streptococcus agalactiae Strain 09mas018883, Isolated from a Swedish Cow.

We announce the complete genome sequence of Streptococcus agalactiae strain 09mas018883, isolated from the milk of a cow with clinical mastitis. The availability of this genome may allow identification of candidate genes, leading to discovery of antigens that might form the basis for development of a vaccine as an alternative means of mastitis control.

Chromosomal distribution of 18S-25S rDNA in four Lupinus species visualized by fluorescence in situ hybridization.

The number and position of 18S-25S rDNA sites in 4 selected Lupinus species are reported for the first time. L. atlanticus, L. subcarnosus and L. paniculatus had two rDNA loci, while L. albus exhibited only one loci. Among these 4 species, all of them exhibited one large pair of strong signals that extends from the short arm to a NOR on a chromosome satellite. L. atlanticus, L. subcarnosus, L. paniculatus had one more locus of 18-25S rDNA, but a pair of weak hybridization signals were observed in L. paniculatus when 18S-25S rDNA was used as probe. The results are discussed in terms of the evolutionary relationships among these species.

Major ampullate spidroins from Euprosthenops australis: multiplicity at protein, mRNA and gene levels.

Spider dragline silk possesses extraordinary mechanical properties. It consists of large fibrous proteins called spidroins that display modular structures. It is known to consist of two proteins: the major ampullate spidroin (MaSp) 1 and MaSp2. This study analyses MaSp sequences from the nursery-web spider Euprosthenops australis. We have identified a previously uncharacterized MaSp2 sequence and a new MaSp-like spidroin, which display distinct homogenous submotifs within their respective Gly-rich repeats. Furthermore, a group of MaSp1 cDNA clones show unexpected heterogeneity. Genomic PCR identified several MaSp1 gene variants within individual spiders, which suggests the presence of a gene cluster in E. australis. Finally, the evolution of spidroin genes is discussed in relation to phylogenetic analysis of nonrepetitive C-terminal domains from diverse species.

Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.

Development of rapeseed with high erucic acid content by asymmetric somatic hybridization between Brassica napus and Crambe abyssinica.

PEG-induced asymmetric somatic hybridization between Brassica napus and Crambe abyssinica was carried out. C. abyssinica is an annual cruciferous oil crop with a high content of erucic acid in the seed oil valuable for technical purposes. UV-irradiated mesophyll protoplasts of C. abyssinica cv 'Carmen' and cv 'Galactica' were fused with hypocotyl protoplasts of different genotypes of B. napus cv 'Maplus' and breeding line '11502'. Shoot regeneration frequency varied between 6.1% and 20.8% among the different doses of UV-irradiation, ranging from 0.05 J/cm(2) to 0.30 J/cm(2). In total, 124 shoots were regenerated, of which 20 asymmetric somatic hybrids were obtained and verified by nuclear DNA content and AFLP analysis. AFLP data showed that some of the characteristic bands from C. abyssinica were present in the hybrids. Cytological analysis of these hybrids showed that 9 out of 20 asymmetric hybrids had 38 chromosomes, the others contained 40-78 chromosomes, having additional chromosomes between 2 and 40 beyond the 38 expected for B. napus. The investigation into the fertility of asymmetric somatic hybrids indicated that the fertility increased with increasing UV-doses ranging from 0.05 J/cm(2) to 0.15 J/cm(2). All of the hybrids were cultured to full maturity, and could be fertilized and set seeds after self-pollination or backcrosses with B. napus. An analysis of fatty acid composition in the seeds was conducted and found to contain significantly greater amounts of erucic acid than B. napus. This study indicates that UV-irradiation could be used as a tool to produce asymmetric somatic hybrids and to promote the fertility of the hybrids.

Colloid and crystalloid resuscitation.

Use of colloid and crystalloid fluids during resuscitation can be a complicated process. An understanding by the veterinarian of the patient's cardiovascular state, the underlying pathologic process, and the characteristics of crystalloid and colloid fluids available is necessary for establishing a fluid therapy plan. Through frequent reassessment and tailoring of the fluid plan according to the patient's response, the risks of fluid overload and fluid deficiency are reduced.

A 1.8kb GFAP-promoter fragment is active in specific regions of the embryonic CNS.

The intermediate filament glial fibrillary acidic protein (GFAP) constitutes the major cytoskeletal protein in astrocytes (J. Neuroimmunol. 8 (1985) 203) and is traditionally referred to as a specific marker for astrocytes. To identify early glial precursors, we created GFAPpromoter-lacZ transgenic mice, using a 1.8kb 5' fragment of human GFAP. The expression of the transgene was first detected in the neuroepithelium at embryonic day 9.5. It was further found in the ventricular zone of the developing telencephalon, in the cerebellar primordium, trigeminal ganglia, and radial glia. Later, scattered beta-gal+ cells were seen in pons, brain stem and glia limitans. The results indicate that GFAP activity is regulated in a region-specific manner during central nervous system (CNS) development and that the gene is turned on in different cell types independently.

Comparative analysis of a BAC contig of the porcine RN region and the human transcript map: implications for the cloning of trait loci.

The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals.

Colloid osmometry.

Complications related to intravascular fluid resuscitation and maintenance can become life-threatening. Overhydration and underhydration can lead to significant perfusion abnormalities and can delay or prevent recovery. A working knowledge of transcapillary fluid dynamics gives the veterinarian the basis for evaluating the cause of alterations that make up Starling's forces. By combining this knowledge with patient assessment and laboratory diagnostics, the veterinarian can make a logical decision about the best treatment to correct the Starling's force alteration. Colloid osmometry is a particularly useful tool for assessing a patient's colloid osmotic pressure. It allows the veterinarian to distinguish between reduced oncotic pressure and increased hydrostatic pressure as a cause for intravascular fluid loss or edema formation.

Tracheal rupture associated with intubation in cats: 20 cases (1996-1998).

OBJECTIVE: To characterize clinical features of tracheal rupture associated with endotracheal intubation in cats and to evaluate the most appropriate treatment for this condition. DESIGN: Retrospective study. ANIMALS: 20 cats with a history of endotracheal intubation that subsequently developed dyspnea or subcutaneous emphysema. PROCEDURE: Medical records of cats with a presumptive diagnosis of tracheal rupture associated with intubation were reviewed. Clinical and clinicopathologic data were retrieved. RESULTS: Cats were evaluated 5 hours to 12 days after a surgical or medical procedure requiring general anesthesia with intubation had been performed. Fourteen (70%) cats were evaluated after dental prophylaxis. All cats radiographed had pneumomediastinum and subcutaneous emphysema. Eighteen of 19 cats were initially treated medically. Duration of medical treatment for cats that did not have surgery ranged from 12 to 72 hours. Cats that had surgery received medical treatment 3 to 24 hours prior to the surgical procedure. Medical treatment alone was administered to 15 cats that had moderate dyspnea, whereas surgical treatment was chosen for 4 cats that had severe dyspnea (open-mouth breathing despite treatment with oxygen) or worsening subcutaneous emphysema. Eighteen cats had improvement of clinical signs, 1 cat died after surgery, and 1 cat died before medical or surgical intervention. CONCLUSIONS AND CLINICAL RELEVANCE: Most cats with tracheal rupture associated with intubation can be treated medically. Cats with worsening clinical signs (severe dyspnea, suspected pneumothorax, or worsening subcutaneous emphysema) should have surgery performed immediately to correct the defect.

Effects of inducible glial fibrillary acidic protein on glioma cell motility and proliferation.

We have studied the effect of induced glial fibrillary acidic protein (GFAP) on motility, cell morphology, and proliferation of two originally GFAP-negative human glioma cell lines. Glioma cell lines U-1242 MG and U-251 MG sp subclone 3A were transfected with a vector system that allows for an inducible GFAP expression. This experimental system creates an "on/off" situation in which GFAP expression is suppressed by tetracycline. Inducible expression of GFAP in the absence of tetracycline was confirmed by immunofluorescence staining and Northern and Western blotting. The study showed that forced GFAP expression resulted in an inhibition of cell motility measured as the phagokinetic track area of individual cells seeded sparsely on a surface covered with gold particles. It also resulted in a change in cell morphology, with extended cell processes, and it was associated with a low fraction of cells in S-phase. We conclude that the down-regulation of GFAP expression that is often seen in gliomas in vivo may be an important parameter of tumor progression related mainly to the motile and thereby invasive properties of malignant glioma cells.

Modulation of phenotype and induction of irregular vessels accompany high tumorigenic potential of clonal human glioma cells xenografted to nude-rat brain.

Three phenotypically different clonal human glioma cell lines were injected stereotactically into nude-rat brains, to determine their individual growth potential and to establish an in vivo system in which different therapeutic modalities could be tested. As assessed by serial sectioning, microscopic evaluation, and computer analysis, the mean approximate tumour volume after 3-7 weeks in vivo was 0.42 mm(3) for U-343 MG, 2.6 mm(3) for U-343 MGa Cl2:6, and 50.3 mm(3) for U-343 MGa 31L. When compared with the initial injected cell volume, only U-343 MGa 31L had increased in size, U-343 MGa Cl2:6 remained approximately the same but showed a certain proliferative potential, and U-343 MG regressed. Thus, only U-343 MGa 31L cells had high tumorigenic potential, invaded and replaced brain tissue in every direction, while U-343 MGa Cl2:6 cells grew in sheet-like tumour extensions along white-matter nerve-fibre tracts, in this respect mimicking foetal astrocytes. The tumorigenic potential of the U-343 MGa 31L cell clone was associated with a variable phenotype, as observed when the in vivo and in vitro characteristics were compared. The in vivo phenotype was characterized by the loss of GFAP immunoreactivity, the gain of heterogeneously distributed cellular tenascin, fibronectin, and laminin, but absence of extracellularly deposited material, and by the formation of irregular vessels. It appears that the intrinsic capacity of glioma cells to adapt to in vivo conditions is decisive for their tumorigenicity in the brain, rather than any single phenotypic property in itself. Moreover, the 2 glioma cell clones best suited for in vitro growth were no longer tumorigenic.

Comparison of esophageal Doppler monitor generated minute distance and cardiac output in a porcine model of ventricular fibrillation.

The primary goal of cardiopulmonary resuscitation (CPR) is to increase cardiac output (CO), providing adequate tissue perfusion and oxygenation to maintain normal organ function. A non-invasive, easy to use, commercially available esophageal doppler monitor (EDM, Deltex) has been found to provide minute distance (MD), which is the distance moved by a column of blood through the aorta in 1 min. The goal of our study was to determine if CO measurements correlate with the EDM MD, before and during cardiac arrest, in a porcine model of ventricular fibrillation. Twenty pigs were anesthetized and an EDM was placed. MD measurement using EDM, and CO measurement using florescent microsphere injections were compared before and during CPR. MD correlated well with CO (r2 = 0.96) before and during CPR. Based on the excellent correlation between MD as determined by EDM and CO by florescent microsphere technique, it appears that the non-invasive use of the EDM may play a valuable role in determination of CO during CPR.

The beta-gal interferon assay: a new, precise and sensitive method.

Cells of a human glioblastoma line were stably transfected with a glial fibrillary acidic protein (GFAP) promoter sequence/lacZ reporter gene. Following this modification, they produced Escherichia coli beta-galactosidase constitutively in amounts that could be measured through their conversion of an added fluorophore into a product readily estimated by fluorimetry. Human interferons (IFN) selectively and in a dose-dependent manner reduce the formation of beta-galactosidase in this system. We have used it as the basis for a novel assay that is sensitive (4-40 pg/ml), precise, completed in 30 h, and applicable to both type I and type II human IFNs. Statistical analysis showed interassay relative standard deviations ranging from 5% to 11%, and most individual assays revealed potencies with limits of error within 85%-115%. Neither partially trypsin-digested IFN nor the other cytokines and mitogens we tested reacted in this system, except for tumor necrosis factor-alpha (TNF-alpha). The high selectivity was further shown by the loss of response to IFN in the presence of the appropriate specific anti-IFN or anti-IFN-gamma receptor antibodies.

PDGF induces reorganization of vimentin filaments.

In this study we demonstrate that stimulation with platelet-derived growth factor (PDGF) leads to a marked reorganization of the vimentin filaments in porcine aortic endothelial (PAE) cells ectopically expressing the PDGF beta-receptor. Within 20 minutes after stimulation, the well-spread fine fibrillar vimentin was reorganized as the filaments aggregated into a dense coil around the nucleus. The solubility of vimentin upon Nonidet-P40-extraction of cells decreased considerably after PDGF stimulation, indicating that PDGF caused a redistribution of vimentin to a less soluble compartment. In addition, an increased tyrosine phosphorylation of vimentin was observed. The redistribution of vimentin was not a direct consequence of its tyrosine phosphorylation, since treatment of cells with an inhibitor for the cytoplasmic tyrosine kinase Src, attenuated phosphorylation but not redistribution of vimentin. These changes in the distribution of vimentin occurred in conjunction with reorganization of actin filaments. In PAE cells expressing a Y740/751F mutant receptor that is unable to bind and activate phosphatidylinositol 3'-kinase (PI3-kinase), the distribution of vimentin was virtually unaffected by PDGF stimulation. Thus, PI3-kinase is important for vimentin reorganization, in addition to its previously demonstrated role in actin reorganization. The small GTPase Rac has previously been shown to be involved downstream of PI3-kinase in the reorganization of actin filaments. In PAE cells overexpressing dominant negative Rac1 (N17Rac1), no change in the fine fibrillar vimentin network was seen after PDGF-BB stimulation, whereas in PAE cells overexpressing constitutively active Rac1 (V12Rac1), there was a dramatic change in vimentin filament organization independent of PDGF stimulation. These data indicate that PDGF causes a reorganization of microfilaments as well as intermediate filaments in its target cells and suggest an important role for Rac downstream of PI3-kinase in the PDGF stimulated reorganization of both actin and vimentin filaments.