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.

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.

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.

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.

Expression of recombinant platelet-derived growth factor A- and B-chain homodimers in rat-1 cells and human fibroblasts reveals differences in protein processing and autocrine effects.

The autocrine effects of platelet-derived growth factor (PDGF) A- and B-chain homodimers (PDGF-AA and PDGF-BB) on rat-1 cells and human fibroblasts have been investigated by using human PDGF A- and B-chain cDNA clones expressed in a retroviral vector. Infection with replication-defective virus carrying the B-chain cDNA resulted in a phenotypical transformation resembling that induced by simian sarcoma virus. The resulting cells were focus forming in monolayer cultures, grew to high saturation densities, and formed large colonies in soft agar. The PDGF A-chain transfectants showed no transformed morphology and lacked focus-forming activity but grew to high saturation density in monolayer culture and formed small colonies in soft agar. A similar but weaker effect was obtained with an A-chain cDNA variant containing a 69-base-pair insertion in the 3' end of the protein-coding domain. A- and B-chain transfectants released PDGF receptor-competing activity into the medium, but only the medium conditioned by the B-chain transfectants possessed potent mitogenic activity on human fibroblasts. Both types of transfectants had downregulated levels of PDGF receptors; however, the B-chain transfectants were downregulated to significantly lower levels. Metabolic labeling and immunoprecipitations with PDGF antiserum showed that the PDGF B-chain protein was processed to a 24-kilodalton cell-associated and a 30-kilodalton secreted dimeric protein. The A-chain protein was rapidly secreted as a 31-kilodalton dimeric protein. The present study shows a marked difference in the autocrine effects of PDGF-AA and -BB expressed under the control of a retroviral promoter and suggests that different biological properties may be assigned to these two PDGF isoforms.

Escape from senescence in hybrid cell clones involves deletions of two regions located on human chromosome 1q.

Human normal cells have been shown to undergo a limited number of cell doublings, a phenomenon termed cellular senescence. Human chromosome 1 has been implicated in this process, and several lines of evidence indicate that there is a senescence-inducing gene or genes on human chromosome 1q. Our approach to analyze the senescence-inducing effect of chromosome 1 includes the use of somatic cell hybrid revertants. We show here that fusion of a hypoxanthine phosphoribosyl transferase-negative mouse cell line (A9) containing a human neo-tagged chromosome 1 with an immortal hamster cell line (10W-2) results in cell hybrids that senesce after a few population doublings. Rare revertants that had escaped senescence were obtained after one large fusion experiment. Thirty-five nonsenescent hybrids were obtained from a total of approximately 1 million hybrids, and 25 of these were subjected to further analysis. The presence of a single copy of human chromosome 1 in the revertant hybrids was confirmed by fluorescence in situ hybridization analysis using a chromosome 1-specific painting probe. No visible translocations or deletions of chromosome 1 were observed in any of the hybrids. Deletion mapping revealed that 11 (56%) of the hybrids analyzed had lost one or more markers on chromosome 1q. Two regions with deletions were detected, one of which has been shown to be implicated in the senescence-inducing effect exerted by chromosome 1 following monochromosome transfer (P. J. Vojta et al., manuscript submitted for publication). The present study suggests that two separate loci on human chromosome 1q may be of importance for the induction of senescence. Moreover, this set of nonsenescent revertants could be useful for future detailed analyses of the senescence-inducing loci.

Cell scattering and migration induced by autocrine transforming growth factor alpha in human glioma cells in vitro.

In the present investigation, we have transfected a human malignant glioma cell line, U-1242 MG, and derived clones that produce transforming growth factor alpha (TGF-alpha) in an inducible manner using the tetracycline suppressible vector system. TGF-alpha expression was confirmed by Northern analysis, by ELISA, and by immunoprecipitation of metabolically labeled cells. The functional activity of the induced protein was proven by the finding of epidermal growth factor receptor (EGFR) tyrosine phosphorylation on induction of TGF-alpha. A clear effect on cell motility, i.e., cell scattering and an increased phagokinetic track area of individual glioma cells, was demonstrated. The fact that the EGFR tyrosine kinase activation was independent of cell density suggests that autocrine activation of the EGFR kinase occurred at the single-cell level. These findings are of interest, because increased cell motility is most likely a requirement for glioma cell invasion in vivo. The results imply that as a result of coexpression of EGFR and its ligand, individual glioma cells are capable of acting as independent autocrine locomotory units.

Human glial fibrillary acidic protein: complementary DNA cloning, chromosome localization, and messenger RNA expression in human glioma cell lines of various phenotypes.

Glial fibrillary acidic protein (GFAP) is a constituent of intermediate filaments of glial cells of the astrocyte lineage. We cloned a human GFAP complementary DNA, deduced the amino acid sequence, and established the chromosomal location (17q21) of the GFAP gene by Southern blot hybridization of somatic cell hybrids and by in situ hybridization. The authenticity of the complementary DNA was proven by expressing it in glioma cells lacking endogenous GFAP; after microinjection of the complementary DNA, such cells became positive for staining with GFAP antibodies. The levels of fibronectin (FN) and GFAP mRNA of ten human glioblastoma cell lines, determined by Northern blot hybridization of RNA, were related to other phenotypic characteristics [cell morphology and expression of the genes encoding platelet-derived growth factor (PDGF) receptors]. A high expression of GFAP mRNA was found only in cells lacking fibronectin mRNA and protein. Glioma cells with a fibroblastic phenotype (bipolar, FN+/GFAP-) were found to express both types of PDGF receptors (alpha and beta). Relatively high levels of PDGF alpha-receptor mRNA, in the absence of beta-receptor expression, were found in cell lines that express GFAP and lack detectable levels of fibronectin mRNA. The findings are compatible with the idea that the genes encoding PDGF receptors in glioma cells are regulated in concert with other genes, the expression of which may reflect the developmental program of normal glia cell lineages.

Molecular cloning and functional characterization of the human platelet-derived growth factor alpha receptor gene promoter.

Expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) is strictly regulated during mammalian development and tumorigenesis. The molecular mechanisms involved in the specific regulation of PDGF alpha R expression are unknown, but transcriptional regulation of the PDGF alpha R gene is most likely to be involved. This study describes the molecular cloning of the non-coding exon 1 and approximately 2 kb of 5' flanking region of the human PDGF alpha R gene. This 5' flanking region is a functional promoter of the PDGF alpha R gene as concluded from its capacity to drive luciferase reporter gene expression in an orientation dependent way. Analysis of 5' promoter deletion mutants revealed that the region from -441 to +118, relative to the transcription initiation site, is sufficient to establish high level promoter activity. In addition, the morphogen retinoic acid, alone or in combination with dibutyryl cAMP, gives a 22-fold induction of PDGF alpha R gene promoter activity in human teratocarcinoma cells. This effect is mediated through specific transcription factor binding within the -52/+118 region of the PDGF alpha R gene.

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.

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.

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.

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.

The role of blood component therapy in the management of canine and feline patients with cancer.

Blood component therapy may be required for a variety of pathologic reasons in the cancer patient. The tumor itself not only affects various cell lines, but treatment with chemotherapeutic agents can induce cytopenias. In addition, chronic and acute hemorrhage can be caused by tumor erosion and rupture or by paraneoplastic coagulopathies. Component replacement will depend on the underlying hematologic deficiencies and should be performed in conjunction with treating the cause of the deficiency.

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.

Hypovolemic shock and resuscitation.

Hypovolemic shock is the manifestation of hypoperfusion from inadequate intravascular volume resulting in cellular hypoxia. Early, effective treatment tailored to the individual patient will minimize morbidity and mortality. The causes and end-organ responses can differ with each patient, requiring an understanding of the underlying physiology and pathophysiology. Treatment always consists of oxygen and isotonic crystalloids and may require the use of colloids, corticosteroids, antibiotics, and vasomotor drugs.

Fluid therapy. Crystalloids and colloids.

Appropriate fluid therapy requires an understanding of fluid dynamics and pathologic alterations during various disease states. In addition, it requires an understanding of the pharmacologic differences of available fluids. Crystalloid fluids and colloid fluids can be used in combination to treat a variety of volume-deficient states. Synthetic colloids are also used to treat hyponcotic states. Blood products are used to replace blood components and coagulation proteins. Frequent monitoring and reassessment are required to prevent volume overload and life-threatening edema.

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.

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.