Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection.

Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED(50) were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.

A case of dissection of intracranial cerebral arteries with segmental mediolytic "arteritis".

The intravital diagnosis of intracranial arterial dissection is not always possible due to atypic and non-specific clinical and radiological presentations. The postmortem pathological examination of cerebral blood vessels is therefore necessary to establish or confirm the presence of a dissecting aneurysm of intracranial arteries. Most of the described cases showed no significant underlying vascular pathology. Here we present the case of a 24-year-old women who died 5 days after admission to the hospital for a rapidly developing right-sided hemisyndrome. Neuroradiological examination had revealed ill-defined bifrontal hypodense lesions and angiographic findings were compatible with a dissection of the left extracranial internal carotid artery with embolic subocclusion of both anterior cerebral arteries. The pathological evaluation ruled out a thromboembolic occlusion of cerebral arteries and an extracranial internal carotid artery dissection but showed an extended dissecting process of variable age in the anterior circulation of the circle of Willis. The dissected vessels showed pathological changes characteristic of segmental mediolytic "arteritis" [Slavin and Gonzalez-Vitale 1976]. To our knowledge this is the first report on intracranial arteries being affected by this pathologic entity. Our case illustrates the importance of a postmortem examination of dissecting aneurysms of intracranial arteries. Careful serial section studies of dissected intracranial arteries in young subjects should be performed and may allow for a better understanding of the vascular pathology underlying the dissection processus.

Acute aortic dissection with carotid and coronary malperfusion: from imaging to pathology.

Postmortem imaging, including postmortem computed tomography angiography, has become an integral tool in forensic investigation in recent years. A relatively new technique, multiphase postmortem computed tomography angiography, allows detailed visualization of the vascular system and makes it possible to evaluate the dynamic perfusion of aortic branches, including the coronary arteries. Here, we report a case of aortic dissection involving the ascending aorta (type A) with coronary and carotid malperfusion. This case illustrates the complementary use of many of the diagnostic tools that are now available in forensic practice, from imaging to conventional autopsy to pathologic techniques such as immunohistochemistry.

Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

Mutations in LACERATA (LCR), FIDDLEHEAD (FDH), and BODYGUARD (BDG) cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis), of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE), which encodes a protein of RNA-processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

MRI visualized neo-intimal dissection and co-localization of novel apoptotic markers apolipoprotein C-1, ceramide and caspase-3 in a Watanabe hyperlipidemic rabbit model

PURPOSE: Apoptotic arterial wall vascular smooth muscle cell death is known to contribute to plaque vulnerability and rupture. Novel apoptotic markers like apolipoprotein C-I have been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway. In vivo relevance of these observations in an animal model of plaque rupture has not been shown. METHODS AND RESULTS: Using Watanabe rabbits, we investigated three different groups (group 1, three normal Watanabe rabbits; group 2, six Watanabe rabbits fed with high cholesterol diet for 3 months; group 3, five Watanabe rabbits with similar diet but additional endothelial denudation). We followed progression of atherosclerosis to pharmacologically induced plaque rupture non-invasively using novel 3D magnetic resonance Fast-Field-Echo angiography (TR=7.2, TE=3.6 ms, matrix=512 x 512) and Fast-Spin-Echo vessel wall imaging methods (TR=3 heart beats, TE=10.5 ms, matrix=304 x 304) on 1.5 T MRI. MRI provided excellent image quality with good MRI versus histology vessel wall thickness correlation (r=0.8). In six animals of group 2/3 MRI detected neo-intimal dissection in the abdominal aorta which was accompanied by immuno-histochemical demonstration of concomitant aforementioned novel apoptotic markers, previously implicated in the apoptotic smooth muscle cell death in vitro. CONCLUSIONS: Our studies suggest a potential role for the signal transduction pathway involving apolipoprotein C-I for in vivo apoptosis and atherosclerotic plaque rupture visualized by MRI.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

Recovery of paraplegia after type B dissection due to spinal collateral recruitment.

Acute paraplegia could be a symptom of aortic dissection due to sudden compromise of arterial spinal cord blood supply. Complete spontaneous neurologic recovery is possible and was observed in the present case 3 hours after symptom onset. Spontaneous spinal cord reperfusion after acute type B dissection was probably due to two main mechanisms. Reperfusion of false lumen and collateral vascular network recruitment, recently confirmed by anatomic animal studies, serve as potential explanations. Favorable evolution of acute paraplegia after aortic dissection exists, but prognosis is uncertain, probably due to individual variable anatomic distribution of spinal cord blood supply.

Intussusception like lesion after fenestration in aortic type B dissection.

We report on a patient with Marfan syndrome who presented a Stanford type B dissection of the descending thoracic aorta in late pregnancy. After a cesarean section, the patient presented a severe obstruction of the mesenteric superior artery. An endovascular fenestration was performed (balloon and guidewire based fenestration). Computed tomography (CT) angiography revealed an intussusception 'like' image of the abdominal aortic layers as a consequence of the fenestration procedure. Because of aneurismal progression in the abdominal aorta, surgical repair of the abdominal aorta and intussusception material removal was achieved six weeks later. The patient is currently in good condition. We conclude that the intussusception could be induced by a guidewire. This fenestration procedure is not recommended in patients with structural aortic disorders.

Functional dissection of the ash2 and ash1 transcriptomes provides insights into the transcriptional basis of wing phenotypes and reveals conserved protein interactions

Background: The trithorax group (trxG) genes absent, small or homeotic discs 1 (ash1) and 2 (ash2) were isolated in a screen for mutants with abnormal imaginal discs. Mutations in either gene cause homeotic transformations but Hox genes are not their only targets. Although analysis of double mutants revealed that ash2 and ash1 mutations enhance each other's phenotypes, suggesting they are functionally related, it was shown that these proteins are subunits of distinct complexes.Results: The analysis of wing imaginal disc transcriptomes from ash2 and ash1 mutants showed that they are highly similar. Functional annotation of regulated genes using Gene Ontology allowed identification of severely affected groups of genes that could be correlated to the wing phenotypes observed. Comparison of the differentially expressed genes with those from other genome-wide analyses revealed similarities between ASH2 and Sin3A, suggesting a putative functional relationship. Coimmunoprecipitation studies and immunolocalization on polytene chromosomes demonstrated that ASH2 and Sin3A interact with HCF (host-cell factor). The results of nucleosome western blots and clonal analysis indicated that ASH2 is necessary for trimethylation of the Lys4 on histone 3 (H3K4).Conclusion: The similarity between the transcriptomes of ash2 and ash1 mutants supports a model in which the two genes act together to maintain stable states of transcription. Like in humans, both ASH2 and Sin3A bind HCF. Finally, the reduction of H3K4 trimethylation in ash2 mutants is the first evidence in Drosophila regarding the molecular function of this trxG gene.

Transcriptional potentiation of the vitellogenin B1 promoter by a combination of both nucleosome assembly and transcription factors: an in vitro dissection.

The Xenopus laevis vitellogenin B1 promoter was assembled into nucleosomes in an oocyte extract. Subsequent RNA polymerase II-dependent transcription from these DNA templates fully reconstituted in chromatin in a HeLa nuclear extract was increased 50-fold compared with naked DNA. Remarkably, under specific conditions, production of a high level of transcripts occurred at very low DNA (1 ng/microliter) and HeLa nuclear protein (1.6 micrograms/microliters) concentrations. When partially reconstituted templates were used, transcription efficiency was intermediate between that of fully reconstituted and naked DNA. These results implicate chromatin in the process of the transcriptional activation observed. Depletion from the oocyte assembly extract of an NF-I-like factor which binds in the promoter region upstream of the TATA box (-114 to -101) or deletion from the promoter of the region interacting with this factor reduced the transcriptional efficiency of the assembled templates by a factor of 5, but transcription of these templates was still 10 times higher than that of naked DNA. Together, these results indicate that the NF-I-like factor participates in the very efficient transcriptional potentiation of the vitellogenin B1 promoter which occurs during nucleosome assembly.

Morbidity and outcome after sentinel lymph node dissection in patients with early-stage malignant cutaneous melanoma.

OBJECTIVE: Prospective analysis of the morbidity and outcome of the sentinel lymph node (SLN) technique in a consecutive series of patients with early-stage melanoma. METHODS: Between 1997 and 1998, 60 patients with stage IB-II malignant melanoma underwent SLN dissection. Preoperative dynamic lymphoscintigraphy with mapping of the lymph vessels and lymph nodes and location of the sentinel node was performed the day before SLN dissection. SLN was identified by use of the blue dye technique. SLN was assessed for histopathological and immunohistochemical examination. Postoperative morbidity and mortality were recorded. Follow-up consisted of repetitive clinical examination with lymph nodes status, laboratory and radiologic findings. RESULTS: Tumor-positive SLN was observed in 18% of the patients and stage II disease was found in 91% of the patients with positive SLN. Breslow thickness was the only significant factor predicting involvement of a SLN (p = 0.02). In 36% of the positive SLN, metastases could be assessed only by immunohistochemical examination. Postoperative complications after SLN dissection were observed in 5% in comparison with 36% after elective lymph node dissection. After a mean follow-up of 32 months, recurrence was observed in 3% with a mean disease-free survival of 8 months. Overall survival was 82% and 90% in patients with positive and negative SLN, respectively. Overall mortality was 15%, due to distant metastases in 78% of the cases. CONCLUSIONS: Staging of early-stage melanoma with the SLN dissection by use of the blue dye technique combined to lymphoscintigraphy and immunohistochemistry is reliable and safe, with less morbidity than elective lymphadenectomy. Long-term follow-up is mandatory to establish the exact reliability of SLN dissection.

Common variation in PHACTR1 is associated with susceptibility to cervical artery dissection.

Cervical artery dissection (CeAD), a mural hematoma in a carotid or vertebral artery, is a major cause of ischemic stroke in young adults although relatively uncommon in the general population (incidence of 2.6/100,000 per year). Minor cervical traumas, infection, migraine and hypertension are putative risk factors, and inverse associations with obesity and hypercholesterolemia are described. No confirmed genetic susceptibility factors have been identified using candidate gene approaches. We performed genome-wide association studies (GWAS) in 1,393 CeAD cases and 14,416 controls. The rs9349379[G] allele (PHACTR1) was associated with lower CeAD risk (odds ratio (OR) = 0.75, 95% confidence interval (CI) = 0.69-0.82; P = 4.46 × 10(-10)), with confirmation in independent follow-up samples (659 CeAD cases and 2,648 controls; P = 3.91 × 10(-3); combined P = 1.00 × 10(-11)). The rs9349379[G] allele was previously shown to be associated with lower risk of migraine and increased risk of myocardial infarction. Deciphering the mechanisms underlying this pleiotropy might provide important information on the biological underpinnings of these disabling conditions.