Non-enhanced CT Maximum Intensity Projections for the Detection of Large Vessel Occlusions.

BACKGROUND: Identification of large vessel occlusions (LVO) is important with recent guidelines supporting endovascular therapy in selected acute ischemic stroke patients. Many stroke centers perform CT angiography (CTA) in patients with suspected LVO, however this requires additional time and contrast administration. Non-enhanced CT maximum intensity projection (NECT-MIPs) may offer a rapid alternative to CTA. METHODS: We retrospectively reviewed acute stroke patients with LVO in the UCSD Stroke Registry, presenting between 6/2014-7/2016. NECT-MIPs were evaluated for presence of LVO. Gold standard comparison was to CTA. Results were stratified by level of training (Faculty, Fellow and Acute Care Practitioners [ACPs]). Inter-rater agreement was assessed using Fleiss' Kappa Coefficient. RESULTS: We reviewed 24 patients using NECT-MIPs for the detection of LVO. Faculty had a sensitivity and specificity of 95% & 92% for ICA/M1, 42% & 100% for M2, and 67% & 96% for basilar occlusions. Fellows and ACPs had a sensitivity and specificity of 61% & 94% for ICA/M1, 19% & 83% for M2, and 75% & 95% for basilar occlusions. Inter-rater agreement among Faculty readers was k=0.75 for ICA/M1, k=0.79 for M2 and k=0.14 for basilar occlusions. Among Fellows and ACPs, k=0.57 for ICA/M1, k=0.40 for M2, and k=0.27 for basilar occlusions. CONCLUSIONS: NECT-MIPs have high sensitivity and specificity for the detection of LVO when compared to CTA. Inter-rater agreement is fair and higher amongst more experienced reviewers. These results suggest that NECT-MIPs may be helpful to streamline the identification of LVO and reduce door to needle and door to intervention times.

Regulated nuclear export of the homeodomain transcription factor Prospero.

Subcellular distribution of the Prospero protein is dynamically regulated during Drosophila embryonic nervous system development. Prospero is first detected in neuroblasts where it becomes cortically localized and tethered by the adapter protein, Miranda. After division, Prospero enters the nucleus of daughter ganglion mother cells where it functions as a transcription factor. We have isolated a mutation that removes the C-terminal 30 amino acids from the highly conserved 100 amino acid Prospero domain. Molecular dissection of the homeo- and Prospero domains, and expression of chimeric Prospero proteins in mammalian and insect cultured cells indicates that Prospero contains a nuclear export signal that is masked by the Prospero domain. Nuclear export of Prospero, which is sensitive to the drug leptomycin B, is mediated by Exportin. Mutation of the nuclear export signal-mask in Drosophila embryos prevents Prospero nuclear localization in ganglion mother cells. We propose that a combination of cortical tethering and regulated nuclear export controls Prospero subcellular distribution and function in all higher eukaryotes.

Roughex mediates G(1) arrest through a physical association with cyclin A.

Differentiation in the developing Drosophila eye requires synchronization of cells in the G(1) phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G(1). We show here that coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A was previously shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, we demonstrate that in interphase cells in the eye imaginal disk cyclin A is present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity.

decapentaplegic is a direct target of dTcf repression in the Drosophila visceral mesoderm.

Drosophila T cell factor (dTcf) mediates transcriptional activation in the presence of Wingless signalling and repression in its absence. Wingless signalling is required for the correct expression of decapentaplegic (dpp), a Transforming Growth Factor (beta) family member, in parasegments 3 and 7 of the Drosophila visceral mesoderm. Here we demonstrate that a dpp enhancer element, which directs expression of a reporter gene in the visceral mesoderm in a pattern indistinguishable from dpp, has two functional dTcf binding sites. Mutations that reduce or eliminate Wingless signalling abolish dpp reporter gene expression in parasegment 3 and reduce it in parasegment 7 while ectopic expression of Wingless signalling components expand reporter gene expression anteriorly in the visceral mesoderm. However, mutation of the dTcf binding sites in the dpp enhancer results in ectopic expression of reporter gene expression throughout the visceral mesoderm, with no diminution of expression in the endogenous sites of expression. These results demonstrate that the primary function of dTcf binding to the dpp enhancer is repression throughout the visceral mesoderm and that activation by Wingless signalling is probably not mediated via these dTcf binding sites to facilitate correct dpp expression in the visceral mesoderm.

Identification of the Drosophila melanogaster homologue of the mammalian signal transducer protein, Vav.

Mammalian Vav signal transducer protein couples tyrosine kinase signals with the activation of the Rho/Rac GTPases, thus leading to cell differentiation and/or proliferation. We have isolated and characterized the DroVav gene, the homologue of hVav in Drosophila melanogaster. DroVav encodes a protein (793 residues) whose similarity with hVav is 47% and with hVav2 and hVav3 is 45%. DroVav preserves the unique, complex structure of hVav proteins, including the 'calponin homology', dbl homology, pleckstrin homology; SH2 and SH3 domains in addition to regions that are acidic rich, proline rich and cysteine rich. DroVav is located on the X chromosome in polytene interval 18A5;18B and is expressed in all stages of development and in all tissues. In mammalian cells, DroVav is tyrosine-phosphorylated in response to epidermal growth factor receptor (EGFR) induction; in vitro, the DroVav SH2 region is associated with tyrosine-phosphorylated EGFR. Thus, DroVav probably plays a pivotal role as a signal transducer protein during fruit fly development.

Drosophila RpS3a, a novel Minute gene situated between the segment polarity genescubitus interruptus and dTCF.

Genetic analysis of the small chromosome 4 of Drosophila has been hampered by the virtual lack of recombination. The segment polarity gene cubitus interruptus (ci) maps to the most intensively studied locus on this chromosome. Up to four complementation groups have been found to be associated with ci. We and others have recently characterized a second segment polarity gene, dTCF or pan, 12 kb upstream of ci, in a head-to-head configuration. During the course of these studies we identified a transcription unit in the intergenic region. We report here the cloning of cDNAs from this transcription unit, which encode the Drosophila homologue of the human ribosomal protein S3a (RpS3a). The RpS3a gene is expressed ubiquitously and throughout development. A Minute allele, M(4)101, linked tightly to ci, was found to harbour an integration of a Doc retroposon in the promotor region of RpS3a. Thus, like other Minute loci, M(4)101 encodes a component of the protein synthesis machinery. These data further unravel the complex genetics surrounding the ci and dTCF loci.

Selection and analysis of rare second-site suppressors of Drosophila RNA polymerase II mutations.

We used a mutagenesis and selection procedure in Drosophila melanogaster to recover rare allele-specific suppressor mutations. More than 11 million flies mutant for one of five recessive-lethal mutations in the two largest subunits of RNA polymerase II were selected for additional mutations that restored viability. Forty-one suppressor mutations were recovered. At least 16 are extragenic, identifying a minimum of three loci, two of which do not map near genes known to encode subunits of RNA polymerase II. At most, 25 are intragenic, 4 reverting the initial altered nucleotide back to wild type. Sequence analysis of interacting mutations in the two largest subunits identified a discrete domain in each subunit. These domains might be contact points for the subunits. Finally, our selections were large enough to allow recovery of multiple independent changes in the same nucleotides yet mutations in other equally likely targets were not recovered. The mutations recovered are not random and might provide insights into possible mechanisms for mutagenesis in eukaryotes.

Genomic organization of the segment polarity gene pan in Drosophila melanogaster.

We previously described the molecular cloning of a mammalian T cell factor 1 (TCF-1)-like protein from Drosophila melanogaster, encoded by the pangolin (pan) locus, and demonstrated that it consists of a DNA binding domain similar to that of other high mobility group proteins and a protein-protein interaction domain that binds beta-catenin (Armadillo in Drosophila) but that it lacks a transcriptional activation domain. Here we show that the pan locus spans approximately 50 kb and the mRNA results from the splicing of 13 exons. We note remarkable conservation of the exon/intron boundaries between the human and D. melanogaster genes, suggesting that they share a common ancestor. Chromosomal in situ hybridization locates pan to the base of chromosome 4, near the cubitus interruptus locus. Restriction map and sequence analyses confirm their close proximity. The small fourth chromosome undergoes little or no recombination and was previously reported to lack DNA polymorphisms; however, we note two DNA polymorphisms occurring in three combinations within the pan locus, demonstrating the presence of synonymous substitutions and the past occurrence of recombination. We present evidence suggesting that the protein encoded by pan is more similar to mammalian TCF-1 and Caenorhabditis elegans POP-1 than to mammalian LEF-1.

Multiple functions of Drosophila heat shock transcription factor in vivo.

Heat shock transcription factor (HSF) is a transcriptional activator of heat shock protein (hsp) genes in eukaryotes. In order to elucidate the physiological functions of HSF in Drosophila, we have isolated lethal mutations in the hsf gene. Using a conditional allele, we show that HSF has an essential role in the ability of the organism to survive extreme heat stress. In contrast to previous results obtained with yeast HSF, the Drosophila protein is dispensable for general cell growth or viability. However, it is required under normal growth conditions for oogenesis and early larval development. These two developmental functions of Drosophila HSF are genetically separable and appear not to be mediated through the induction of HSPs, implicating a novel action of HSF that may be unrelated to its characteristic function as a stress-responsive transcriptional activator.

Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF.

The vertebrate transcription factors TCF (T cell factor) and LEF (lymphocyte enhancer binding factor) interact with beta-catenin and are hypothesized to mediate Wingless/Wnt signaling. We have cloned a maternally expressed Drosophila TCF family member, dTCF. dTCF binds a canonical TCF DNA motif and interacts with the beta-catenin homolog Armadillo. Previous studies have identified two regions in Armadillo required for Wingless signaling. One of these interacts with dTCF, while the other constitutes a transactivation domain. Mutations in dTCF and expression of a dominant-negative dTCF transgene cause a segment polarity phenotype and affect expression of the Wingless target genes engrailed and Ultrabithorax. Epistasis analysis positions dTCF downstream of armadillo. The Armadillo-dTCF complex mediates Wingless signaling as a bipartite transcription factor.

Transcriptional competition and homeosis in Drosophila.

Interference between different classes of RNA polymerase II alleles causes a mutant phenotype called the "Ubx effect" that resembles one seen in flies haploinsufficient for the transcription factor, Ultrabithorax (Ubx). Flies carrying the mutation in the largest subunit of Drosophila RNA polymerase II, RpII215(4), display the Ubx effect when heterozygous as in RpII215(4)/+ but not when homozygous mutant or wild type. In this report we demonstrate that the interaction between alleles in different classes of polymerase occurs even in the absence of transcription by the wild-type polymerase. We utilized the resistance to the transcriptional inhibitor alpha-amanitin conferred by RpII215(4) to show that RpII215(4)/+ flies raised on alpha-amanitin-containing food still show the Ubx effect and are indistinguishable from flies raised on normal food. We demonstrate using HPLC that the intracellular concentration of alpha-amanitin in the developing larvae is sufficient to inhibit transcription by alpha-amanitin-sensitive polymerase. Furthermore, fluorescein-labeled alpha-amanitin accumulates in imaginal discs, which are the precursor cells for the tissue showing the homeotic transformation in adults. We conclude that the interaction between different classes of RNA polymerase II alleles resulting in the Ubx effect occurs prior to the block in transcription caused by alpha-amanitin.

Molecular cloning of cDNA encoding the small subunit of Drosophila transcription initiation factor TFIIF.

Transcription initiation factor TFIIF is a tetramer consisting of two large subunits (TFIIF alpha or RAP74) and two small subunits (TFIIF beta or RAP30). We report here the molecular cloning of a Drosophila cDNA encoding TFIIF beta. The cDNA clone contains an open-reading frame encoding a 277 amino acid polypeptide having a calculated molecular mass of 32,107 Da. Comparison of the deduced amino acid sequence with the corresponding sequences from vertebrates showed only 50% identity, with four insertion/deletion points. For transcription activity in a TFIIF-depleted Drosophila nuclear extract, both TFIIF alpha and TFIIF beta are essential. Moreover, Drosophila TFIIF beta interacts with both Drosophila and human TFIIF alpha in vitro. Thus we conclude that isolated cDNA encodes bona fide TFIIF beta. The structural domains of TFIIF beta and its sequence similarity to bacterial delta factors are discussed.

Molecular modeling of RNA polymerase II mutations onto DNA polymerase I.

Genetic and molecular analysis in Drosophila melanogaster identifies eight suppressor mutations in the second largest subunit of RNA polymerase II. The suppressor mutations fall into two classes: five are strong, result from the same serine to cysteine amino acid residue substitution and rescue one conditional lethal allele in the largest subunit of RNA polymerase II; three are mild, result from a change in the same methionine residue to either isoleucine or valine, are located seven amino acid residues away from the strong suppressors and rescue two conditional lethal alleles in the largest subunit. Sequence analysis of the three regions around these mutations demonstrates that they are located within highly conserved domains but fails to explain the observed genetic interactions. One of the conditional lethal alleles maps within a region previously reported to share sequence similarity to Escherichia coli DNA polymerase I. As the gross structure of RNA polymerase II and DNA polymerase I is similar, even though their primary sequence is not, we predict that more similarities exist but may be too highly divergent to be detected by normal homology searches. We identify the most similar regions between each of the three conserved domains of RNA polymerase II, identified as functionally important because of the mutations we isolated, and DNA polymerase I. Molecular modeling these regions of RNA polymerase II onto the tertiary structure of DNA polymerase I predicts that all lie adjacent to the DNA binding cleft in positions such that they could interact with the phosphate backbone of DNA. This juxtaposition of mutations in the two largest subunits of RNA polymerase II suggest a mechanism for their genetic interactions.

Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

Reverse genetics of Drosophila RNA polymerase II: identification and characterization of RpII140, the genomic locus for the second-largest subunit.

We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus.

Mutations in the second-largest subunit of Drosophila RNA polymerase II interact with Ubx.

Specific mutations in the gene encoding the largest subunit of RNA polymerase II (RpII215) cause a partial transformation of a structure of the third thoracic segment, the capitellum, into the analogous structure of the second thoracic segment, the wing. This mutant phenotype is also caused by genetically reducing the cellular concentration of the transcription factor Ultrabithorax (Ubx). To recover mutations in the 140,000-D second-largest subunit of RNA polymerase II (RpII140) and determine whether any can cause a mutant phenotype similar to Ubx we attempted to identify all recessive-lethal mutable loci in a 340-kilobase deletion including this and other loci. One of the 13 complementation groups in this region encodes RpII140. Three RpII140 alleles cause a transformation of capitellum to wing but unlike RpII215 alleles, only when the concentration of Ubx protein is reduced by mutations in Ubx.

The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster.

A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster. Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II. The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species. Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains. The gene is expressed at all developmental stages and in all tissues. Two deletions within the RpII15 gene are multiphasic lethal deletions, with accumulation of dead animals commencing at the second larval instar. Ovary transplantation experiments indicate that survival of mutant animals to this stage is due to the persistence of maternal gene product throughout embryogenesis and early larval development. The RpII15 gene product is thus necessary for viability of D. melanogaster.

Use of second-site suppressor mutations in Drosophila to identify components of the transcriptional machinery.

Isolation of second-site suppressor mutations provides a powerful method for identifying (i) genes that encode proteins that interact and (ii) domains within the interacting proteins that contact each other. Flies conditionally lethal because they carry mutations in the largest subunit of RNA polymerase II were mutagenized; ten million progeny were then screened for compensatory mutations. Eight intragenic and 10 extragenic suppressor mutations were recovered. Both the conditional lethality and premature termination of transcription caused by one mutation in the largest subunit of RNA polymerase II are compensated by an allele-specific suppressor mutation in the second-largest subunit of the enzyme.

Antagonistic interactions between alleles of the RpII215 locus in Drosophila melanogaster.

The RpII215 locus encodes the large subunit of RNA polymerase II (polII). Three of 22 RpII215 alleles cause a synergistic enhancement of the mutant phenotype elicited by mutations in the Ultrabithorax (Ubx) locus. We have recovered and analyzed three new mutations that suppress this enhancement. All three mutations map to the RpII215 locus. In addition to suppressing the Ubx enhancement of other RpII215 alleles, two of the new mutations, JH1 and WJK2, themselves enhance Ubx. RpII215 alleles can be placed into three classes based on their ability to enhance Ubx. Class I alleles, including Ubl, C4, C11, JH1, and WJK2, enhance Ubx when heterozygous with class II alleles, which include wild-type RpII215. Class III alleles, which include amorphic alleles, do not enhance Ubx. The third new mutation, WJK1, is a conditional amorphic allele, which behaves like a class III allele at 29 degrees but like a class II allele at 19 degrees. Another mutant phenotype is caused by certain RpII215 alleles, including all class I alleles. This phenotype is a synergistic enhancement of a mutant phenotype elicited by mutations at the Delta (Dl) locus. Unlike the enhancement of Ubx, the enhancement of Dl is not dependent upon antagonistic interactions between different classes of RpII215 alleles.

Genetic interactions of modifier genes and modifiable alleles in Drosophila melanogaster.

We have examined the effects of mutations in the six allele-specific modifier genes su(Hw), e(we), su(f), su(s), su(wa), and su(pr) on the expression of 18 modifiable alleles, situated at 11 loci. Ten of the modifiable alleles are associated with insertions of the gypsy retrotransposon and the others include alleles associated with insertions of copia and 412. We tested or retested 90 of the 108 possible combinations and examined the expression of modifiable alleles in flies mutant for pairs of modifier genes in various heterozygous and homozygous configurations. Our principal findings are: (1) a screen of 40,000 mutagenized X chromosomes yielded three new mutations in known modifier genes, but revealed no new modifier genes; (2) the modification effects of different mutations in a given modifier gene were qualitatively similar; (3) each of the six modifiers suppressed some modifiable alleles, enhanced others, and had no noticeable effect on still others; (4) the modifier genes could be placed in four classes, according to their effects on the gypsy-insertion alleles; and (5) the effects of mutations in different modifier genes combined additively. Implications of these results for models of modifier gene action are discussed.