Morphological Parameters Associated with Ruptured Posterior Communicating Aneurysms

The rupture risk of unruptured intracranial aneurysms is known to be dependent on the size of the aneurysm. However, the association of morphological characteristics with ruptured aneurysms has not been established in a systematic and location specific manner for the most common aneurysm locations. We evaluated posterior communicating artery (PCoA) aneurysms for morphological parameters associated with aneurysm rupture in that location. CT angiograms were evaluated to generate 3-D models of the aneurysms and surrounding vasculature. Univariate and multivariate analyses were performed to evaluate morphological parameters including aneurysm volume, aspect ratio, size ratio, distance to ICA bifurcation, aneurysm angle, vessel angles, flow angles, and vessel-to-vessel angles. From 2005–2012, 148 PCoA aneurysms were treated in a single institution. Preoperative CTAs from 63 patients (40 ruptured, 23 unruptured) were available and analyzed. Multivariate logistic regression revealed that smaller volume (p = 0.011), larger aneurysm neck diameter (0.048), and shorter ICA bifurcation to aneurysm distance (p = 0.005) were the most strongly associated with aneurysm rupture after adjusting for all other clinical and morphological variables. Multivariate subgroup analysis for patients with visualized PCoA demonstrated that larger neck diameter (p = 0.018) and shorter ICA bifurcation to aneurysm distance (p = 0.011) were significantly associated with rupture. Intracerebral hemorrhage was associated with smaller volume, larger maximum height, and smaller aneurysm angle, in addition to lateral projection, male sex, and lack of hypertension. We found that shorter ICA bifurcation to aneurysm distance is significantly associated with PCoA aneurysm rupture. This is a new physically intuitive parameter that can be measured easily and therefore be readily applied in clinical practice to aid in the evaluation of patients with PCoA aneurysms.     

Rat heart cannot synthesize docosahexaenoic acid from circulating alpha-linolenic acid because it lacks elongase-2

The extent to which the heart can convert alpha-linolenic acid (alpha-LNA, 18:3n-3) to longer chain n-3 PUFAs is not known. Conversion rates can be measured in vivo using radiolabeled alpha-LNA and a kinetic fatty acid model. [1-(14)C]alpha-LNA was infused intravenously for 5 min in unanesthetized rats that had been fed an n-3 PUFA-adequate [4.6% alpha-LNA, no docosahexaenoic acid (DHA, 22:6n-3)] or n-3 PUFA-deficient diet (0.2% alpha-LNA, nor DHA) for 15 weeks after weaning. Arterial plasma was sampled, as was the heart after high-energy microwaving. Rates of conversion of alpha-LNA to longer chain n-3 PUFAs were low, and DHA was not synthesized at all in the heart. Most alpha-LNA within the heart had been beta-oxidized. In deprived compared with adequate rats, DHA concentrations in plasma and heart were both reduced by >90%, whereas heart and plasma levels of docosapentaenoic acid (DPAn-6, 22:5n-6) were elevated. Dietary deprivation did not affect cardiac mRNA levels of elongase-5 or desaturases Delta6 and Delta5, but elongase-2 mRNA could not be detected. In summary, the rat heart does not synthesize DHA from alpha-LNA, owing to the absence of elongase-2, but must obtain its DHA entirely from plasma. Dietary n-3 PUFA deprivation markedly reduces heart DHA and increases heart DPAn-6, which may make the heart vulnerable to different insults.     

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (R_Select) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of R_Select proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.     

Assay of the activity of malonyl-coenzyme A decarboxylase by gas chromatography-mass spectrometry

We developed a gas chromatography-mass spectrometry (GC-MS) assay to measure the activity of malonyl-coenzyme A (CoA) decarboxylase (MCD) in crude tissue homogenates. Liver extracts are incubated with [U-(13)C(3)]malonyl-CoA to form [U-(13)C(2)]acetyl-CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2-(13)C(2)]acetyl-CoA by acetyl-CoA hydrolase present in the extracts. Newly formed [U-(13)C(2)]acetyl-CoA and internal standard of [(2)H(3),1-(13)C]acetyl-CoA are analyzed as thiophenol derivatives by GC-MS. This assay was applied to a study of the kinetics of MCD in rat liver. Using the Lineweaver-Burke plot of MCD kinetics, K(m) of 202microM and V(max) of 3.3micromol min(-1) (g liver)(-1) were calculated. The liver MCD activities (micromol min(-1) g(-1)+/-SD) in three groups of rats with different nutritional statuses-fed, 1-day fasted, and 2-day fasted-were 1.80+/-0.41, 2.59+/-0.37 (P<0.05), and 3.07+/-0.70 (P<0.05), respectively. We report a practical, nonradioactive, sensitive assay of MCD in crude tissue extract.     

Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas,Magicicada septendecim and M. cassini

Nodulation is the first and quantitatively most important cellular defense reaction to bacterial infections in insects. Treating adults of the 17-year periodical cicadas, Magicicada septendecim and M. cassini, with eicosanoid biosynthesis inhibitors immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a multi-step process in which individual steps are separately mediated by lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent by 2 h after injection, and nodulation was significantly reduced, relative to control insects, over the following 14 h. The dexamethasone effects were reversed by treating bacteria-challenged insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids. These findings in adults of an exopterygote insect species with an unusual life history pattern broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.     

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide

In a rat model of acute neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo-oxygenase-1 and -2, and quantified other aspects of brain phospholipid and fatty acid metabolism. The 6-day intracerebral ventricular infusion increased lectin-reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles. LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non-esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2. LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5- to 2.8-fold, without changing these rates in phosphatidylserine or phosphatidylinositol. These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation.     

Identification of a conserved RNA replication element (cre) within the 3Dpol-coding sequence of hepatoviruses

Internally located, cis-acting RNA replication elements (cre) have been identified within the genomes of viruses representing each of the major picornavirus genera (Enterovirus, Rhinovirus, Aphthovirus, and Cardiovirus) except Hepatovirus. Previous efforts to identify a stem-loop structure with cre function in hepatitis A virus (HAV), the type species of this genus, by phylogenetic analyses or thermodynamic predictions have not succeeded. However, a region of markedly suppressed synonymous codon variability was identified in alignments of HAV sequences near the 5′ end of the 3D^pol-coding sequence of HAV, consistent with noncoding constraints imposed by an underlying RNA secondary structure. Subsequent MFOLD predictions identified a 110-nucleotide (nt) complex stem-loop in this region with a typical AAACA/G cre motif in its top loop. A potentially homologous RNA structure was identified in this region of the avian encephalitis virus genome, despite little nucleotide sequence relatedness between it and HAV. Mutations that disrupted secondary RNA structure or the AAACA/G motif, without altering the amino acid sequence of 3D^pol, ablated replication of a subgenomic HAV replicon in transfected human hepatoma cells. Replication competence could be rescued by reinsertion of the native 110-nt stem-loop structure (but not an abbreviated 45-nt stem-loop) upstream of the HAV coding sequence in the replicon. These results suggest that this stem-loop is functionally similar to cre elements of other picornaviruses and likely involved in templating VPg uridylylation as in other picornaviruses, despite its significantly larger size and lower free folding energy.     

Chemical sex recognition in the harvestman <Emphasis Type="Italic">Discocyrtus prospicuus</Emphasis> (Arachnida: Opiliones)

Several arachnid species use chemicals to detect sexual partners. In harvestmen, there are evidences that chemicals may play a role in intraspecific communication. Using the behavior of Discocyrtus prospicuus (Holmberg 1876), whose males expose the penis to females before they engage in mating posture, we tested if males detect females by contact chemoreception (chemicals left on the substrate) and if males detect females by olfaction. First, we exposed males to three experimental groups, where males had to choose between two substrates: female chemicals/blank control; male chemicals/blank control; female/male chemicals. Then, we gave males access to volatiles of males, females, and control simultaneously. We predicted that males would expose the penis when approaching volatiles and chemicals deposited on the substrate by females. We also tested if males spent more time close to the source of female volatiles and on the substrate with female chemicals and if males tapped the substrate with female chemicals for more time than the others. Finally, we put males and females together to observe if males would expose the penis upon touching the female’s cuticle. Most of our predictions were not supported, though males did tap for more time when exposed to female cues instead of male cues and exposed the penis in 70% of the observations when interacting with the female but only after touching her. Our data does not support olfaction as a way to detect females and corroborate the idea that contact chemicals, either on the substrate or on female’s cuticle, play an important role in the detection and recognition of the opposite sex. This is the first evidence in harvestmen that males may react differently to female/male chemicals.     

Viability of cotton and canola pollen on the proboscis of Helicoverpa armigera: implications for spread of transgenes and pollination ecology

Abstract.  1. Pollen can be transported thousands of kilometres by insects but its viability after long-distance transport is not known. Knowing the potential for this mechanism to cause outcrossing of transgenes from genetically modified (GM) plants is important for risk assessments.
2. The viability of pollen from cotton ( Gossypium hirsutum L.) and canola ( Brassica napus L.) was determined after placing it on the proboscis of Helicoverpa armigera moths for intervals of up to 32 h. Viability of both cotton and canola pollen declined at a much greater rate when in contact with the moth proboscis. Most was non-viable by 8 h compared with 16 h for control cotton pollen or 32 h for canola pollen.
3. There was no significant difference in the rate of decline of pollen viability between the five conventional cotton varieties, or between these and the one GM cotton variety used in these experiments.
4. The number of canola pollen grains remaining on the proboscis declined over time. Very few cotton pollen grains were retained on the proboscis.
5. The reduction in pollen viability during contact with the proboscis might indicate partial ingestion of the pollen via the proboscis.
6. The points above suggest that pollen is unlikely to remain attached or remain viable when carried over large distances by H. armigera . The implications for spread of pollen from transgenic plants and for pollination ecology in general are discussed.