Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species.

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Isomer-specific comparisons of the hydrolysis of synthetic pyrethroids and their fluorogenic analogues by esterases from the cotton bollworm Helicoverpa armigera.

The low aqueous solubility and chiral complexity of synthetic pyrethroids, together with large differences between isomers in their insecticidal potency, have hindered the development of meaningful assays of their metabolism and metabolic resistance to them. To overcome these problems, Shan and Hammock (2001) [7] therefore developed fluorogenic and more water-soluble analogues of all the individual isomers of the commonly used Type 2 pyrethroids, cypermethrin and fenvalerate. The analogues have now been used in several studies of esterase-based metabolism and metabolic resistance. Here we test the validity of these analogues by quantitatively comparing their hydrolysis by a battery of 22 heterologously expressed insect esterases with the hydrolysis of the corresponding pyrethroid isomers by these esterases in an HPLC assay recently developed by Teese et al. (2013) [14]. We find a strong, albeit not complete, correlation (r = 0.7) between rates for the two sets of substrates. The three most potent isomers tested were all relatively slowly degraded in both sets of data but three esterases previously associated with pyrethroid resistance in Helicoverpa armigera did not show higher activities for these isomers than did allelic enzymes derived from susceptible H. armigera. Given their amenability to continuous assays at low substrate concentrations in microplate format, and ready detection of product, we endorse the ongoing utility of the analogues in many metabolic studies of pyrethroids.

Statistical shape modeling of proximal femoral shape deformities in Legg-Calvé-Perthes disease and slipped capital femoral epiphysis.

INTRODUCTION: The current understanding of morphological deformities of the hip such as femoroacetabular impingement (FAI), Legg-Calvé-Perthes disease (LCPD), and slipped capital femoral epiphysis (SCFE) is based on two-dimensional metrics, primarily involving the femoral head, that only partially describe the complex skeletal morphology. OBJECTIVE: This study aimed to improve the three-dimensional (3-D) understanding of shape variations during normal growth, and in LCPD and SCFE, through statistical shape modeling. DESIGN: Thirty-two patients with asymptomatic, LCPD, and SCFE hips, determined from physical and radiographic examinations, were scanned using 3-D computed tomography (CT) at a voxel size of (0.5-0.9 mm)(2) in-plane and 0.63 mm slice thickness. Statistical shape modeling was performed on segmented proximal femoral surfaces to determine modes of variation and shape variables quantifying 3-D shape. In addition, conventional variables were determined for all femora. RESULTS: Proximal femur shape was described by eight modes of variation and corresponding shape variables. Statistical shape variables were distinct with age and revealed coordinated, growth-associated differences in neck length-to-width ratio, femoral head medialization, and trochanter protrusion. After size and age-based shape adjustment, diseased proximal femora were characterized by shape variables distinct from those of asymptomatic hips. The shape variables defined morphology in health and disease, and were correlated with certain conventional variables of shape, including neck-shaft angle, head diameter, and neck diameter. CONCLUSION: 3-D quantitative analyses of proximal femoral bone shape during growth and in disease are useful for furthering the understanding of normal and abnormal shape deviations which affect cartilage biomechanics and risk of developing osteoarthritis.

Identification of geranylgeranyl-modified proteins in HeLa cells.

Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analyzed by gas-liquid chromatography (GC) and mass spectrometry (MS). This approach revealed that an all-trans geranylgeranyl group was a major isoprenoid modification.

Prenyl proteins in eukaryotic cells: a new type of membrane anchor.

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.

Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP.

We have previously demonstrated that substitution of Asn for Ser at position 17 of RasH yields a dominant inhibitory protein whose expression in cells interferes with endogenous Ras function (L. A. Feig, and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988). Subsequent structural studies have shown that the hydroxyl group of Ser-17 contributes to the binding of Mg2+ associated with bound nucleotide. In this report, we show that more subtle amino acid substitutions at this site that would be expected to interfere with complexing Mg2+, such as Cys or Ala, also generated dominant inhibitory mutants. In contrast, a Thr substitution that conserves a reactive hydroxyl group maintained normal Ras function. These results argue that the defect responsible for the inhibitory activity is improper coordination of Mg2+. Preferential affinity for GDP, observed in the original Asn-17 mutant, was found exclusively in inhibitory mutants. However, this binding specificity did not completely block the mutant proteins from binding GTP in vivo since introduction of the autophosphorylation site, Thr-59, in 17N Ras resulted in the phosphorylation of the double mutant in cells. Furthermore, inhibitory mutants failed to activate a model downstream target, yeast adenylate cyclase, even when bound to GTP. Thus, the consequence of improper complexing of Mg2+ was to lock the protein in a constitutively inactive state. A model is presented to explain how these properties could cause the mutant protein to inhibit the activation of endogenous Ras by competing for a guanine nucleotide-releasing factor.

A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.

Patient-specific 3D models aid planning for triplane proximal femoral osteotomy in slipped capital femoral epiphysis.

PURPOSE: Slipped capital femoral epiphysis (SCFE) can result in a complex three-dimensional (3D) deformity of the proximal femur. A three-plane proximal femoral osteotomy (TPFO) has been described to improve hip mechanics. The purpose of this study was to evaluate the benefits of using 3D print technology to aid in surgical planning. PATIENTS AND METHODS: Fifteen children treated with TPFO for symptomatic proximal femoral deformity due to SCFE were included in this study. Ten patients were treated by a single surgeon with (model group, n = 5) or without (no-model group, n = 5) a 3D model for pre-operative planning, and compared with patients treated by two senior partners without the use of a model (senior group, n = 5) to evaluate for a learning curve. Peri-operative data including patient body mass index (BMI), surgical time and fluoroscopy time were recorded. RESULTS: Children in all three groups had similar BMIs at the time of the TPFO. Post-operative radiographic parameters were equally improved in all three groups. On average, surgical time decreased by 45 minutes and 38 minutes, and fluoroscopy time decreased by 50% and 25%, in the model group compared with the no-model and senior groups, respectively. CONCLUSIONS: Patient-specific 3D models aid in surgical planning for complex 3D orthopaedic deformities by enabling practice of osteotomies. Results suggest that 3D models may decrease surgical time and fluoroscopy time while allowing for similar deformity correction. These models may be especially useful to overcome steep learning curves for complex procedures or in trainee education through mock surgical procedures.

Oxidative damage of retinal rod outer segment membranes and the role of vitamin E.

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N2 atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin. We have found a large seasonal variation in the endogenous level of alpha-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of alpha-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol. This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.

Effects of vitamin E and selenium deficiency on the fatty acid composition of rat retinal tissues.

The fatty acid composition of retinal tissues was measured in rats maintained for 26--32 weeks on each of the following diets: a purified basal diet deficient in alpha-tocopherol and selenium, an identical control diet supplemented with alpha-tocopherol and selenium, and a commerical laboratory rat chow. Dietary deficiencies of antioxidant nutrients were found to cause a large decrease in total polyunsaturated fatty acids in the retinal pigment epithelium, a small decrease in the retinal rod outer segments, but no change in the whole retina or liver when compared to tissues from animals fed the vitamin E- and selenium-supplemented control diet. The polyunsaturated fatty acid content which we have observed for the retinal pigment epithelium from rats fed commerical lab chow is similar to that which we observed for bovine retinal pigment epithelium. Our results indicate that changes in fatty acid composition are not generalized to all tissues in severely antioxidant-deficient animals, but that changes do occur in some tissues, such as the retinal pigment epithelium, which appears to be particularly sensitive to in vivo lipid peroxidation.

A mouse homologue of FAST-1 transduces TGF beta superfamily signals and is expressed during early embryogenesis.

The transcription factor FAST-1 has recently been shown to play a key role in the specification of mesoderm by TGF beta superfamily signals in the early Xenopus embryo. We have cloned Fast1, a mouse homologue of Xenopus FAST-1, and characterized its expression during embryogenesis and function in activin/TGF beta signal transduction. In vitro, Fast1 associates with Smads in response to an activin/TGF beta signal to form a complex that recognizes the Xenopus activin responsive element (ARE) targeted by Xenopus FAST-1. In intact cells, introduction of Fast1 confers activin/TGF beta regulation of an ARE-luciferase reporter. In embryos, Fast1 is expressed predominantly throughout the epiblast before gastrulation and declines as development progresses. We propose that mouse Fast1, like Xenopus FAST-1, mediates TGF beta superfamily signals specifying developmental fate during early embryogenesis.

Measuring the effects of problem-based learning on the development of veterinary students' clinical expertise.

PURPOSE: To investigate whether repeated exposure to simulated clinical cases, as employed in problem-based learning (PBL), accelerates the development of clinical expertise in veterinary students. METHOD: In 1995 all 122 second-year students at the Texas A&M University College of Veterinary Medicine were organized into 24 self-selected groups to complete four computer-based case simulations in veterinary neuroanatomy. A scoring rubric of four categories (poor, neutral, good, and excellent) was used to assign quality classifications for physical examination questions selected by the students. Each group's diagnostic efficiency was calculated for each case on the basis of these classifications. An analysis of variance (ANOVA) using a one-factor repeated-measures procedure was employed to examine the cumulative effects of the case simulations on diagnostic efficiency. Post hoc procedures involved the use of contrasts to determine the trend of diagnostic efficiency with repeated use of case simulations. A Pearson product-moment correlation coefficient was calculated to determine the strength of the relationship between the change in diagnostic efficiency for each group and the selected group characteristics, total hands-on veterinary experience, and average grade-point average (GPA). RESULTS: Because six of the 24 groups did not perform a physical examination in one of the cases, a rating of zero was given when no physical examination was done; the data were then analyzed with and without the six groups included, and compared. The ANOVA yielded a significant result for all 24 groups (F(3,69) = 2.75, p = .0491) and for the 18 groups that performed physical examinations in all four cases (F(3,51) = 3.03, p = .0377). Significant linear contrasts were also found for all 24 groups (F(1,69) = 7.21, p = .009) and for the 18 groups (F(1,51) = 4.25, p = .044). Improvement in diagnostic efficiency could not be correlated with GPAs and was only somewhat correlated with the total amount of prior clinical experience reported by the students. CONCLUSION: Study findings suggest that there is a significant relationship between the repeated use of case simulations in PBL and the accelerated development of clinical expertise.

Primary choriocarcinoma of the pulmonary artery mimicking pulmonary embolism.

Due to their rarity, primary tumours of the pulmonary arteries are often incorrectly diagnosed as more common diseases such as pulmonary thromboembolism and are seldom diagnosed during a patient's lifetime. Surgery or potentially curative chemotherapy may therefore be withheld. We report a patient with a primary choriocarcinoma of the left pulmonary artery, which was first suspected on a CT scan. The neoplastic nature of the obstruction of the pulmonary arteries was confirmed by positron emission tomography.

A biomechanical comparison of open and thoracoscopic anterior spinal release in a goat model.

STUDY DESIGN: A biomechanical assessment of anterior release and discectomy in the thoracic spine was performed on an animal model using thoracoscopic and open thoracotomy techniques. OBJECTIVES: To compare the relative efficacy of these two techniques of release in achieving increased spinal mobility. BACKGROUND DATA: The clinical use of video-assisted thoracoscopy in the correction of spinal deformity is increasing. The effectiveness of thoracoscopic anterior spinal release with discectomy has not been evaluated biomechanically. METHODS: Anterior release with discectomy was performed on six midthoracic motion segments in five mature goats. The thoracoscopic technique was used for three levels on one side, and an open thoracotomy was used for the alternating three levels of the contralateral side. The duration of surgery for disc excision and the amount of blood loss for each technique were recorded. The intact cranial and caudal motion segments served as controls. The motion segments were individually subjected to nondestructive biomechanical testing. Torsional, sagittal, and coronal bending torques were applied, and the resulting angular displacement was measured. RESULTS: The duration of surgery to remove a disc thoracoscopically decreased as experience was gained by the surgeon. The amount of intraoperative blood loss was comparable using the two methods. There was significantly increased flexibility in the released segments with both techniques, compared with the flexibility in the intact levels for all three loading directions. There was no difference in the motion obtained after release between the two techniques. CONCLUSION: Open and thoracoscopic anterior release and discectomy have been demonstrated, through biomechanical in vitro testing, to increase the flexibility of the spine to a similar extent.

[MR tomography of the bone marrow changes after high-dosage chemotherapy and autologous peripheral stem-cell transplantation]. / MR-Tomographie von Knochenmarkveränderungen nach Hochdosis-Chemotherapie und autologer peripherer Stammzell-Transplantation.

PURPOSE: Evaluation of MR standard imaging and short time inversion recovery (STIR) imaging to assess changes in red bone marrow cellularity after high-dose chemotherapy (HDC) and peripheral blood stem cells transplantation (PBSCT). MATERIALS AND METHODS: Sixty-one magnetic resonance (MR) studies were performed in 15 patients (8 female and 7 male, average age 45 years) who received HDC and PBSCT for therapy of either a solid tumor or multiple myeloma. All patients underwent MR examinations of the lumbar region and both femora with T1- and T2-weighted turbo spin-echo (TSE) and STIR sequences at predefined time intervals. Qualitative analysis of the signal intensity was performed by consensus reading of four radiologists. MR results were correlated with results of blood smears and marrow histology. RESULTS: STIR sequences demonstrated marked changes in signal intensity not only until the aplasia occurred but also during bone marrow repopulation. An increased signal intensity was observed after HDC in 13/15 patients (87%), followed by a decrease in signal intensity immediately after aplasia in 14/15 patients (93%). Signal intensity further changed parallel to marrow engraftment in 11/15 patients (73%). T2-TSE only showed clear changes during repopulation in 8/15 patients (53%). The individual course of the signal in T1-TSE was markedly inhomogeneous. CONCLUSIONS: STIR sequences show bone marrow edema during aplasia and marrow cellularity during reconstitution and are suitable for characterisation of red bone marrow after HDC and autologous PBSCT.