Larval dietary wheat germ oil influences age-specific protein expression in adults of the oriental fruit fly.

Changes in essential dietary components alter global gene expression patterns in animals. We reported on a proteomics study designed to identify molecular markers of deficiencies in culture media developed for the oriental fruit fly, Bactrocera dorsalis. In that study, we found significant changes in expression of 70 proteins in adults of larvae reared on media lacking wheat germ oil (WGO), compared to media supplemented with WGO. Of these, a gene encoding an insect chitin-binding protein was expressed at about 120-fold higher levels in adult males reared on media supplemented with WGO. We inferred it may be feasible to develop the gene as a molecular marker of dietary lipid deficiency. The work was focused, however, on analysis of 11 day old adults. We have no information on expression of the chitin-binding protein, nor on any other proteins at other adult ages. In this paper we address the idea that the whole animal proteome changes dynamically with age. We reared separate groups of fruit fly larvae on media with and without WGO supplementation and analyzed protein expression in adult males and females age 0, 4, 8 and 12 days old using 2D electrophoresis. Gel densitometry revealed significant increases (by >2-fold) and decreases (by >50%) in expression levels of 29 proteins in females and 10 in males. We identified these proteins by mass spectrometry on MALDI TOF/TOF and bioinformatic analyses of the protein sequences. Two proteins, peroxiredoxin (26-fold increase) and vitellogenin 1 (15-fold increase) increased in expression in day 8 females. The key finding is that most changes in protein expression occurred in day 8 females. We infer that the fruit fly proteome changes with adult age. The natural changes in proteome with adult age is a crucial aspect of developing these and other proteins into molecular markers of lipid deficiency in fruit flies and possibly other insect species.

Short-term repeatability of joint space width measurements using a magnetic resonance imaging compatible knee positioning device.

The purpose of this study was to evaluate a magnetic resonance imaging (MRI) compatible knee positioning device to aid in minimizing intratechnologist and intertechnologist differences of minimum joint space width (JSW) measurements. Five subjects were scanned by two separate technologists, with and without an MRI-compatible positioning device. A semi-automated program calculated the minimum JSW of the tibiofemoral and patellofemoral joints. The scan-to-scan repeatability was evaluated from measurements between serial scans without subject repositioning, and the intratechnologist and intertechnologist repeatabilities were evaluated when the subject was removed from the magnet and repositioned by an individual technologist. The root mean square (RMS) error of the JSW measurements was also calculated. All measures of scan-to-scan repeatability and intratechnologist repeatability were unchanged with the MRI-compatible positioning device. The intertechnologist repeatability decreased from 0.70 to 0.42 mm, and the RMS error was significantly reduced (P = 0.0006) from 0.26 to 0.15 mm for the tibiofemoral joint. The variability of patellofemoral JSW measurements increased when using the positioning device; however, the increases were not statistically significant. The intertechnologist repeatability increased from 1.55 to 1.79 mm, and the RMS error increased from 0.58 to 0.73 mm. The MRI-compatible positioning device was successful at reducing JSW measurement variability at the tibiofemoral joint. The increase in measurement variability at the patellofemoral joint may be due to local incongruities of the articular surfaces. An MRI-compatible positioning device may be beneficial for quantitative longitudinal studies evaluating knee joint health.

The role of nasal CPAP in obstructive sleep apnoea syndrome due to mandibular hypoplasia.

Melnick Needles syndrome (MNS), Treacher Collins syndrome (TCS) and Pierre Robin syndrome (PRS) are congenital abnormalities with characteristic facial appearances that include micrognathia. A 20-year-old girl with MNS, a 16-year-old boy with TCS and a 12-year-old girl with PRS attended the sleep apnoea clinic at our institution at different times. Diagnostic sleep studies were initially performed on all three patients to confirm the diagnosis of obstructive sleep apnoea syndrome (OSAS). They subsequently commenced nasal CPAP (nCPAP) treatment and their progress was followed. A limited sleep study on the patient with MNS demonstrated moderate/severe OSAS with an AHI of 33 events/h. Commencement of nCPAP resulted in symptomatic improvement. Overnight oximetry in the patient with TCS showed repeated desaturation to SpO2<90%. Subsequent treatment by nCPAP almost completely abolished the desaturation events. Overnight polysomnography in the patient with PRS demonstrated severe OSAS with an AHI of 49 events/h. After 3 years of nCPAP therapy, this patient requested discontinuation of treatment. Subsequent polysomnography without nCPAP revealed an AHI of <5 events/h. The use of nCPAP in the patients with MNS and TCS resulted in effective control of their sleep abnormalities. Mandibular growth and enlargement of the posterior airway space led to resolution of OSAS in the patient with PRS. There is a definite role for nCPAP therapy in patients with congenital micrognathia and OSAS. The use of nCPAP may obviate the need for more invasive corrective surgery for OSAS and is not necessarily a life-long requirement.

Predictive validity of measurements of clinical competence using the team objective structured bedside assessment (TOSBA): assessing the clinical competence of final year medical students.

BACKGROUND: The importance of valid and reliable assessment of student competence and performance is gaining increased recognition. Provision of valid patient-based formative assessment is an increasing challenge for clinical teachers in a busy hospital setting. A formative assessment tool that reliably predicts performance in the summative setting would be of value to both students and teachers. AIM: This study explores the utility of the team objective structured bedside assessment (TOSBA), a novel ward-based formative assessment tool, in predicting student performance in the final clinical examination. METHODS: The performance of a cohort of final year students (n = 191) in the TOSBA was compared with their subsequent performance in the final examination. A comparison was also made between student performance in the existing formative assessment tool, the objective structured long examination record (OSLER) and the final examination. We also examined the relationship between the TOSBA and the components of the final examination using clustering around latent variables analysis. RESULTS: There was a clear relationship between student performance in the TOSBA and performance in the final examination (r(2) = 0.35). Student performance in the OSLER showed a poor relationship with performance in the final examination (r(2) = 0.15) compared with the TOSBA. The TOSBA results showed particular correlation with specific components of the final examination which were clinically based. CONCLUSION: TOSBA performance is a strong predictor of subsequent performance in the final examination. The clustering of the TOSBA with other assessments of clinical skills underlines its utility. Further research is required to determine whether performance in the TOSBA is predictive of subsequent performance during internship.

Tauroursodeoxycholic acid inhibits apoptosis induced by Z alpha-1 antitrypsin via inhibition of Bad.

UNLABELLED: Z alpha-1 antitrypsin (AAT) deficiency is a genetic disease associated with accumulation of misfolded AAT in the endoplasmic reticulum (ER) of hepatocytes. ZAAT-expressing cells display ER stress responses including nuclear factor kappaB activation and apoptosis. Using an in vitro model of ZAAT ER accumulation, we investigated the mechanism of ZAAT-mediated ER-induced apoptosis and evaluated methods to inhibit this process. Here we demonstrate that expression of ZAAT, but not normal MAAT, in HEK293 cells leads to cleavage and activation of caspase-4 and induces apoptosis that is characterized by activation of caspase-3 and caspase-7 and DNA fragmentation. Similar effects are also induced using the ER agonist thapsigargin. A caspase-4-specific short interfering RNA (siRNA) does not impair ZAAT-induced caspase-3/7 activation or cell death in these cells. However, inhibition studies performed using tauroursodeoxycholic acid (TUDCA) demonstrate its ability to inhibit caspase-4 and caspase-3/7 activation, mitochondrial cytochrome c release, and caspase-3 cleavage induced by ZAAT and to promote cell survival. The mechanism by which TUDCA (tauroursodeoxycholic acid) promotes cell survival in ZAAT-expressing cells involves phosphorylation and inactivation of the proapoptotic factor Bad. TUDCA is unable to rescue cells from apoptosis or phosphorylate Bad in the presence of LY294002, a selective P-I-3-kinase inhibitor. CONCLUSION: These data show that caspase-4 is not essential for ZAAT-induced apoptosis in HEK293 cells and implicates P-I-3-kinase and Bad as potential therapeutic targets for the liver disease associated with ZAAT deficiency.

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers.

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.

Long-term changes in watershed nutrient inputs and riverine exports in the Neuse River, North Carolina.

We compared patterns of historical watershed nutrient inputs with in-river nutrient loads for the Neuse River, NC. Basin-wide sources of both nitrogen and phosphorus have increased substantially during the past century, marked by a sharp increase in the last 10 years resulting from an intensification of animal production. However, this recent increase is not reflected in changes in river loading over the last 20 years. Temporal patterns in river loads more closely parallel short-term changes in point sources and cropland nutrient application despite their overall lower magnitude. Total phosphorus loads have declined at all stations considered, corresponding to a 1988 phosphate detergent ban. Nitrogen load temporal patterns vary by location and the nitrogen fraction considered. The furthest upstream station exhibited nitrogen decreases after the completion of a dam in 1983. At a station just downstream of a rapidly growing urban area, the total nitrogen load has increased since the mid-1980s, primarily as a nitrate concentration increase. This is consistent with concurrent increases in chemical fertilizer use and point source discharges, as well as increased nitrification at treatment plants. This increase in nitrate loading is not reflected at the most downstream station, where no clear nitrogen trends are discernable. The lack of clear downstream nutrient increases suggests that current water quality impairment in the lower river and estuary may result from chronic nutrient overload rather than recent changes in the watershed. If this is true, then the impact of a planned 30% nitrogen loading reduction may not be immediately apparent. We calculate that, given annual variability, detecting a load reduction of this magnitude will take at least four years, and, should nutrients accumulated in the watershed become a significant source, detecting the resulting ecological improvements is likely to take substantially longer.

Insects in hypoxia.

Insects exhibit a remarkable array of adaptations that allow them to handle more or less severe hypoxia associated with numerous aquatic and terrestrial habitats. We consider these habitats and then discuss physiological, behavioral and morphological mechanisms that facilitate insect life under oxygen deprivation. Actually or potentially hypoxic habitats include aquatic systems, flood-prone soils and burrows, intertidal zones, ice encasement and high altitudes. Some microhabitats, including dung, carrion, mammalian alimentary canals, grain and wood, also are subject to hypoxia. Adaptations to hypoxia include the ability to switch from aerobic to anaerobic metabolic pathways (with attendant generation of end products), the ability to drastically attenuate basal metabolic rates, altered behaviors and enlarged tracheal system volumes. Research into the biology of hypoxia seems to be progressing from early observations of the abilities of some insects to withstand exposure to hypoxia or anoxia through studies of organismal mechanisms operating in hypoxia to detailed investigations of cellular and intracellular signaling processes. Our hope is this essay will help crystallize the emergent picture of this area for those interested in contributing to future research.

Prostaglandin biosynthesis by fat body from true armyworms, Pseudaletia unipuncta.

We describe prostaglandin (PG) biosynthesis by microsomal-enriched fractions of fat body prepared from true armyworms, Pseudaletia unipuncta. PG biosynthesis was sensitive to experimental conditions, including incubation time, temperature, pH, substrate and protein concentration. Optimal PG biosynthesis conditions included 1 mg of microsomal-enriched protein, incubated at 28 degrees C for 7.5 min at pH 8. These preparations yielded four major PGs: PGA(2), PGE(2), PGD(2) and PGF(2alpha). PGA(2) and PGE(2) were the predominant eicosanoids produced under these conditions. Two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, effectively inhibited PG biosynthesis. Unlike other invertebrate PG biosynthetic systems studied so far, the true armyworm system appeared to be independent of the usual exogenous co-factors required by mammalian and other invertebrate systems. These findings are discussed with respect to PG biosynthesis in other invertebrate and vertebrate systems.

Arachidonic and eicosapentaenoic acids in tissues of the firefly, Photinus pyralis (Insecta: Coleoptera).

We report on the presence of high proportions of arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) in the tissue lipids of adult fireflies, Photinus pyralis. Arachidonic acid typically occurs in very small proportions in phospholipids (PLs) of terrestrial insects, ranging from no more than traces to less than 1% of PL fatty acids, while 20:5n-3 is often missing entirely from insect lipids. Contrarily, 20:4n-6 made up approximately 21% of the PL fatty acids prepared from whole males and females, and from heads and thoraces prepared from males. Proportions of 20:4n-6 associated with PLs varied among tissues, including approximately 8% for male gut epithelia, 13% for testes, and approximately 25% for light organs and body fat from males. Substantial proportions of 20:5n-3 were also associated with PLs prepared from male firefly tissues, including 5% for body fat and 8% for light organs. Because 20:4n-6 and 20:5n-3 are precursors for biosynthesis of prostaglandins and other eicosanoids, we considered the possibility that firefly tissues might produce eicosanoids at exceptionally high rates. Preliminary experiments indicated otherwise. Hence, fireflies are peculiar among terrestrial insects with respect to maintaining high proportions of PL 20:4n-6 and 20:5n-3.

Eicosanoids mediate microaggregation reactions to bacterial challenge in isolated insect hemocyte preparations.

Nodule formation is the quantitatively predominant insect cellular defense reaction to bacterial challenges, responsible for clearing the largest proportion of infecting bacteria from circulation. It has been suggested that eicosanoids mediate several steps in the nodulation process, including formation of hemocyte microaggregates, an early step in the process. While fat body and hemocytes are competent to biosynthesize eicosanoids, the source of the nodulation-mediating eicosanoids remains unclear. To investigate this issue, we studied hemocyte microaggregation reactions to bacterial challenge in vitro. Hemocyte suspensions from the tobacco hornworm, Manduca sexta, were treated with the phospholipase A(2) inhibitor, dexamethasone, then challenged with the bacterium Serratia marcescens. Preparations treated with dexamethasone yielded fewer hemocyte microaggregations than untreated, control preparations. Furthermore, the influence of dexamethasone was reversed by amending experimental (dexamethasone-treated) preparations with the eicosanoid biosynthesis precursor, arachidonic acid. Palmitic acid, which is not a substrate for eicosanoid biosynthesis, did not reverse the influence of dexamethasone on the microaggregation reaction. The influence of dexamethasone was also reversed by adding filtered media from challenged hemocyte preparations to dexamethasone-treated preparations. Finally, most hemocyte preparations treated with selected eicosanoid biosynthesis inhibitors formed fewer hemocyte microaggregations than control preparations. The 5- and 12-lipoxygenase inhibitor, esculetin, did not influence the formation of hemocyte microaggregations in this system. These results are consistent with similar investigations performed in vivo, and we infer that hemocytes are responsible for forming and secreting eicosanoids, which subsequently initiate nodulation by mediating hemocyte microaggregation.

Innate immune reactions stimulated by a lipopolysaccharide-like component of the alga Prototheca (strain 289).

We report on the influence of an LPS-like molecule (aLPS) from the pathogenic alga, Prototheca (strain 289) on insect and murine innate immune reactions. Insect innate reactions to infection include nodule formation, a process of entrapping bacterial cells in aggregates of hemocytes. We recorded eicosanoid-dependent, dose-related nodulation reactions to aLPS in hornworms (Manduca sexta). The insect reaction was attenuated by pre-incubating the aLPS with polymyxin-B. Conversely, the murine macrophages reacted to challenge with Escherichia coli LPS by secreting cytokines, but did not react to aLPS. We infer that, while highly conserved with respect to intracellular mechanisms of interaction, insect and mammalian immune surveillance systems differ in recognition of LPS molecular types.

Anoxia tolerance of con-familial tiger beetle larvae is associated with differences in energy flow and anaerobiosis.

In this study, we compared survivorship, heat dissipation and biochemical features of anaerobiosis of two tiger beetle species (Coleoptera: Cicindelidae) exposed to anoxia. One species commonly experiences environmental immersion from rainfall and snowmelt (Cicindela togata), and the habitat of the other (Amblycheila cylindriformis) is not prone to flooding. The ancestral genus, A. cylindriformis, survives anoxia for only 2 days at 25 degrees C. In response to anoxia, these larvae immediately lose locomotory abilities, tissue concentrations of ATP fall precipitously within 12 h, and significant amounts of lactate are quickly produced. In contrast, C. togata larvae tolerate anoxia for 5 days. Heat dissipation is downregulated to a greater degree than that seen in A. cylindriformis (3.4% versus 14% of standard normoxic rate, respectively), the ability for locomotion is maintained and normoxic levels of ATP are defended for at least 24 h. Lactate is not accumulated until well into anoxic bout, and significant amounts of alanine are also produced. This study provides evidence that tiger beetles differ in physiological responses to anoxia, and that these differences are correlated with flooding risk and with species distribution.

Eicosanoids mediate nodulation reactions to bacterial infections in larvae of the butterfly, Colias eurytheme.

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. Treating larvae of the butterfly Colias eurytheme with the eicosanoid biosynthesis inhibitor dexamethasone, strongly impaired nodulation reactions to bacterial infections. The influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. An eicosanoid biosynthesis system in C. eurytheme larvae is documented. Specifically, the presence of eicosanoid-precursor polyunsaturated fatty acids in tissue phospholipids was determined, an intracellular phospholipase A2 that can release arachidonic acid from tissue phospholipids was recorded, and eicosanoid biosynthesis, registered as conversion of exogenous radioactive 20:4n-6 into eicosanoids, was observed. These findings support the hypothesis that eicosanoids mediate cellular immune responses to bacterial infections in these butterfly larvae, and more broadly, in most, if not all, insects.

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.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis.

Nodulation is the temporally and quantitatively most important cellular defense reaction to bacterial infections in insects. Inhibition of eicosanoid biosynthesis in adults of the cricket, Gryllus assimilis, immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a complex process involving lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 2h of injection, and nodulation was significantly reduced, relative to control crickets, over 22h. The dexamethasone effects were reversed by treating bacteria-injected insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids, and fat body preparations were shown to be competent to biosynthesize eicosanoids from exogenous radioactive arachidonic acid. These findings in a hemimetabolous insect broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

The influence of bacterial species and intensity of infections on nodule formation in insects.

Nodulation is the predominant cellular immune reaction to bacterial infection in insects. Nodulation is a complex process involving an unknown number of discrete cellular actions. Currently, there is only limited information on the signal transduction mechanisms that result in nodulation. In older larvae of the tobacco hornworm, Manduca sexta, and of the tenebrionid beetle, Zophobas atratus, eicosanoids are involved in one or more steps in the overall process, and treating these insects with inhibitors of eicosanoid biosynthesis prior to bacterial infection severely impairs their ability to form nodules. In this paper we address more detailed questions on eicosanoid-mediated nodulation. The nodulation reaction to bacterial infection occurs in all larval stages we examined, specifically, second, third, and fourth instars of M. sexta. In both species, the number of nodules formed in response to bacterial infection is related in an exponential way to the number of bacterial cells in the infection. Nodulation is also not related to larval size. We also found that nodulation intensity varies according to the species of infecting bacteria.

A digestive phospholipase A(2) in midguts of tobacco hornworms, Manduca sexta L.

We hypothesized that phospholipase A(2) (PLA(2)) is a common feature of insect digestive physiology. PLA(2) hydrolyzes polyunsaturated fatty acids (PUFAs) associated with the sn-2 position of phospholipids (PLs). We describe here a PLA(2) from midgut contents of the tobacco hornworm, Manduca sexta. Our results indicate that the enzyme is sensitive to pH (inactivated at low pH), protein concentration (up to 1.6&mgr;g/&mgr;l), substrate concentration (up to 1.4nmoles/reaction), temperature (up to 30 degrees C), and incubation time. We also found that PLA(2) activity is higher in fed than in starved larvae, and enzyme activity is associated with the midgut contents, rather than the midgut epithelium of fed larvae. All known secretory PLA(2)s, except for a PLA(2) in venom of the marine snail, Conus magus, require high calcium concentrations for catalysis, but the Manduca PLA(2) appears to be calcium-independent, and it exhibits increased PLA(2) activity in the presence of a calcium-chelator, EGTA. In addition, the partially purified Manduca PLA(2) is not inhibited by the phospholipid analog, oleyloxyethylphosphorylcholine. These findings suggest that the Manduca digestive PLA(2) may represent another novel form of PLA(2).