Lucifensin, the long-sought antimicrobial factor of medicinal maggots of the blowfly Lucilia sericata.

A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.

Diapause hormone in the corn earworm, Helicoverpa zea: optimum temperature for activity, structure-activity relationships, and efficacy in accelerating flesh fly pupariation.

Diapause hormone (DH) effectively terminated pupal diapause in Helicoverpa zea. This effect was temperature-dependent, with an optimum of 21 degrees C. The dose-response curve indicated an ED50 of DH for diapause termination of approximately 100 pmol. The core sequence and essential amino acids were determined by bioassays using modified and truncated DH analogs. A C-terminal hepta-peptide, LWFGPRLa, was the core sequence required for diapause termination. Activity was lost when Alanine was substituted for any of the amino acids in the hepta-peptide, with the exception of Glycine. A fragment series of analogs suggested that the amide and Arginine were the most important components needed for terminating diapause. Leucine, Tryptophan, and Phenylalanine at the N-terminus of the hepta-peptide were also critical for activity. The C-terminal Leucine was less important: deletion resulted in decreased activity, although it could not be substituted by Alanine. The fact that a portion of the DH sequence is similar to the pyrokinin that accelerates fly pupariation prompted us to also evaluate the capability of DH to accelerate development in the flesh fly, Sarcophaga bullata. The threshold dose of DH essential to accelerate fly pupariation was 5 pmol for immobilization/retraction and longitudinal contraction and 10 pmol for tanning, approximately one or two orders of magnitude lower than the effective dose required for diapause termination in H. zea. Tensiometric measurements revealed that DH affected neuromuscular patterns of pupariation behavior and associated cuticular changes in a manner similar to that of the fly pyrokinins and their analogs.

A comparison of the pupariation acceleration activity of pyrokinin-like peptides native to the flesh fly: Which peptide represents the primary pupariation factor?

Five native pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, [L9]Neb-PVK-2, [I9]Neb-PVK-2) identified in the neuropeptidome of the flesh fly Neobellieria bullata were compared for their quantitative and/or qualitative effects on puparium formation (pupariation). In a standard pupariation bioassay, both Neb-PVK-1 and [I9]Neb-PVK-2 proved inactive, whereas [L9]Neb-PVK-2 demonstrated only weak activity. In contrast, both Neb-PK-1 and Neb-PK-2 demonstrated potent threshold doses, with Neb-PK-2 about 10-fold more active than Neb-PK-1. Analysis of neuromuscular activity during pupariation using a tensiometric technique demonstrates that the two native Neb-PKs accelerate the onset of immobilization and cuticular shrinkage more than motor programs associated with retraction of the anterior segments and longitudinal body contraction. It was further determined that the sensitivity of various components of the pupariation process to these peptides decreases in the following order: immobilization>cuticular shrinkage>motor program for anterior retraction>motor program for longitudinal contraction congruent to tanning of cuticle of the newly formed puparium. A paradoxical situation was observed whereby the motor programs of pupariation are temporally dissociated from actual morphogenesis of the puparium. The tensiometric data suggest that the most likely candidate for a primary pupariation factor is Neb-PK-2, rather than Neb-PK-1.

Detection and discrimination of tachycardia in ICDs manufactured by St. Jude Medical.

Modern implantable cardioverter/defibrillator (ICD) systems offer a multitude of algorithms to optimize performance in sensing and tachycardia detection even in difficult circumstances (e. g., ventricular tachycardia during supraventricular tachycardia, fine ventricular fibrillation with intermittent undersensing), to reliably discriminate sustained ventricular tachyarrhythmia from noise, nonsustained and supraventricular tachyarrhythmia, and to limit shock therapy only to those arrhythmias that definitely need to be treated by a shock. A disadvantage of these multiple algorithms is the complexity of annotated tracings that makes it sometimes difficult to understand why the ICD did what it did. If a tachycardia classification was wrong, it may be thus difficult to find the best way to reprogram the device to avoid another misclassification. This review explains in detail the algorithms used for tachycardia detection, discrimination, and prevention of inappropriate therapy in single- and dual-chamber ICDs manufactured by St. Jude Medical. Knowledge of these features may help to optimize ICD treatment in patients fitted with these devices.

Comparison of the effects of pyrokinins and related peptides identified from arthropods on pupariation behaviour in flesh fly (Sarcophaga bullata) larvae (Diptera: Sarcophagidae).

Peptides from the pyrokinin/PBAN family and some structurally related compounds identified in various arthropods were tested for acceleration of puparial contraction in flesh fly larvae. Modifications of behavioural patterns of pupariation were further studied for the active compounds using a behavioural analysis based on the recording of changes in tension of the cuticle. Nine peptides belonging to the pyrokinin/PBAN family (Lem-PK, Pea-PK-5, Lom-PK II, Hez-PBAN, Bom-DH-I), identified in five different insect species, two pyrokinin peptides derived from the genome of Drosophila melanogaster (capa-3, and hugin), and two pyrokinins identified from the white shrimp Penaeus vannamei were very active in the pupariation assay, with threshold doses within the range of 0.1-5.0 pmol larva(-1). High activity was also detected for a related peptide ETH1 from Drosophila. All of these peptides share a C-terminal PRLamide, which is essential and sufficient for the activity. Interestingly, two other structurally related peptides from Drosophila--ETH2 and capa-1--which feature conservative changes (Ile and Val, respectively) at the C-terminal Leu position, were inactive within a physiological range of concentrations. It is clear that the receptor mediating the acceleration of puparial contraction behaviour is sensitive to the introduction of greater steric bulk at the C-terminal Leu position. The peptides that accelerated pupariation showed very similar patterns of muscular and cuticular activity.

Intraoperative characterization of interventricular mechanical dyssynchrony using electroanatomic mapping system--a feasibility study.

BACKGROUND: Interventricular mechanical dyssynchrony (VVMD) is a strong predictor of cardiac resynchronization therapy (CRT) response. However, no simple and reliable clinical method of measuring VVMD during CRT implant is currently available. We tested the hypothesis that the EnSite™ NavX™ system (St. Jude Medical, St. Paul, MN, USA) can be used intraoperatively to determine VVMD, thereby facilitating CRT optimization. METHODS: During CRT implant, the leads in the right atrium (RA), right ventricle (RV), and left ventricle (LV) were connected to the EnSite™ NavX™ system to record the real-time 3D motion of the lead electrodes. The distances from RA to RV lead electrodes (RA-RV) and RA to LV lead electrodes (RA-LV) were computed over ten cardiac cycles during each of RV pacing and biventricular (BiV) pacing, respectively. The degree of synchrony was computed from the distance waveforms between RA-RV and RA-LV by a cross-covariance method to characterize VVMD. Septal-to-posterior wall motion delay (SPWMD) from M-mode echocardiography (echo) was measured for reference at each pacing intervention. VVMD was present in all five patients undergoing CRT implant. RESULTS: Four of the five patients demonstrated clear improvement in EnSite™ NavX™-derived VVMD during BiV versus RV pacing, which corresponded to the SPWMD results by echo. CONCLUSIONS: It is feasible to characterize VVMD and resynchronization in CRT patients with the EnSite™ NavX™ system during implant, demonstrating its potential as a tool for intraoperative CRT optimization.

THE EVALUATION OF THE "CALLIPHORA TEST" AS AN ASSAY FOR ECDYSONE.

1. The effect of ligation on pupariation in the front or hind parts of larvae of four species of flies, Calliphora erythrocephala, Phormia regina, Sarcophaga bullata, and S. argyrostoma was investigated. Ligation causes effects of delay or inhibition of pupariation which are very differently expressed in the four species. A large proportion of pre- or postcritically ligated specimens of P. regina and S. bullata altogether fail to pupariate in the anterior part. This makes these species unsuitable test subjects for the pupariation test for ecdysone. 2. Test abdomens of C. erythrocephala required significantly less ecdysone for a given pupariation effect when also injected with a CNS-extract. Tanning was also considerably accelerated in this case. 3. The value of the pupariation unit of ecdysone is influenced by a number of factors, such as age at the time of ligation, the waiting period between ligation and injection, the dilution effect of the solvent, and the simultaneous action of a neurohormone. The requirements for natural ecdysone in normal larvae at the time of pupariation are probably substantially lower than the values which have been determined by others with test abdomens and the use of synthetic ecdysones. 4. In confirmation of older data, and contrary to recent claims, tanning was induced in test abdomens of the larvae of C. erythrocephala, P. regina, and S. argyrostoma by the injection of hemolymph from pupariating larvae. Calliphora blood induced tanning in specimens of S. argyrostoma, and vice versa. The conclusions are drawn that differences between the different species in the action of ecdysone are of a quantitative rather than qualitative nature.

Disruption of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae), by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

The action of venom from the ectoparasitic wasp, Nasonia vitripennis, was monitored by examining alterations in patterned muscular movements characteristic of pupariation and eclosion behavior in the flesh fly, Sarcophaga bullata. Venom injected into larvae prior to pupariation caused a dose-dependent delay in pupariation. Eventually, such larvae did pupariate, but puparia were abnormally formed. Barographic records revealed that all elements of pupariation behavior were present in venom-injected larvae, but pupariation behavior was not well synchronized with tanning, thus implying that the venom caused disruption in the temporal organization of central motor programs. When larvae were ligated and injected with venom posterior to the ligature, no response was evident in the posterior region, suggesting that the venom does not directly stimulate muscles or neuromuscular junctions. Injection of exogenous ecdysteroid into venom-injected larvae restored some elements of pupariation behavior, consistent with ecdysone's role in stimulating the release of anterior retraction factor and puparium tanning factor, two factors that are released from the CNS to regulate pupariation. When the venom was injected into newly emerged imagoes, the duration of extrication behavior was shortened, whereas all phases of post-eclosion behavior were lengthened. These observations imply that the venom affects CNS centers that regulate the muscular systems engaged in extrication and post-eclosion behavior.

Fraenkel's pupariation factor identified at last.

Thirty-five years ago, Zdarek and Fraenkel demonstrated that nervous tissue extracts influenced development by accelerating pupariation in the grey flesh fly, Neobellieria bullata. We have now identified this pupariation factor as SVQFKPRLamide, designated Neb-pyrokinin-2 (Neb-PK-2). To achieve this, the central nervous system of N. bullata wandering stage larvae, that is, preceding pupariation, were dissected and extracted before HPLC separation. Chromatographic fractions were screened with a bioassay for pupariation accelerating activity. Only one fraction showed huge pupariation activity. Mass spectrometry revealed the presence of a pyrokinin, whose primary sequence could not be unequivocally determined by tandem mass spectrometry. However, this Neb-pyrokinin appeared to be very prominent in the ring gland from which it was subsequently purified and identified. Synthetic Neb-PK-2 accelerates pupariation with a threshold dose of only 0.2 pmol and therefore, Neb-pyrokinin is considered to be the genuine pupariation factor. The immunohistochemical distribution pattern of Neb-PK-2 is very similar to that of Drosophila pyrokinin-2, from which it differs by only one amino acid residue. Hence, the recently identified G-protein coupled receptors (CG8784, CG8795) for Drosophila pyrokinin-2 might play an important role in puparium formation.