Inter- and intra-observer variability of visual fragmented QRS scoring in ischemic and non-ischemic cardiomyopathy.

BACKGROUND: Fragmented QRS (fQRS) on a 12-lead ECG has been linked with adverse outcome. However, the visual scoring of ECGs is prone to inter- and intra-observer variability. METHODS: Five observers, two experienced and three novel, assessed fQRS in 712 digital ECGs, 100 were re-evaluated to assess intra-observer variability. Fleiss and Cohen's Kappa were calculated and compared between subgroups. RESULTS: The inter-observer variability for assessing fQRS in all leads combined was substantial with a Kappa of 0.651. Experienced observers only had a better agreement with a Kappa of 0.823. Intra-observer variability ranged from 0.736 to 0.880. In the subgroup with ventricular pacing the inter-observer variability was even significantly larger when compared to ECGs with normal QRS duration (Kappa 0.493 vs 0.664, p<0.001). CONCLUSION: The visual assessment of QRS fragmentation is prone to inter- and intra-observer variability, mainly influenced by the experience of the observers, the underlying rhythm and QRS morphology.

Blood pressure response to graded bicycle exercise in males and females across the age and fitness spectrum.

AIM: Blood pressure (BP) responses to exercise are frequently measured, with the concern that greater increases are a marker of disease. We sought to characterize the normal exercise BP response in healthy adults and its relationships with age, sex, and fitness. METHODS: 589 participants (median age 46 [IQR 24-56] years, 81% male) underwent cardiopulmonary exercise testing with repeated, automated BP measures. An exaggerated maximal systolic BP (SBPmax) was defined from current guidelines as ≥210 mmHg in males and ≥190 mmHg in females. Individual linear regression analyses defined the relationship between BP and workload (SBP/W-slope and DBP/W-slope). Participants with or without an exaggerated SBPmax and above or below median SBP/W-slope were compared. RESULTS: An exaggerated SBPmax was found in 51% of males and 64% of females and was more prevalent in endurance-trained athletes (males 58%, females 72%, p<0.001). The mean SBP/W-slope was lower in males (0.24±0.10 mmHg/W) than females (0.27±0.12 mmHg/W), p=0.031. In both sexes, peak oxygen uptake (VO2peak) was inversely correlated with SBP/W-slope (p<0.01). Those with an exaggerated SBPmax and below-median SBP/W-slope were 10 years younger and had a 20% higher VO2peak, on average (p<0.001). A non-exaggerated SBPmax and above-median SBP/W-slope was observed in older individuals with the lowest VO2peak. CONCLUSION: In a large cohort of healthy individuals, an exaggerated SBPmax was common and associated with higher fitness. In contrast, higher SBP indexed to workload was associated with older age, lower fitness, and female sex. Thus, sex, age and fitness should be considered when evaluating BP response to exercise.

We evaluated the predictors of blood pressure responses to exercise in 589 healthy individuals. We showed that there is a strong, positive relationship between the increase in systolic blood pressure during exercise with cardiorespiratory fitness and exercise workload.During intensive exercise, high maximal systolic blood pressures are more prevalent in young fit individuals than older, less fit individuals. Systolic blood pressure measures are higher in females than males when indexed to workload.Previous diagnostic cut-offs for peak exercise blood pressure are frequently exceeded in healthy individuals and are likely to have poor disease specificity. Workload-indexed exercise blood pressure is therefore a more informative metric than peak exercise blood pressure.

Myoinhibiting peptides are the ancestral ligands of the promiscuous Drosophila sex peptide receptor.

Male insects change behaviors of female partners by co-transferring accessory gland proteins (Acps) like sex peptide (SP), with their sperm. The Drosophila sex peptide receptor (SPR) is a G protein-coupled receptor expressed in the female's nervous system and genital tract. While most Acps show a fast rate of evolution, SPRs are highly conserved in insects. We report activation of SPRs by evolutionary conserved myoinhibiting peptides (MIPs). Structural determinants in SP and MIPs responsible for this dual receptor activation are characterized. Drosophila SPR is also expressed in embryonic and larval stages and in the adult male nervous system, whereas SP expression is restricted to the male reproductive system. MIP transcripts occur in male and female central nervous system, possibly acting as endogenous SPR ligands. Evolutionary consequences of the promiscuous nature of SPRs are discussed. MIPs likely function as ancestral ligands of SPRs and could place evolutionary constraints on the MIP/SPR class.

Identification, distribution and molecular evolution of the pacifastin gene family in Metazoa.

BACKGROUND: Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. Structural and biochemical characteristics of insect pacifastin-like peptides have been studied intensively, but only one inhibitor has been functionally characterised. Recent sequencing projects of metazoan genomes have created an unprecedented opportunity to explore the distribution, evolution and functional diversification of pacifastin genes in the animal kingdom. RESULTS: A large scale in silico data mining search led to the identification of 83 pacifastin members with 284 inhibitor domains, distributed over 55 species from three metazoan phyla. In contrast to previous assumptions, members of this family were also found in other phyla than Arthropoda, including the sister phylum Onychophora and the 'primitive', non-bilaterian Placozoa. In Arthropoda, pacifastin members were found to be distributed among insect families of nearly all insect orders and for the first time also among crustacean species other than crayfish and the Chinese mitten crab. Contrary to precursors from Crustacea, the majority of insect pacifastin members contain dibasic cleavage sites, indicative for posttranslational processing into numerous inhibitor peptides. Whereas some insect species have lost the pacifastin gene, others were found to have several (often clustered) paralogous genes. Amino acids corresponding to the reactive site or involved in the folding of the inhibitor domain were analysed as a basis for the biochemical properties. CONCLUSION: The absence of the pacifastin gene in some insect genomes and the extensive gene expansion in other insects are indicative for the rapid (adaptive) evolution of this gene family. In addition, differential processing mechanisms and a high variability in the reactive site residues and the inner core interactions contribute to a broad functional diversification of inhibitor peptides, indicating wide ranging roles in different physiological processes. Based on the observation of a pacifastin gene in Placozoa, it can be hypothesized that the ancestral pacifastin gene has occurred before the divergence of bilaterian animals. However, considering differences in gene structure between the placozoan and other pacifastin genes and the existence of a 'pacifastin gene gap' between Placozoa and Onychophora/Arthropoda, it cannot be excluded that the pacifastin signature originated twice by convergent evolution.

Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions.

BACKGROUND: To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of these genes. RESULTS: We have identified seven orthologs of commonly used housekeeping genes in the desert locust. The selected genes were the orthologs of actin, EF1a, GAPDH, RP49, TubA1, Ubi, and CG13220. By employing real time RT-PCR we have analysed the expression of these housekeeping genes in brain tissue of fifth instar nymphs and adults. In the brain of fifth instar nymphs geNorm indicated Sg-EF1a, Sg-GAPDH and Sg-RP49 as most stable genes, while Normfinder ranked Sg-RP49, Sg-EF1a and Sg-ACT as most suitable candidates for normalization. The best normalization candidates for gene expression studies in the brains of adult locusts were Sg-EF1a, Sg-GAPDH and Sg-Ubi according to geNorm, while Normfinder determined Sg-GAPDH, Sg-Ubi and Sg-ACT as the most stable housekeeping genes. CONCLUSION: To perform transcript profiling studies on brains of the desert locust, the use of Sg-RP49, Sg-EF1a and Sg-ACT as reference genes is proposed for studies of fifth instar nymphs. In experiments with adult brains, however, the most preferred reference genes were Sg-GAPDH, Sg-Ubi and Sg-EF1a. These data will facilitate transcript profiling studies in desert locusts and provide a good starting point for the initial selection of genes for validation studies in other insects.

Pacifastin-related peptides: structural and functional characteristics of a family of serine peptidase inhibitors.

Members of the pacifastin family are serine peptidase inhibitors, found in arthropods and have many members within different insect orders. Based on their structural characteristics, inhibitors of this peptide family are divided into two groups (I and II). Members of both groups exhibit specificity towards different types of serine peptidases. In addition, group I inhibitors display species selectivity. The specificity and selectivity of these inhibitors depends on the nature of their P1 residue and on additional interaction sites at the inhibitor's surface. Functional analysis studies have shown that crustacean pacifastin plays a key role in the immune response, whereas insect pacifastin-like peptides have multiple regulatory functions in processes involved in immunity, reproduction, phase transition, etc.

The role of hemocytes, serine protease inhibitors and pathogen-associated patterns in prophenoloxidase activation in the desert locust, Schistocerca gregaria.

The prophenoloxidase-activating system is an important component of the innate immune response of insects, involved in wound healing and melanotic encapsulation. In this paper we show that in the desert locust, Schistocerca gregaria, hemocytes, challenged with microbial elicitors, are indispensable for the limited proteolytic activation of prophenoloxidase (proPO) in plasma. In addition, we assessed the influence of serine protease inhibitors on the induction of PO-activity in plasma. While soybean Bowman-Birk inhibitor (SBBI) inhibited the PO activation by laminarin-treated hemocytes, the endogenous pacifastin-related inhibitors, SGPI-1 (S. gregaria pacifastin-related inhibitor-1) and SGPI-2 did not affect the PO-activity under similar conditions. On the other hand, real-time PCR analysis revealed that the transcripts, encoding SGPI-1-3, were more abundant in the fat body of immune challenged animals, as compared to control animals.

Development of a real-time PCR assay for measurement of yellow protein mRNA transcription in the desert locust Schistocerca gregaria: a basis for isolation of a peptidergic regulatory factor.

A major unresolved issue in insect endocrinology concerns the question of whether or not insects have sex hormones. Conclusive evidence in favor of the presence of such hormones awaits the establishment of appropriate bioassays in males. The cuticle of sexually mature males of the desert locust Schistocerca gregaria turns yellow in gregarious conditions only. Neither females nor isolated males ever turn yellow. The yellowing is due to the deposition in the cuticle of a male-specific Yellow Protein (YP), of which the amino acid sequence is known. In this paper, we describe the partial cloning of the cDNA encoding this Yellow Protein. The tissue distribution and temporal expression of the YP-mRNA is studied in detail using RT-PCR. Furthermore, an RT-PCR based bioassay was developed, which may serve as a reliable tool to help identify the hormones controlling the yellowing process. In addition to juvenile hormone, we have shown that a factor present in the brain-corpora cardiaca is involved in the yellow coloration, as injection of an extract induces the expression of YP-mRNA in isolated gregarious males.

Molecular identification of SGPP-5, a novel pacifastin-like peptide precursor in the desert locust.

Recently, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from locusts and 13 peptides have been identified by cDNA cloning. The peptides share a conserved cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa(3)-Cys) with nine inhibitory domains (PLDs) of the light chain of the crayfish protease inhibitor, pacifastin. A molecular identification of a pacifastin-related precursor (SGPP-5) with three novel PLD-related peptides is presented in this study. This is a first report, identifying the presence of a SGPP-transcript in the brain, fore- and hindgut, including a 100-fold difference in fat body SGPP-transcript level of male as compared with female locust.

In vitro activity of pacifastin-like inhibitors in relation to their structural characteristics.

Information on the structural characteristics and inhibitory activity of the pacifastin family is restricted to a handful of locust pacifastin-related inhibitors. In this report the optimization of a bacterial recombinant expression system is described, resulting in the high yield production of pacifastin-like inhibitors of the desert locust. Subsequently, the relative inhibitory activity of these peptides towards mammalian, locust and caterpillar digestive peptidases has been compared. In general, the enzyme specificity of locust pacifastin-like inhibitors towards trypsin- or chymotrypsin-like peptidases corresponds to the nature of the P1-residue at the reactive site. In addition, other structural characteristics, including specific core interactions, have been reported to result in a different affinity of pacifastin members towards digestive trypsin-like enzymes from mammals and arthropods. One remarkable observation in this study is a specifically designed pacifastin-like peptidase inhibitor, which, unlike other inhibitors of the same family, does not display this specificity and selectivity towards digestive enzymes from different animals.

Role of the Halloween genes, Spook and Phantom in ecdysteroidogenesis in the desert locust, Schistocerca gregaria.

The functional characterization of the Halloween genes represented a major breakthrough in the elucidation of the ecdysteroid biosynthetic pathway. These genes encode cytochrome P450 enzymes catalyzing the final steps of ecdysteroid biosynthesis in the dipteran Drosophila melanogaster and the Lepidoptera Manduca sexta and Bombyx mori. This is the first report on the identification of two Halloween genes, spook (spo) and phantom (phm), from a hemimetabolous orthopteran insect, the desert locust Schistocerca gregaria. Using q-RT-PCR, their spatial and temporal transcript profiles were analyzed in both final larval stage and adult locusts. The circulating ecdysteroid titers in the hemolymph were measured and found to correlate well with changes in the temporal transcript profiles of spo and phm. Moreover, an RNA interference (RNAi)-based approach was employed to study knockdown effects upon silencing of both transcripts in the fifth larval stage. Circulating ecdysteroid levels were found to be significantly reduced upon dsRNA treatment.

Differential quantitative zinc-induced expression of human metallothionein isogenes in haematopoietic precursor cell lines.

The expression pattern of functional members of the metallothionein (MT) gene family was studied in the haematopoietic precursor cell lines, K562, DAMI, MEG-01, and ELF-153 in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 with 10% (v/v) foetal calf serum, with or without different zinc supplements. Expression of MT isogenes was analysed by quantitative real-time PCR (RT-PCR) using mRNA extracted from cultured cells. The more mature K562, DAMI, and MEG-01 cell lines exhibited transcription of all MT isogenes, except MT-3 and MT-4. Relative quantitative expression of MT isogenes in the mature cell lines such as K562, DAMI, and MEG-01 was higher than in the immature ELF-153 cell line. Immunohistochemical staining (IHC) reveals an increased MT protein biosynthesis in more mature cell lines such as K562, DAMI and MEG-01 greater than in the immature ELF-153 cell line. Real-time PCR and immunohistochemical staining for investigating the effect of phorbol ester and hemin (haematopoietic differentiation stimuli) on expression of MT isogenes in K562 cells reveals that phorbol ester induces increased MT transcription and biosynthesis. Therefore, to our knowledge, the role of MT in differentiation in human haematopoietic precursor cell lines is here reported for the first time.

A lepidopteran pacifastin member: cloning, gene structure, recombinant production, transcript profiling and in vitro activity.

Members of the pacifastin family have been characterized as serine peptidase inhibitors (PI), but their target enzyme(s) are unknown in insects. So far, the structural and biochemical characteristics of pacifastin-like PI have only been studied in locusts. Here we report the molecular identification and functional characterization of a pacifastin-like precursor in a lepidopteran insect, i.e. the silkworm Bombyx mori. The bmpp-1 gene contains 17 exons and codes for two pacifastin-related precursors of different length. The longest splice variant encodes 13 inhibitor domains, more than any other pacifastin-like precursor in arthropods. The second transcript lacks two exons and codes for 11 inhibitor domains. By studying the expression profile of the Bombyx pacifastin-like gene a different expression pattern for the two variants was observed suggesting functional diversification. Next, several PI domains of BMPP-1 were produced and, contrary to locust pacifastin peptides, they were found to be potent inhibitors of both bovine trypsin and chymotrypsin. Surprisingly, the same Bombyx PI are only weak inhibitors of endogenous digestive peptidases, indicating that other peptidases are the in vivo targets. Interestingly, the Bombyx PI inhibit a fungal trypsin-like cuticle degrading enzyme, suggesting a protective function for BMPP-1 against entomopathogenic fungi.

Purification and characterization of an insulin-related peptide in the desert locust, Schistocerca gregaria: immunolocalization, cDNA cloning, transcript profiling and interaction with neuroparsin.

Members of the insulin superfamily are not restricted to vertebrates, but have also been identified in invertebrate species. In the current report, we present the characterization of Scg-insulin-related peptide (IRP), an insulin-related peptide in the desert locust, Schistocerca gregaria. This peptide was isolated from corpora cardiaca (CC) extracts by means of a high-performance liquid chromatography (HPLC)-based purification strategy. Subsequent cloning and sequencing of the corresponding cDNA revealed that the encoded Scg-IRP precursor displays the structural organization that is typical for members of the insulin superfamily. Moreover, immunocytochemistry on brain tissue sections demonstrated the presence of Scg-IRP in median neurosecretory cells of the pars intercerebralis and their projections towards the storage part of the CC. Quantitative real-time RT-PCR studies revealed the presence of Scg-IRP transcripts in a variety of tissues, including nervous tissue and fat body. Furthermore, these transcripts showed a tissue- and phase-dependent, temporal regulation during the reproductive cycle of adult males and females. Finally, we demonstrated that Scg-IRP interacts in vitro with a recombinant neuroparsin, a locust protein displaying sequence similarity with vertebrate IGF binding proteins.

Regulation of Schistocerca gregaria neuroparsin transcript levels by juvenile hormone and 20-hydroxyecdysone.

Neuroparsins (NPs) are small proteins that were originally discovered in the pars intercerebralis-corpus cardiacum neurosecretory complex of the migratory locust brain. From the desert locust, Schistocerca gregaria, we recently cloned four different transcripts, each coding for a distinct NP-related peptide. In addition to the brain, some NP-like precursor (Scg-NPP) transcripts also occur in a number of peripheral tissues, and their expression levels are controlled in a gender- and stage-dependent manner. Previous studies revealed a close correlation between Scg-NPP transcript levels and the gonotrophic cycle. In the present report, we demonstrate that certain Scg-NPP transcript levels are significantly altered upon injection of juvenile hormone (JH) or 20-hydroxyecdysone (20E) in adult gregarious desert locusts (five days after final ecdysis). While Scg-NPP1 transcript levels did not significantly change as a result of hormone treatment (animals were analyzed 24 h after injection), Scg-NPP2, Scg-NPP3, and Scg-NPP4 displayed hormone-dependent regulation in various tissues. Scg-NPP2 and Scg-NPP3 transcript levels significantly increased in the brain of JH-treated locusts. In addition, JH induction of Scg-NPP3 and Scg-NPP4 transcripts was observed in male fat body and in male and female gonads. Furthermore, 20E injection also induced Scg-NPP2, Scg-NPP3, and Scg-NPP4 transcripts in desert locust gonads. This is the first report showing NP-like precursor gene expression in insect ovaries. Our study indicates that the expression levels of some Scg-NPP transcripts are regulated by developmental hormones, suggesting a close correlation between NP expression and the endocrine control of the reproductive cycle.

Neuroparsin transcripts as molecular markers in the process of desert locust (Schistocerca gregaria) phase transition.

Desert locust swarms occasionally cause severe ecological and economic damage, particularly in countries of northwest Africa. However, the physiological mechanisms underlying locust phase transition, the switch of the solitarious to the gregarious phase, remain elusive. Therefore, identification of molecular changes linked to this phenomenon represents a primary requirement to start unraveling this enigma. The present paper provides novel information on phase-related molecular markers for locust phase transition. We present a detailed quantitative real-time RT-PCR analysis of two distinct neuroparsin precursor transcripts (Scg-NPP3 and Scg-NPP4) in the brain and in abdominal tissues of gregarious and solitarious desert locusts (Schistocerca gregaria). Our data reveal different temporal changes of these transcripts in the fat body during the adult stage of both phases. We, hereby, present novel scientific evidence for a phase-dependent regulation of these particular peptide hormone encoding transcripts and assign them as possible molecular markers in the process of locust phase transition.

Transcript profiling of pacifastin-like peptide precursors in crowd- and isolated-reared desert locusts.

Locusts have fascinated researchers for several decades, because they have the remarkable ability to undergo phase transition from the harmless solitary to the swarm-forming gregarious phase. However, the physiological and endocrine mechanisms, underlying phase polymorphism, are only partially unravelled. Nevertheless, besides the 'classical' hormones, pacifastin-related peptides have been suggested to play a role in phase transition. Here, we present the first quantitative and comparative analysis of locust transcripts, in particular pacifastin-related precursor (SGPP-1-3) mRNAs, between isolated-reared (solitary) and crowd-reared (gregarious) desert locusts, revealing a phase-dependent transcriptional regulation of the corresponding genes. While the SGPP-1 and SGPP-3 transcripts were most abundant in fat body from crowd-reared males, corresponding to significantly higher levels than in isolated-reared males, the SGPP-2 transcript was detected most abundantly in brain from crowd-reared male locusts. Furthermore, SGPP-2 transcript levels in brain, testes, fat body, and accessory glands from crowd-reared males significantly exceeded the levels in solitary locusts.