Search

Hans De Loof,1 Ellen De Win,1 Nathalie Moens,1 Veronique Verhoeven,2 Paul Van Royen,2 Elke O Kreps,3 Hilde Philips2 1Laboratory of Physiopharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium; 2Research Group, Primary and Interdisciplinary Care (ELIZA), Department Family Medicine and Population Health (FAMPOP) University of Antwerp, Antwerp, Belgium; 3Department of Ophthalmology, Ghent University Hospital, Ghent, BelgiumCorrespondence: Hans De LoofLaboratory of Physiopharmacology, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, Antwerp, B-2610, BelgiumTel +32 3 265 91 39Email hans.deloof@uantwerpen.beAbstract: Prescribing patterns by primary care physicians concerning ophthalmic problems were studied using the iCAREdata, a database containing information from the out-of-hours care setting in the Flanders region of Belgium. A very high percentage of prescribed ophthalmic medication was topical antibiotics (89.4%) with tobramycin as the most prevalent substance and in clear conflict with the prevailing guidelines. In addition, a very substantial fraction of prescribed medication contained corticosteroids (30.4%). This is a potentially unsafe option within the technical infrastructure of this setting, which limits the diagnostic possibilities concerning viral infections or preexisting glaucoma risk. We conclude that more efforts are required to limit unnecessary and inappropriate prescribing behavior to further promote patient safety.Keywords: antibiotics, conjunctivitis, drug utilization research, referral process

Loss of the insulin-stimulated glucose uptake in muscle is a crucial event participating in the defect of whole-body metabolism in type 2 diabetes. Therefore, identification by Pavarotti et al. (Biochem. J (2021) 478 (2): 407–422) of complexin-2 as an important contributor to glucose transporter 4 (GLUT4) translocation to muscle cell plasma membrane upon insulin stimulation is essential. The present commentary discusses the biological importance of the findings and proposes future challenges and opportunities.

Background High uptake of antiretroviral treatment (ART) is essential to reduce human immunodeficiency virus (HIV) transmission and related mortality; however, gaps in care exist. We aimed to construct the continuum of HIV care (CoC) in 2016 in 11 European Union (EU) countries, overall and by key population and sex. To estimate progress toward the Joint United Nations Programme on HIV/AIDS (UNAIDS) 90-90-90 target, we compared 2016 to 2013 estimates for the same countries, representing 73% of the population in the region. Methods A CoC with the following 4 stages was constructed: number of people living with HIV (PLHIV); proportion of PLHIV diagnosed; proportion of those diagnosed who ever initiated ART; and proportion of those ever treated who achieved viral suppression at their last visit. Results We estimated that 87% of PLHIV were diagnosed; 92% of those diagnosed had ever initiated ART; and 91% of those ever on ART, or 73% of all PLHIV, were virally suppressed. Corresponding figures for men having sex with men were: 86%, 93%, 93%, 74%; for people who inject drugs: 94%, 88%, 85%, 70%; and for heterosexuals: 86%, 92%, 91%, 72%. The proportion suppressed of all PLHIV ranged from 59% to 86% across countries. Conclusions The EU is close to the 90-90-90 target and achieved the UNAIDS target of 73% of all PLHIV virally suppressed, significant progress since 2013 when 60% of all PLHIV were virally suppressed. Strengthening of testing programs and treatment support, along with prevention interventions, are needed to achieve HIV epidemic control., Standardized definitions were used to estimate a 4-stage continuum of human immunodeficiency virus (HIV) care in 11 European Union (EU) countries in 2016. The EU is close to the 90-90-90 target, with the main challenge being the percentage of undiagnosed infections.

Trypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation. ispartof: FRONTIERS IN PHYSIOLOGY vol:12 ispartof: location:Switzerland status: published

Introduction Pharmaceutical reimbursement policies should aim for satisfactory health care at reasonable prices and assure treatment adherence while avoiding wasteful pharmaceutical spending. In Belgium, the maximum billing system, also called “Maximum Amount Fixed” system (MAF), ensures that out-of-pocket payments beyond a ceiling are fully reimbursed to guarantee pharmaceutical treatment in vulnerable population groups (e.g. low-income people and chronic patients). Starting from 2015, a policy change occurred and these expenses were waived during the same calendar year once this ceiling was reached instead of reimbursing these expenses in the next fiscal year. Each subsequent fiscal year, out-of-pocket payments were reintroduced in January. Methods Longitudinal dispensing trends, from early 2013 to mid-2020, for 13 prevalent reimbursed opioids were investigated. Results For MAF patients, significant seasonal increases in opioid dispensing reoccurred towards the end of the year. This pattern was absent for non-MAF patients and was only observed after the 2015 policy change. Periodic changes in out-of-pocket expenses, a characteristic of the reimbursement policy in Belgium, clearly influences opioid availability in an already at-risk group. Conclusions Out-of-pocket reimbursement policies should be urgently re-evaluated to minimize unnecessary opioid exposure, while preserving the affordability of pain treatment for vulnerable patients.

Introduction Oesophageal surgery recently became centralised in Belgium. This study aims to evaluate surgical outcomes and service delivered one year after implementation of centralisation. Patients and methods All patients undergoing an oesophagectomy between the start of the centralisation; 1st of June 2019 and 31st of May 2020, were included from a prospectively maintained database. Results 53 patients (41 male, 12 female) underwent an oesophagectomy during the study period. Most oesophagectomies were performed through an open left thoracoabdominal approach (64.2%), 30.2% via a minimally invasive approach and hybrid approaches were carried out in 5.7% of patients. In this study population, the 30 day mortality rate was 0% and the 90 day mortality rate was 3.8%, equating to 2 deaths. The overall 30 day readmission rate was 7.5%. Clinically significant anastomotic leaks occurred in 4 patients, (7.5%). Pneumonia and atrial fibrillation were the most frequent complications, both having a prevalence of 32.1%. The median length of stay was 11 days (IQR 9.5-14.5). Conclusion The results from our centre are comparable to those from international registers which demonstrate that centralisation of complex cancer services can be safely implemented.

Pharmacogenetic tests in Belgian care: (how) do we get started? Personalized medicine attempts to take all the information about an individual into account, and this also includes characteristics that differ from the presumed ‘average patient’. This approach includes pharmacogenetics, where the influence of genetic variation in various biomolecules on drug response is studied. By performing preemptive pharmacogenetic testing, drug therapies can be optimized, and serious side effects can be avoided. In order to implement pharmacogenetic testing in practice, some hurdles still need to be overcome. For example, scientific information needs to be translated into practical clinical guidelines that are applicable in the local context and reimbursement issues also need to be resolved. In this paper, a current list of gene-drug interactions is presented that could be prioritized during the implementation process in Belgium. The list only contains clinically relevant interactions for which there is sufficient scientific evidence. In addition, a tool is described that takes into account the drug consumption in a specific healthcare environment, to prioritize the most interesting gene-drug interactions. International implementation initiatives show that the obstacles are surmountable. It is therefore time to start a dialogue on accelerating the implementation of pharmacogenetic testing in Belgium. We hope that this prioritized list, together with a discussion of some hurdles that need to be overcome, can inform this debate.

Objective Examining the implementation barriers and facilitators of this service as provided by Belgian community pharmacists in collaboration with general practitioners. Setting Community pharmacies in Flanders. Method Qualitative study through interviews of pharmacists and general practitioners. Main outcome measure Opinions and experiences of pharmacists and general practitioners about type 3 medication review. Results Sixteen community pharmacists and thirteen general practitioners were interviewed and generally gave a positive assessment of the project. The general practitioners saw the pharmaceutical and pharmacotherapeutic recommendations of the pharmacists as an added value for the patients. The pharmacists indicated that performing an medication review was time-consuming, but that it improved their professional relationship with general practitioners and patients. They reported obstacles in obtaining information: cumbersome access to individual patient data (laboratory values) and difficulties in finding and choosing adequate medical information sources. Moreover, pharmacists indicated that there is a need for adequate reimbursement and additional training to make the implementation sustainable. Conclusion Both pharmacists and general practitioners were enthusiastic about medication reviews. The implementation improved the interprofessional collaboration. However, important barriers remain, such as the considerable investment of time and the difficulty in gathering all the necessary information. The sustainable implementation of type 3 medication review in Belgium requires adequate reimbursement and additional training.

In recent years, several hierarchical extensions of well-known learning algorithms have been proposed. For example, when stimulus-action mappings vary across time or context, the brain may learn two or more stimulus-action mappings in separate modules, and additionally (at a hierarchically higher level) learn to appropriately switch between those modules. However, how the brain mechanistically coordinates neural communication to implement such hierarchical learning, remains unknown. Therefore, the current study tests a recent computational model that proposed how midfrontal theta oscillations implement such hierarchical learning via the principle of binding by synchrony (Sync model). More specifically, the Sync model employs bursts at theta frequency to flexibly bind appropriate task modules by synchrony. 64-channel EEG signal was recorded while 27 human subjects (Female: 21, Male: 6) performed a probabilistic reversal learning task. In line with the Sync model, post-feedback theta power showed a linear relationship with negative prediction errors, but not with positive prediction errors. This relationship was especially pronounced for subjects with better behavioral fit (measured via AIC) of the Sync model. Also consistent with Sync model simulations, theta phase-coupling between midfrontal electrodes and temporo-parietal electrodes was stronger after negative feedback. Our data suggest that the brain uses theta power and synchronization for flexibly switching between task rule modules, as is useful for example when multiple stimulus-action mappings must be retained and used.Significance StatementEveryday life requires flexibility in switching between several rules. A key question in understanding this ability is how the brain mechanistically coordinates such switches. The current study tests a recent computational framework (Sync model) that proposed how midfrontal theta oscillations coordinate activity in hierarchically lower task-related areas. In line with predictions of this Sync model, midfrontal theta power was stronger when rule switches were most likely (strong negative prediction error), especially in subjects who obtained a better model fit. Additionally, also theta phase connectivity between midfrontal and task-related areas was increased after negative feedback. Thus, the data provided support for the hypothesis that the brain uses theta power and synchronization for flexibly switching between rules.

Recent evidence suggests that reward prediction errors (RPEs) play an important role in declarative learning, but its neurophysiological mechanism remains unclear. Here, we tested the hypothesis that RPEs modulate declarative learning via theta-frequency oscillations, which have been related to memory encoding in prior work. For that purpose, we examined the interaction between RPE and transcranial Alternating Current Stimulation (tACS) in declarative learning. Using a between-subject (real versus sham stimulation group), single-blind stimulation design, 76 participants learned 60 Dutch-Swahili word pairs, while theta-frequency (6 Hz) tACS was administered over the medial frontal cortex (MFC). Previous studies have implied MFC in memory encoding. We replicated our previous finding of signed RPEs (SRPEs) boosting declarative learning; with larger and more positive RPEs enhancing memory performance. However, tACS failed to modulate the SRPE effect in declarative learning and did not affect memory performance. Bayesian statistics supported evidence for an absence of effect. Our study confirms a role of RPE in declarative learning, but also calls for standardized procedures in transcranial electrical stimulation.

Background The healthcare system is faced by an ageing population, increase in chronic conditions and multimorbidity. Multimorbid patients are faced with multiple parallel care processes leading to a risk of fragmented care. These problems relate to the disease-oriented paradigm. In this paradigm the treatment goals can be in contrast with what patients value. The concept of goal-oriented care is proposed as an alternative way of providing care as meeting patients’ goals could have potential benefits. Though, there is a need to translate this concept into tangible knowledge so providers can better understand and use the concept in clinical practice. The aim of this study is to address this need by means of a concept analysis. Method This concept analysis using the method of Walker and Avant is based on a literature search in PubMed, Embase, Cochrane Library, PsychInfo, CINAHL, OTSeeker and Web of Science. The method provides eight iterative steps: select a concept, determine purpose, determine defining attributes, identify model case, identify additional case, identify antecedents and consequences and define empirical referents. Results The analysis of 37 articles revealed that goal-oriented care is a dynamic and iterative process of three stages: goal-elicitation, goal-setting, and goal-evaluation. The process is underpinned by the patient’s context and values. Provider and patient preparedness are required to provide goal-oriented care. Goal-oriented care has the potential to improve patients’ experiences and providers’ well-being, to reduce costs, and improve the overall population health. The challenge is to identify empirical referents to evaluate the process of goal-oriented care. Conclusion A common understanding of goal-oriented care is presented. Further research should focus on how and what goals are set by the patient, how this knowledge could be translated into a tangible workflow and should support the development of a strategy to evaluate the goal-oriented process of care.

Recent behavioral evidence implicates reward prediction errors (RPEs) as a key factor in the acquisition of episodic memory. Yet, important neural predictions related to the role of RPEs in episodic memory acquisition remain to be tested. Humans (both sexes) performed a novel variable-choice task where we experimentally manipulated RPEs and found support for key neural predictions with fMRI. Our results show that in line with previous behavioral observations, episodic memory accuracy increases with the magnitude of signed (i.e., better/worse-than-expected) RPEs (SRPEs). Neurally, we observe that SRPEs are encoded in the ventral striatum (VS). Crucially, we demonstrate through mediation analysis that activation in the VS mediates the experimental manipulation of SRPEs on episodic memory accuracy. In particular, SRPE-based responses in the VS (during learning) predict the strength of subsequent episodic memory (during recollection). Furthermore, functional connectivity between task-relevant processing areas (i.e., face-selective areas) and hippocampus and ventral striatum increased as a function of RPE value (during learning), suggesting a central role of these areas in episodic memory formation. Our results consolidate reinforcement learning theory and striatal RPEs as key factors subtending the formation of episodic memory.SIGNIFICANCE STATEMENTRecent behavioral research has shown that reward prediction errors (RPEs), a key concept of reinforcement learning theory, are crucial to the formation of episodic memories. In this study, we reveal the neural underpinnings of this process. Using fMRI, we show that signed RPEs (SRPEs) are encoded in the ventral striatum (VS), and crucially, that SRPE VS activity is responsible for the subsequent recollection accuracy of one-shot learned episodic memory associations.

Background: Seborrheic keratosis (SK) of the outer ear canal is rarely described in literature. Etiological risk factors involved in SK such as exposure to human papillomavirus (HPV) and ultraviolet (UV) light are established but must still be confirmed. In recent years, new insights into the pathogenesis of SKs occurred in the area of molecular pathogenesis. Fibroblast growth factor receptor 3 (FGFR3) gene and p110 subunit of phosphoinositide 3-kinase (PIK3CA) oncogene mutations are known to be involved. Methods: We describe two cases of SK of the outer ear canal. We conducted a review of literature and examined the role of etiological risk factors involved in our cases. The lesions were primarily treated with surgical resection. Postoperatively, in both patients, the lesions recurred after a considerably long disease-free interval. We tested both FGFR3 and PIK3CA genes for mutations, in the primary and recurrent lesions. Results: We did not find any mutations in both genes in all samples. Conclusion: Additional research is needed to further establish possible etiological risk factors and to clarify the involvement of PIK3CA and FGFR3 genes in the pathogenesis of seborrheic keratosis of the outer ear canal. These cases underscore the need for meticulous diagnosis, treatment, and sufficient long-term follow-up.

Wastewater-based epidemiology (WBE) employs the analysis of human metabolic biomarkers in influent wastewater (IWW) to estimate community-wide exposure to xenobiotics (e.g. prescription opioids). The low ng/L range of concentrations of these biomarkers and the complex matrix composition pose bioanalytical challenges related to sample preparation and detection/quantification. Therefore, a sensitive analytical method for the detection and analysis of 19 opioid biomarkers was optimized and validated according to the European Medicines Agency guidelines. Oasis HLB cartridges were used for sample concentration and an Atlantis T3 column with gradient elution resulted in sufficient separation of the analytes. Absolute recoveries (RE) were highly reproducible and ranged between 50 and 93% with the exception of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The lower limit of quantification (LLOQ) ranged between 1 and 100 ng/L and was based on the analyte concentrations found in IWW. Process efficiency was acceptable for all biomarkers for which an isotope-labelled deuterated analogue was available. All biomarkers showed high benchtop stability with the exception of buprenorphine, EDDP, fentanyl and normorphine. Apart from buprenorphine and hydrocodone, all analytes under investigation were detected at least once above LLOQ levels in five locations in Belgium. The highest population-normalized mass loads were found for tramadol, O-desmethyltramadol and codeine. The proposed methodology was able to evaluate spatial differences in opioid use.

Introduction A recent meta-analysis showed that 20% of the world's population suffers from non-alcoholic fatty liver disease (NAFLD) without obesity. The progression of NAFLD involves cardiac manifestations, but the underlying mechanisms are not well established. Objective Our objective is to characterize the cardiometabolic manifestations of NAFLD and evaluate the presence of a sexual dimorphism. Method The cardiac phenotype was investigated in a mouse model of hepatic Lrpprc deficiency (H-Lrpprc−/−), as these mice develop microvesicular steatosis in absence of obesity and a NAFLD lipidomic profile secondary to mitochondrial bioenergetic deficiency. The cardiac repercussions were studied using molecular (qPCR, immunoblots), lipidomic (mass spectrometry) and functional (oximetry on isolated mitochondria) approaches. Results Loss of Lrpprc was specific to the liver and residual expression was similar in both sexes. In female mice, Lrpprc deficiency in the liver was associated with a decreased cardiac expression of gene markers for mitochondrial biogenesis (Tfam) and fusion (Mfn1-2), as well as genes related to fatty acid (FA) metabolism, including transcriptional regulator Pparα, FA transporters (Cd36, Cpt1b, Cpt2), and s-oxidation enzymes (Vlcad, Lcad, Mcad). FA-dependent mitochondrial respiration was also reduced while cardiac triglyceride levels were elevated. In contrast, these alterations were almost completely absent in male mice. Conclusion Collectively these results indicate that metabolic defects in the liver consequent to mitochondrial dysfunction in absence of obesity result in significant abnormalities in mitochondrial and metabolic phenotype of the heart, particularly in females, thus highlighting the existence of a major sexual dimorphism. This experimental model may prove useful to better understand the mechanisms underlying the sex-related variability in the progression of NAFLD in humans.

Introduction Plasmalogens belong to a subclass of phospholipids called ether lipids. The peroxisome synthesize these lipids and involve the limiting AGPS enzyme. In addition, mutations in the Agps gene are associated with cardiac manifestations. We hypothesized that decreased plasmalogens in cells negatively affect mitochondrial function and cell survival. Objective We aimed to evaluate the metabolic and functional consequences of plasmalogens deficiency in a cardiomyoblastic cell line (H9c2 cells) modified using a CRISPR-Cas9 strategy targeting the Agps gene. Methods We characterized this cell line using qPCR and immunoblotting to explore the influence on markers related to mitochondrial function [biogenesis, fatty acid metabolism (FA)], endoplasmic reticulum (ER) stress and apoptosis. Results AGPS reduction was validated at the gene (−58%) and protein (−78%) level, and is associated with decreased plasmalogens (−76%) that we analysed using mass spectrometry. This deficiency leaded to mitochondrial perturbations as revealed by changes on the expression of genes involved in biogenesis: Pgc-1α and s (−27%; −61%), dynamics: Opa1 and Mff (−15%) as well as in the use of FA: Cd36 and Cpt1 (−52%; −51%) and in the oxidation of FA: Vlcad and Lcad (−22%; −13%). Consistently, using mass spectrometry, we observed decreased acylcarnitines (75%) used as markers of mitochondrial FA metabolism. This is accompanied with signs of increased ER stress and apoptosis as suggested by the increase in Chop gene expression (+31%) and the decreased uncleaved form of the Caspase-3 protein (−32%), respectively. Finally, we were able to normalize the levels of plasmalogen in our cells using alkylglycerols. Conclusion Collectively, these results demonstrate that lowering plasmalogens affects mitochondrial function and cell stress in H9c2 cells. The normalisation of plasmalogens using alkylglycerols is encouraging to further characterize their metabolic and functional benefits.

Item does not contain fulltext Abstract Based on reward and difficulty information, people can strategically adjust proactive cognitive control. fMRI research shows that motivated proactive control is implemented through fronto-parietal control networks that are triggered by reward and difficulty cues. Here, we investigate electrophysiological signatures of proactive control. Previously, the contingent negative variation (CNV) in the ERPs and oscillatory power in the theta (4–8 Hz) and alpha band (8–14 Hz) have been suggested as signatures of control implementation. However, experimental designs did not always separate control implementation from motor preparation. Critically, we used a mental calculation task to investigate effects of proactive control implementation on the CNV and on theta and alpha power, in absence of motor preparation. In the period leading up to task onset, we found a more negative CNV, increased theta power, and decreased alpha power for hard versus easy calculations, showing increased proactive control implementation when a difficult task was expected. These three measures also correlated with behavioral performance, both across trials and across subjects. In addition to scalp EEG in healthy participants, we collected intracranial local field potential recordings in an epilepsy patient. We observed a slow-drift component that was more pronounced for hard trials in a hippocampal location, possibly reflecting task-specific preparation for hard mental calculations. The current study thus shows that difficulty information triggers proactive control in absence of motor preparation and elucidates its neurophysiological signatures.

Objective: The accessibility of contraceptives varies greatly from country to country. Because unintended pregnancies have a considerable impact, programmes have been initiated in some countries to make certain contraceptives available without a prescription. We therefore investigated whether or not Flanders, the Dutch-speaking part of Belgium, is ready for such an initiative. Method: We used a mixed-methods approach with a mainly qualitative methodology. The opinions of pharmacists, general practitioners (GPs) and gynaecologists, the three types of health care provider most closely involved in the prescription and delivery of contraception, were examined. Results: A majority of pharmacists supported the idea. Moreover, a large majority occasionally dispensed hormonal contraception without a prescription. Pharmacists expected negative responses from physicians. Among GPs and gynaecologists, a small majority supported the idea conditionally. A minority either fully supported the idea or found it completely unacceptable. Conclusion: Economic aspects were clearly important in forming an opinion on the topic, although medical arguments were often used when they happened to point in the same direction. Flemish pharmacists were willing to train for and implement a new service that would provide contraceptives without a prescription. The majority of GPs and gynaecologists expressed reservations about such a service and doubted that it would reduce unintended pregnancies. If this service were to be implemented, caution would be needed to avoid giving contraceptive users conflicting information.

Drastic changes in hormone titers, in particular of steroid hormones, are intuitively interpreted as necessary and beneficial for optimal functioning of animals. Peaks in progesterone- and estradiol titers that accompany the estrus cycle in female vertebrates as well as in ecdysteroids at each molt and during metamorphosis of holometabolous insects are prominent examples. A recent analysis of insect metamorphosis yielded the view that, in general, a sharp rise in sex steroid hormone titer signals that somewhere in the body some tissue(s) is undergoing programmed cell death/apoptosis. Increased steroid production is part of this process. Typical examples are ovarian follicle cells in female vertebrates and invertebrates and the prothoracic gland cells, the main production site of ecdysteroids in larval insects. A duality emerges: programmed cell death-apoptosis is deleterious at the cellular level, but it may yield beneficial effects at the organismal level. Reconciling both opposites requires reevaluating the probable evolutionary origin and role of peptidic brain hormones that direct steroid hormone synthesis. Do e.g. Luteinizing Hormone in vertebrates and Prothoracicotropic Hormone (PTTH: acting through the Torso receptor) in insects still retain an ancient role as toxins in the early immune system? Does the functional link of some neuropeptides with Ca(2+)-induced apoptosis make sense in endocrine archeology? The endocrine system as a remnant of the ancient immune system is undoubtedly counterintuitive. Yet, we will argue that such paradigm enables the logical framing of many aspects, the endocrine one inclusive of both male and female reproductive physiology.

Reward prediction errors (RPEs) are crucial to learning. Whereas these mismatches between reward expectation and reward outcome are known to drive procedural learning, their role in declarative learning remains underexplored. Earlier work from our lab addressed this, and consistently found that signed reward prediction errors (SRPEs; “better-than-expected” signals) boost declarative learning. In the current EEG study, we sought to explore the neural signatures of SRPEs. Participants studied 60 Dutch-Swahili word pairs while RPE magnitudes were parametrically manipulated. Behaviorally, we replicated our previous findings that SRPEs drive declarative learning, with increased recognition for word pairs accompanied by large, positive RPEs. In the EEG data, at the start of reward feedback processing, we found an oscillatory (theta) signature consistent with unsigned reward prediction errors (URPEs; “different-than-expected” signals). Slightly later during reward feedback processing, we observed oscillatory (high-beta and high-alpha) signatures for SRPEs during reward feedback, similar to SRPE signatures during procedural learning. These findings illuminate the time course of neural oscillations in processing reward during declarative learning, providing important constraints for future theoretical work.

Dye-coupling of lucifer yellow between follicle cells and germ cells of the vitellogenic polytrophic follicles of Sarcophaga bullata, Drosophila melanogaster, and Manduca sexta does not always occur. The electrical field around the follicles of Sarcophaga bullata can be modified by altering the ionic composition of the Ringer solution. We propose general model that suggests possible pathways for both intracellular and extracellular current loops.

Introduction Sodium-glucose transporter 1 (SGLT1) is the major SGLT isoform expressed in the heart. Under baseline condition, its absence does not affect heart function and structure. Using the pharmacological SGLT1 inhibitor, phlorizin, several data suggested that SGLT1 could contribute to overall glucose transport under physiological as well as under pathological conditions, i.e. in the ischemic heart. However, SGL1 role in glucose transport has never been formerly demonstrated. Purpose This study aimed to evaluate, in physiological and pathological conditions, the consequence of SGLT1 transporter invalidation on cardiomyocyte glucose uptake. Methods Glucose uptake was measured in isolated cardiomyocytes using 2,3[H]-glucose and 3[H]-2-deoxyglucose. SGLT1 contribution was evaluated by pharmacologic (phlorizin) and genetic means (KO mice). GLUT transporters expression was examined by RT-qPCR and Western Blot. Results Under basal and hyperglycemic conditions as well as under insulin or oligomycin stimulation, glucose uptake was similar in SGLT1 + / + and SGLT1 − / − cardiomyocytes. We verified that SGLT1 absence was not compensated by an increase in GLUT1 or GLUT4 expression. Phlorizin inhibited insulin-stimulated glucose transport in a dose dependent manner, leading to its complete inhibition at high concentration (1 mM). However, this effect was independent of SGLT1 as phlorizin impact on glucose transport was similar in SGLT1 + / + and SGLT1 − / − cardiomyocytes. Furthermore, the phlorizin-induced glucose uptake inhibition was not explained by any change on insulin signaling or GLUT4 translocation, suggesting that phlorizin directly affected the GLUTs facilitated glucose transporters. Conclusion Phlorizin effect on glucose transport does not involve SGLT1 but rather a direct effect on GLUTs. We also demonstrated that SGLT1 does not significantly contribute to glucose uptake in the cardiomyocyte in physiological and pathological conditions.

Intractable aspiration is a serious, often life-threatening condition due to its potential impact on pulmonary function. Aspiration requires therapeutic measures, starting with conservative management but often necessitating surgical treatment. The basic surgical principle is to separate the alimentary and respiratory tracts through a variety of procedures which, unfortunately, nearly all result in the loss of phonation, with the exception of total laryngectomy (TL) which includes the placement of an indwelling voice prosthesis. In this study, we present a modified laryngotracheal separation (LTS) technique that, we believe, offers multiple advantages compared to standard TL. After reviewing the medical records of 35 patients with intractable aspiration who have undergone LTS, we describe the surgical technique and present the postoperative result. In a second surgical procedure about two months following LTS, we aimed to achieve voice restoration by placement of an indwelling voice prosthesis. Intractable aspiration was successfully treated in all patients. Placement of an indwelling voice prosthesis during a second operation was successful in 15 patients, representing the largest reported cohort thus far. LTS is a reliable surgical technique to treat intractable aspiration, with restoration of oral intake, thereby improving the general condition and quality of life of these unfortunate patients. Furthermore, voice restoration can be achieved in selected patients, by placement of a voice prosthesis.

Ca(2+) is the most omnipresent pollutant on earth, in higher concentrations a real threat to all living cells. When [Ca(2+)]i rises above 100 nM (=resting level), excess Ca(2+) needs to be confined in the SER and mitochondria, or extruded by the different Ca(2+)-ATPases. The evolutionary origin of eggs and sperm cells has a crucial, yet often overlooked link with Ca(2+)-homeostasis. Because there is no goal whatsoever in evolution, gametes did neither originate "with the purpose" of generating a progeny nor of increasing fitness by introducing meiosis. The explanation may simply be that females "invented the trick" to extrude eggs from their body as an escape strategy for getting rid of toxic excess Ca(2+) resulting from a sex-hormone driven increased influx into particular cells and tissues. The production of Ca(2+)-rich milk, seminal fluid in males and all secreted proteins by eukaryotic cells may be similarly explained. This view necessitates an upgrade of the role of the RER-Golgi system in extruding Ca(2+). In the context of insect metamorphosis, it has recently been (re)discovered that (some isoforms of) Ca(2+)-ATPases act as membrane receptors for some types of lipophilic ligands, in particular for endogenous farnesol-like sesquiterpenoids (FLS) and, perhaps, for some steroid hormones as well. A novel paradigm, tentatively named "Calcigender" emerges. Its essence is: gender-specific physiotypes ensue from differential Ca(2+)-homeostasis enabled by genetic differences, farnesol/FLS and sex hormones. Apparently the body of reproducing females gets temporarily more poisoned by Ca(2+) than the male one, a selective benefit rather than a disadvantage.

Background In October 2013, a New Medicines Service (NMS) was introduced in community pharmacies in Belgium to support asthma patients who are novice users of inhaler devices with corticosteroids. The protocol-based intervention used the Asthma Control Test (ACT) and the Medication Adherence Report Scale (MARS) to assess asthma control and medication adherence. The NMS is the first initiative that puts advanced pharmaceutical care into practice in Belgium. The present study evaluated the degree to which the NMS program is delivered as intended, drawing on the concept of implementation fidelity (IF). Methods The main dimensions of IF and potential moderating and facilitating factors for the implementation of NMS in community pharmacies were evaluated using telephone interviews with pharmacists ( n = 497), semi-structured interviews with patients eligible for NMS ( n = 30), focus groups among general practitioners ( n = 72) and lung specialists ( n = 5), and a work system analysis in community pharmacies ( n = 19). Results The uptake of NMS in Belgian community pharmacies remains low. In addition to practical barriers, pharmacists found it difficult to identify new asthmatic patients when they were not informed about the diagnosis. A lack of commitment from physicians, patients and pharmacists was noted in the early start-up phase of the program. Many pharmacists did not see how NMS differed from existing pharmaceutical care. Physicians considered this service as part of their own tasks and discouraged ACT for asthma follow-up in the community pharmacy. Conclusions The introduction of the NMS program was not sufficiently embedded in the Belgian health care organization, causing low uptake and resistance to its implementation by pharmacists, patients, and other health care professionals. To increase the uptake of this type of service and its possible extension to other patient groups, more collaboration among the different health care professionals during design and implementation is necessary, as well as systematic data collection to monitor the quality of the service, better training of pharmacists, and more information for patients and physicians.

Purpose The aim is to describe the process, principles and results of the International Nonproprietary Name (INN) prescribing project in Belgium. The purpose of this project was to operationalize electronic INN prescribing for outpatient care in daily medical practice and to develop a factual database which can be used in electronic INN prescribing applications. Methods The operationalization process consisted of three phases: (1) expert consultation, (2) review by regulatory authorities and (3) test phase with stakeholders and end-users. Results The INN prescribing project resulted into (1) operational rules for electronic INN prescribing and (2) a reference database to be implemented in commercial medical software. The operational rules for electronic INN prescribing define valid INN groups as sets of equivalent medicinal products, described by three elements: the therapeutic moiety (the active part of the therapeutic ingredient) or combination of therapeutic moieties, the strength (with standardized denominators), and the method of administration (with simplified but standardized options). The operational rules also define two categories of exemptions for INN prescribing: INN groups where the first choice of treatment should be continued throughout the therapy period (NO SWITCH) and medicinal product groups not suitable for INN prescribing (NO INN). The reference database is the result of the virtual classification of the Belgian therapeutic arsenal into INN groups, according to the operational rules. Conclusions Defining the operational rules for INN prescribing for and with different stakeholders was a difficult yet feasible assignment. The INN prescribing project resulted into explicit operational rules and a reference database. The Belgian experience may provide important information for other countries planning to operationalize or refine electronic INN prescribing. It can also be used for a thorough evaluation of the impact of the new concept of INN prescribing on daily practice and on medical education.

Introduction At rest, the heart favors fatty acids to produce its energy switching to glucose in response to insulin at postprandial state. Diabetic heart features metabolic adaptation defaults and is no longer able to switch, producing its energy exclusively through fatty acid oxidation. This metabolic inflexibility is a major factor of cardiac dysfunction. Observations made in high-fat diet mouse model lead to an increase in cardiac protein acetylation. We hypothesized that modulation of acetylation impacts the ability of the heart to uptake glucose with a special focus on tubulin. Objectives Microtubules playing a key role in GLUT4 translocation to the plasma membrane, we evaluated the impact of tubulin acetylation on cardiac glucose uptake. Methods The model is primary cultured rat cardiomyocytes. Acetylation levels and signaling pathways are evaluated by western blotting. The impact of long term incubation (20 h) with different pharmacological reducers of acetylation (garcinol 10 μM and anacardic acid 25 μM) on basal and insulin (3 nM, 30 min)-stimulated glucose uptake are measured following the detritiation rate of 2-3H-glucose. Tubacin, the inhibitor of tubulin deacetylase and an adenovirus overexpressing a non-acetylable form of tubulin are used to modulate tubulin acetylation. Results Insulin induces a 6-fold increase in glucose transport. Pharmacologically inhibiting global acetylation increases basal glucose transport similarly to insulin stimulation. By contrast, increasing specifically tubulin acetylation using tubacin inhibits 40% of basal and insulin-induced glucose transport. Accordingly, the overexpression of a dominant negative form and non-acetylable form of tubulin decreases endogenous tubulin acetylation while increasing basal glucose transport. Finally, targeting tubulin acetylation partially restores fatty acid-mediated inhibition of glucose transport. Conclusion There is a clear correlation between tubulin acetylation and glucose uptake level.

In some insect species, two sites of juvenile hormone (JH) synthesis have been reported: the very well documented corpora allata that secrete JH for "general use", and the reproductive system, in particular the male accessory glands, in which the function of the sometimes huge amounts of JH (e.g. in Hyalophora cecropia) remains to be clarified. A recent finding in Schistocerca gregaria, namely that suppression of the ecdysteroid peak preceding a molt by RNAi of the Halloween genes spook, phantom and shade does not impede normal molting, challenges the (never experimentally proven) classical concept that such a peak is causally linked to a molt. Recent developments in epigenetic control of gene expression in both the honey bee and in locusts suggest that, in addition to the classical scheme of hormone-receptor (membrane- and/or nuclear) mode of action, there may be a third way. Upon combining these and other orphan data that do not fit in the commonly accepted textbook schemes, we here advance the working hypothesis that both JH and ecdysone might be important but overlooked players in epigenetic control of gene expression, in particular at extreme concentrations (peak values or total absence). In this review, we put forward how epi-endocrinology can complement classical arthropod endocrinology.

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata. In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE. Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal–induced trypsin peak in the midgut and elevated levels of protein meal–induced yolk polypeptides in the hemolymph. The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. Arch. Insect Biochem. Physiol. 47:161–167, 2001. © 2001 Wiley-Liss, Inc.

Angiotensin converting enzyme (ACE) activity, defined as a captopril-inhibitable dipeptidyl carboxypeptidase activity towards 3H-hippurylglycylglycine, was demonstrated in haemolymph, testes and ovaries of the grey fleshfly Neobellieria bullata, hereby suggesting a physiological role for ACE in these particular tissues. While the ACE activity in haemolymph and testes reached relatively high levels, only minute ACE activity could be detected in ovaries throughout the entire vitellogenic cycle. Ovarian extracts of Neobellieria bullata do contain, however, in addition to Neb-TMOF, the Neobellieria bullata trypsin modulating oostatic factor which is an in vitro and a putative in vivo substrate of ACE in circulation, several other heat-stable molecules which individually function either as an ACE substrate or ACE inhibitor. Presumably these ACE interactive factors mask ACE activity in the fly ovaries, as measured by a classic substrate-binding assay. Purification and characterisation of these ACE substrates/inhibitors is in progress and is likely to facilitate the elucidation of the enigmatic physiological relevance of ACE in insects.

Endothelin-converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest-derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin-1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT-PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.

Vertebrate releasing hormones include gonadotropin releasing hormone (GnRH), growth hormone releasing hormone (GHRH), corticotropin releasing hormone (CRF), and thyrotropin-releasing hormone (TRH). They are synthesized in the hypothalamus and stimulate the release of pituitary hormones. Here we review the knowledge on hormone releasing systems in the protostomian lineage. We address the question: do insects have peptides that may be phylogenetically related to an ancestral GnRH, GHRH, TRH, and CRF? Such endocrine archeology has become possible thanks to the growing list of fully sequenced genomes as well as to the continuously improving bioinformatic tool set. It has recently been shown that the ecdysozoan (nematodes and arthropods) adipokinetic hormones (AKHs), the lophotrochozoan (annelids and mollusks) GnRHs as well as the protochordate GnRHs are structurally related. The adipokinetic hormone precursor-related peptides (APRPs), in locusts encoded by the same gene that contains the AKH-coding region, have been forwarded as the structural counterpart of GHRH of vertebrates. CRF is relatively well conserved in insects, in which it functions as a diuretic hormone. Members of TRH-receptor family seem to have been conserved in some arthropods, but other elements of the thyroid hormone signaling system are not. A challenging idea is that in insects the functions of the thyroid hormones were taken over by juvenile hormone (JH). Our reconstruction suggests that, perhaps, the ancestral releasing hormone precursors played a role in controlling energy metabolism and water balance, and that releasing hormone functions as present in extant vertebrates were probably secondarily acquired.

The only well established function of the prothoracic glands (PGs) of insects is the production of ecdysteroids. In gregarious locusts, like in most insect species, the PGs degenerate soon after the adult molt. In this way they resemble the thymus of mammals, a gland with an important role in the build up of the immune system in young animals. In adult solitarious locusts the PGs persist much longer, however without producing substantial amounts of ecdysteroids. In the literature the existence of a well developed rough endoplasmic reticulum and Golgi complex system has been repeatedly reported, suggesting an active role in peptide or/and protein synthesis and release. The nature of the secreted products remained unknown. Our pepdidomic analysis of an acidified methanolic extract of PGs of last instar gregarious nymphs did not yield any indication for the presence of known locust or other peptides. The peptide release assay was also negative. For our proteomic analysis, we developed an EST-based identification strategy. We successfully identified 50 protein spots on a two dimensional map. In addition to typical protein synthesis-related proteins, a number of proteins with a role in detoxification processes were found, suggesting some role of the PGs in the defense system.

Changes in the color of the cuticle, days after the completion of hardening, are rare in adult insects. Even more so when such changes are specific to one sexual form and coincide with sexual maturation. Adult males of the desert locust Schistocerca gregaria deposit a well characterized ‘yellow protein’ in their cuticle about 10 days after the adult molt, but only if they live under crowded (gregarious) conditions. Isolated-reared (solitarious) males do not turn yellow, neither do the females. Upon regrouping, yellowing is quickly induced, but again, only in the males. Juvenile hormone (JH) is involved, but its sex- and phase-specific effect suggests that other factors are also involved. We analyzed the recent and classical literature to find out what should be added or changed to the classical way of thinking on sex differentiation in insects so that a comprehensive conceptual framework could emerge. Undervalued and/or new data on male accessory glands as a possible second site of JH synthesis, on ecdysteroids as possible sex steroids, on the transcription factor fruitless in insects and on the evolutionarily highly conserved transcription factor Foxl2 that, when ablated in mice is responsible for the transdifferentiation of the ovaries into testes, are considered.

The storage at home of medicines is a poorly researched topic, but it can be a major source of medication errors and other unsafe practices. In this pilot-study, we wanted to get an idea of the scope of the problem and research the feasibility and acceptability of a home-based intervention by a pharmacist. In a convenience sample of 48 households, we encountered numerous problems in a sizable percentage of households. Medicines were frequently not stored out of reach of children, usage instructions and indications were unknown, organization was absent, and there were a plethora of expired medicines present. Refrigeration was less of a problem. Acceptability and perception of utility of the intervention were generally very high. We developed a protocol-based intervention to be used in future research to increase the safe use of medicines at home.

The haemolymph titre of the phase-related 6-kDa peptide is determined in fourth- and fifth-instar larvae, as well as in adults, by high-performance liquid chromatography. In larvae, the concentration of this peptide slowly increases towards the end of the instar to reach a maximum just before the moult. The titre is slightly higher in the fifth instar than in the fourth instar. In adults, it reaches an average concentration of 0.8 μg μL−1 of the haemolymph. In females, a peak is found at day 15 whereas, in males, there are two peaks, the first at day 9 and the second at day 15 after fledging. The role of the peptide in inducing yellow protein mRNA synthesis is investigated, as is its effect on the production of the pheromonal compound phenylacetonitrile (PAN). The peptide stimulates yellow protein mRNA synthesis, but its injection causes no change in yellow colouration. No effect on PAN production is found.

A 6-kDa phase-related peptide (PRP) was recently identified from the hemolymph of the desert locust Schistocerca gregaria. Its presence in much higher concentrations in the crowd-reared (gregarious) phase than in the isolated-reared (solitarious) one suggests a role in phase polyphenism. However, when tested in a variety of classical bioassays, no activity could be found. We hoped that uncovering its site(s) of synthesis might yield hints as to possible functions. An antiserum was raised against the C-terminal 16 aa part of PRP for use in immunocytochemistry. No immunoreactivity was recorded in the fat body, midgut, or Malpighian tubules. The strongest positive immunostaining was observed in the follicle cells of the ovary and in the seminal vesicle tubes of the male accessory gland complex. Also, positive were a pair of large neurosecretory cells in the subesophageal ganglion, the storage part of the corpora cardiaca and some nerve fibers in the brain- and abdominal regions. An additional mass spectrometric analysis was successfully done in combination with a BLAST search to detect possible false positive staining. This confirmed the presence of genuine PRP in most of the immunopositive tissues. Additional experiments are needed to unravel the role of PRP.

In the recent decade, tremendous progress has been realized in insect endocrinology as the result of the application of a variety of advanced methods in neuropeptidome- and receptor research. Hormones of which the existence had been shown by bioassays four decades ago, e.g. bursicon (a member of the glycoprotein hormone family) and pupariation factor (Neb-pyrokinin 2, a myotropin), could be identified, along with their respective receptors. In control of diurnal rhythms, clock genes got company from the neuropeptide Pigment Dispersing Factor (PDF), of which the receptor could also be identified. The discovery of Inka cells and their function in metamorphosis was a true hallmark. Analysis of the genomes of Caenorhabditis elegans, Drosophila melanogaster and Apis mellifera yielded about 75, 100 and 200 genes coding for putative signaling peptides, respectively, corresponding to approximately 57, 100 and 100 peptides of which the expression could already be proven by means of mass spectrometry. The comparative approach invertebrates-vertebrates recently yielded indications for the existence of counterparts in insects for prolactin, atrial natriuretic hormone and Growth Hormone Releasing Hormone (GRH). Substantial progress has been realized in identifying the Halloween genes, a membrane receptor(s) for ecdysteroids, a nuclear receptor for methylfarnesoate, and dozens of GPCRs for insect neuropeptides. The major remaining challenges concern the making match numerous orphan GPCRs with orphan peptidic ligands, and elucidating their functions. Furthermore, the endocrine control of growth, feeding-digestion, and of sexual differentiation, in particular of males, is still poorly understood. The finding that the prothoracic glands produce an autocrine factor with growth factor-like properties and secrete proteins necessitates a reevaluation of their role in development.

Literature on the question whether the juvenile stage of vertebrates is hormonally regulated is scarce. It seems to be intuitively assumed that this stage of development is automated, and does not require any specific hormone(s). Such reasoning mimics the state of affairs in insects until it was shown that surgical removal of a tiny pair of glands in the head, the corpora allata, ended larval life and initiated metamorphosis. Decades later, the responsible hormone was found and named "juvenile hormone" (JH) because when present, it makes a larva molt into another larval stage. JH is a simple ester of farnesol, a sesquiterpenoid present in all eukaryotes. Whereas vertebrates do not have an anatomical counterpart of the corpora allata, their tissues do contain farnesol-like sesquiterpenoids (FLS). Some display typical JH activity when tested in appropriate insect bioassays. Some FLS are intermediates in the biosynthetic pathway of cholesterol, a compound that insects and nematodes (=Ecdysozoa) cannot synthesize by themselves. They ingest it as a vitamin. Until a recent (2014) reexamination of the basic principle underlying insect metamorphosis, it had been completely overlooked that the Ca(2+)-pump (SERCA) blocker thapsigargin is a sesquiterpenoid that mimics the absence of JH in inducing apoptosis. In our opinion, being in the juvenile state is primarily controlled by endogenous FLS that participate in controlling the activity of Ca(2+)-ATPases in the sarco(endo)plasmic reticulum (SERCAs), not only in insects but in all eukaryotes. Understanding the control mechanisms of being in the juvenile state may boost research not only in developmental biology in general, but also in diseases that develop after the juvenile stage, e.g., Alzheimer's disease. It may also help to better understand some of the causes of obesity, a syndrome that holometabolous last larval insects severely suffer from, and for which they found a very drastic but efficient solution, namely metamorphosis. ispartof: Frontiers in Endocrinology vol:5 issue:DEC ispartof: location:Switzerland status: published

The paradigm, still around in textbooks, that ‘in insects sex is strictly genetic, thus that they do not have sex hormones’, is mainly based on a wrong interpretation of the ‘gynandromorph argument’. It is no longer tenable. Given the fact that vertebrates and invertebrates probably had a common, sexually reproducing ancestor, there is no reason to assume that only vertebrates need sex hormones. The major function of sex hormones is to inform the somatoplasm about developmental changes that take place in the gonads. In contrast to juvenile hormone and neuropeptides, ecdysteroids meet all criteria to act as sex hormones, which was probably their ancient role. Their much better documented role in moulting and metamorphosis was a secondary acquisition that enabled arthropods to cope with growth problems, imposed by a rigid cuticle. Female insects use 20-hydroxyecdysone (20E), secreted by the follicle cells of the ovary, in a similar way as females of egg-laying vertebrates use estrogens. For a variety of reasons, the possibility that ecdysteroids, in particular ecdysone (E), might also act as sex hormones in male insects, thus as the counterpart of testosterone of vertebrates, has been very much overlooked. Thanks to the recent discovery of the molecular basis of the haploid-diploid system of sex determination in the honeybee, the characterization of Halloween genes, proteomics, RNAi and so on, it now becomes possible to verify whether in insects, as with vertebrates, males are the endocrinologically default gender form.

In the last decade, a new serine protease inhibitor family has been described in arthropods. Eight members of the family were purified from locusts and share a conserved cysteine array (Cys-Xaa9–12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2–3-Gly-Xaa3–6-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). Using cDNA cloning, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different insect species. In the present study, two isoforms of a novel pacifastin-related precursor (SGPP-4) have been identified in the desert locust, predicting the previously identified SGPI-5 (Schistocerca gregaria PLD-related inhibitor-5) peptide and two novel PLD-related peptide sequences. One novel peptide (SGPI-5A) was synthesized chemically, and its inhibitory activity was assessed in vitro. Although proteases from a locust midgut extract were very sensitive to SGPI-5A, the same peptide proved to be a relatively poor inhibitor of bovine trypsin. By an in silico datamining approach, a novel pacifastin-related precursor with seven PLD-related domains was identified in the mosquito, Aedes aegypti. As in other insect pacifastin-related precursors, the Aedes precursor showed a particular domain architecture that is not encountered in other serine protease inhibitor families. Finally, a comparative real-time RT-PCR analysis of SGPP-4 transcripts in different tissues of isolated- (solitarious) and crowded-reared (gregarious) locusts was performed. This showed that SGPP-4 mRNA levels are higher in the brain, testes and fat body of gregarious males than of solitarious males. These results have been compared with data from a similar study on SGPP-1–3 transcripts and discussed with respect to a differential regulation of serine-protease-dependent pathways as a possible mechanism underlying locust phase polymorphism.

The insecticidal activity of the antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was assessed in in vivo assays against the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera, Noctuidae). BVDU, mixed into an artificial diet, caused a variety of effects, depending on the concentration used. Compared with controls, food intake was lower, larval growth was retarded and larval development was prolonged. The treated larvae formed smaller pupae and the hatching moths often showed morphogenetic defects. A higher mortality could be found in larval and pupal stages and was generally caused by moult disruption. A choice assay showed that BVDU has very slight feeding-deterrent properties, which only partly explain the toxic effects. The agent most probably acts through its cytostatic activity that has been described previously using cell lines of different insect species.

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5). The light chain of the heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of six conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-6)-Cys-Thr-Xaa(3)-Cys) with the locust inhibitors. Via cDNA cloning, eight pacifastin-related precursors have been identified in locusts. Interestingly, additional pacifastin-related precursors have been identified in Diptera, Lepidoptera and Coleoptera utilising an in silico data mining approach.

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning. Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa3-Cys) with the locust inhibitors. So far, for most of the PLD-related peptides the biological functions remain obscure. To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system. The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide). As a consequence, both peptides were expressed as fusion proteins (2-3 mg/l) and targeted to the periplasmic space. Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity. Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively.