Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes

The Treatment In Morning versus Evening (TIME) study:Analysis of recruitment, follow-up and retention rates post-recruitment

Abstract Background The use of information technology (IT) is now the preferred method of capturing and storing clinical research data. The Treatment In Morning versus Evening (TIME) study predominantly uses electronic data capture and IT to compare morning dosing of hypertensive medication against evening dosing. Registration, consent, participant demographics and follow-up data are all captured via the study website. The aim of this article is to assess the success of the TIME methodology compared with similar studies. Methods To assess the TIME study, published literature on similar clinical trials was reviewed and compared against TIME recruitment, follow-up and email interaction data. Results The TIME website registered 31,695 individuals, 21,116 of whom were randomised. Recruitment cost per randomised participant varied by strategy: £17.40 by GP practice, £3.08 by UK Biobank and £58.82 for GoShare. Twelve-month follow-up retention rates were 96%. A total of 1089 participants have withdrawn from their assigned time of dosing, 2% of whom have declined follow-up by record linkage or further contact. When the TIME data are compared with similar study data, study recruitment is very successful. However, TIME suffers difficulties with participant follow-up and withdrawal rates similar to those of conventional studies. Conclusions The TIME study has been successful in recruitment. Follow-up, retention rates and withdrawal rates are all acceptable, but ongoing work is required to ensure participants remain engaged with the study. Various recruitment strategies are necessary, and all viable options should be encouraged to maintain participant engagement throughout the life of studies using IT

Aged garlic has more potent antiglycation and antioxidant properties compared to fresh garlic extract in vitro

Protein glycation involves formation of early (Amadori) and late advanced glycation endproducts (AGEs) together with free radicals via autoxidation of glucose and Amadori products. Glycation and increased free radical activity underlie the pathogenesis of diabetic complications. This study investigated whether aged garlic has more potent antiglycation and antioxidant properties compared to fresh garlic extract in vitro in a cell-free system. Proteins were glycated by incubation with sugars (glucose, methylglyoxal or ribose) ±5–15 mg/mL of aged and fresh garlic extracts. Advanced glycation endproducts were measured using SDS-PAGE gels and by ELISA whereas Amadori products were assessed by the fructosamine method. Colorimetric methods were used to assess antioxidant activity, free radical scavenging capacity, protein-bound carbonyl groups, thiol groups and metal chelation activities in addition to phenolic, total flavonoid and flavonol content of aged and fresh garlic extracts. Aged garlic inhibited AGEs by 56.4% compared to 33.5% for an equivalent concentration of fresh garlic extract. Similarly, aged garlic had a higher total phenolic content (129 ± 1.8 mg/g) compared to fresh garlic extract (56 ± 1.2 mg/g). Aged garlic has more potent antiglycation and antioxidant properties compared to fresh garlic extract and is more suitable for use in future in vivo studies

The cognitive-affective-conative model of destination image: a confirmatory analysis

Destination image influences tourist behaviour before, during and after travel, as it is an important instrument which contributes to tourists’ loyalty. Although Gartner (1993) advocates that the cognitive, affective and conative dimensions of destination image are hierarchically interrelated, there is no empirical evidence to support the complete model. This study aims to test the hierarchical nature of the relationship between the dimensions of destination image. The results of structural equation modelling confirm Gartner’s theoretical model, validating the theory that the influence of the cognitive component on the conative dimension is higher when mediated by the affective component, raising managerial implications

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

Some structural studies by deuterium magnetic resonance

Single crystals of ND 4 DS0 4 and Sr(DCOO)2.2D 2 0 have been studied by deuteron magnetic resonance spectroscopy; the apparatus and techniques used are described in some detail. ND 4 DS0 4 has considerable interest because of its ferroelectric properties: spectra were recorded at 300 K and 230 K, within the paraelectric and ferroelectric phases respectively. Symmetry relationships were used in solving the structure, particularly for refining the orientation data. At room temperature, the two sets of bisu1phate deuterons were found to have (within xperimental error) the same quadrupole coupling constants and asymmetry parameters: h = 191.9 ± 3KHz, T1 = 0.094 ± 0.02 ~ h = 193.2 ± 3KHz, T1 = 0.070 ± 0.0l. The quadrupolar constants appear somewhat larger than expected from the known 0····0 distances. No evidence of disorder is apparent from the room temperature data. At 230 K, both groups of deuterons appear to have suffered small angular displacements, essentially planar and antisymmetric with relation to the room temperature positions. One type of deuteron undergoes movements of larger amplitude in a plane more favourably oriented with respect to the polar axis, [Cl], which suggests a definite contribution from these deuterons to the appearance of ferroelectricity. The Curie point has been determined independently by use of the radio frequency spectrometer; a value of 266 ± 2K was obtained, in reasonable agreement with the literature value from dielectric constant measurements. The ND4+ group is reorienting rapidly both at 230 K and 300 K. Little change seems to occur upon transition. Some distortion of the tetrahedral structure, a possible difference in the reorientation rate for the two kinds of ND4+ ions, and a slight difference in their respective environments could be the main causes for the fine structure observed in the ammonium resonance. The study of Sr(DCOO)2.2D 2 0 gave the following average values for the quadrupolar constants and asymmetry parameters at 300 K: 2 formate ion [DC(1)0(1)0(2)]- ~ = 167.6 + 3KHz 'T] = 0.043 ± 0.01, DB h = 170.3 ± 3KHz 'T] = 0.025 ± 0.01. formate ion [DC(2)0(3)0(4)]- DB= h 242.8 + 3KHz 'T] = O. 100 ± 0.005 2 ~ = 216.8 + 3KHz 'T] = O. 113 ± 0.005 Both formate ions give e.f.g. tensors very close to axial symmetry. For [DC(1)0(1)0(2)]-, the deuteron is approximately aligned with the C(1) 0(10(2) bisectrix in an essentially planar arrangement. For [DC(2)0(3)0(4)]- the deuteron shows a surprising deviation of 22̊ from the plane of the COO group, as determined from Osaki's X-ray data. A weak C-D---O bonding interaction is tentatively suggested to account for this anomaly. D 2 0(1) was easily localised in the surrounding structure as known from X-ray data; the electric field gradient constants indicate that this molecule is 'static' at room temperature. A-D-0-D angle of 107.8̊ is in good agreement with the value for H2O D 2 D0(2) gave rise to broad and rather weak resonances with marked fine structure: some poorly defined curves were analysed only approximately. The high quadrupolar constants (330 ± 30 KHz) and asymmetry parameters (0.2 ± 0.1) derived for one set of the curves suggest that this D 2 0 molecule is weakly hydrogen bonded (if at all), and may have a rather high degree of motional freedom. analysed only approximately. The high quadrupolar constants (330 ± 30 KHz) and asymmetry parameters (0.2 ± 0.1) derived for one set of the curves suggest that this D 2 0 molecule is weakly hydrogen bonded (if at all), and may have a rather high degree of motional freedom

Recent advances in understanding hypertension development in sub-Saharan Africa

Consistent reports indicate that hypertension is a particularly common finding in black populations. Hypertension occurs at younger ages and is often more severe in terms of blood pressure levels and organ damage than in whites, resulting in a higher incidence of cardiovascular disease and mortality. This review provides an outline of recent advances in the pathophysiological understanding of blood pressure elevation and the consequences thereof in black populations in Africa. This is set against the backdrop of populations undergoing demanding and rapid demographic transition, where infection with the Human Immunodeficiency Virus predominates, and where under and over-nutrition coexist. Collectively, recent findings from Africa illustrate an increased lifetime risk to hypertension from foetal life onwards. From young ages black populations display early endothelial dysfunction, increased vascular tone and reactivity, microvascular structural adaptions, as well as increased aortic stiffness resulting in elevated central and brachial blood pressures during the day and night, when compared to whites. Together with knowledge on the contributions of sympathetic activation and abnormal renal sodium handling, these pathophysiological adaptations result in subclinical and clinical organ damage at younger ages. This overall enhanced understanding on the determinants of blood pressure elevation in blacks encourages (a) novel approaches to assess and manage hypertension in Africa better, (b) further scientific discovery to develop more effective prevention and treatment strategies, and (c) policymakers and health advocates to collectively contribute in creating health-promoting environments in Africa

Insect pathogens as biological control agents: back to the future

The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 15 years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of MCAs, particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for medically important pests including dipteran vectors,. These pathogens combine the advantages of chemical pesticides and microbial control agents (MCAs): they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systemics (i.e. in transgenic plants). Unlike broad spectrum chemical pesticides, B. thuringiensis toxins are selective and negative environmental impact is very limited. Of the several commercially produced MCAs, B. thuringiensis (Bt) has more than 50% of market share. Extensive research, particularly on the molecular mode of action of Bt toxins, has been conducted over the past two decades. The Bt genes used in insect-resistant transgenic crops belong to the Cry and vegetative insecticidal protein families of toxins. Bt has been highly efficacious in pest management of corn and cotton, drastically reducing the amount of broad spectrum chemical insecticides used while being safe for consumers and non-target organisms. Despite successes, the adoption of Bt crops has not been without controversy. Although there is a lack of scientific evidence regarding their detrimental effects, this controversy has created the widespread perception in some quarters that Bt crops are dangerous for the environment. In addition to discovery of more efficacious isolates and toxins, an increase in the use of Bt products and transgenes will rely on innovations in formulation, better delivery systems and ultimately, wider public acceptance of transgenic plants expressing insect-specific Bt toxins. Fungi are ubiquitous natural entomopathogens that often cause epizootics in host insects and possess many desirable traits that favor their development as MCAs. Presently, commercialized microbial pesticides based on entomopathogenic fungi largely occupy niche markets. A variety of molecular tools and technologies have recently allowed reclassification of numerous species based on phylogeny, as well as matching anamorphs (asexual forms) and teleomorphs (sexual forms) of several entomopathogenic taxa in the Phylum Ascomycota. Although these fungi have been traditionally regarded exclusively as pathogens of arthropods, recent studies have demonstrated that they occupy a great diversity of ecological niches. Entomopathogenic fungi are now known to be plant endophytes, plant disease antagonists, rhizosphere colonizers, and plant growth promoters. These newly understood attributes provide possibilities to use fungi in multiple roles. In addition to arthropod pest control, some fungal species could simultaneously suppress plant pathogens and plant parasitic nematodes as well as promote plant growth. A greater understanding of fungal ecology is needed to define their roles in nature and evaluate their limitations in biological control. More efficient mass production, formulation and delivery systems must be devised to supply an ever increasing market. More testing under field conditions is required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly, greater attention must be paid to their use within integrated pest management programs; in particular, strategies that incorporate fungi in combination with arthropod predators and parasitoids need to be defined to ensure compatibility and maximize efficacy. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are potent MCAs. Substantial progress in research and application of EPNs has been made in the past decade. The number of target pests shown to be susceptible to EPNs has continued to increase. Advancements in this regard primarily have been made in soil habitats where EPNs are shielded from environmental extremes, but progress has also been made in use of nematodes in above-ground habitats owing to the development of improved protective formulations. Progress has also resulted from advancements in nematode production technology using both in vivo and in vitro systems; novel application methods such as distribution of infected host cadavers; and nematode strain improvement via enhancement and stabilization of beneficial traits. Innovative research has also yielded insights into the fundamentals of EPN biology including major advances in genomics, nematode-bacterial symbiont interactions, ecological relationships, and foraging behavior. Additional research is needed to leverage these basic findings toward direct improvements in microbial control

Characterization of α-Glucosidases From Lutzomyia longipalpis Reveals Independent Hydrolysis Systems for Plant or Blood Sugars

Lutzomyia longipalpis is the main vector of Leishmania infantum and exploits different food sources during development. Adults have a diet rich in sugars, and females also feed on blood. The sugar diet is essential for maintaining longevity, infection, and Leishmaniasis transmission. Carbohydrases, including α-glucosidases, are the main enzymes involved in the digestion of sugars. In this context, we studied the modulation of α-glucosidase activities in different feeding conditions and compartments of Lutzomyia longipalpis females, in order to characterize in detail their roles in the physiology of this insect. All tissues showed activity against MUαGlu and sucrose, with highest activities in the midgut and crop. Activity was 1,000 times higher on sucrose than on MUαGlu. Basal activities were observed in non-fed insects; blood feeding induced activity in the midgut contents, and sugar feeding modulated activity in midgut tissues. α-glucosidase activity changed after female exposure to different sugar concentrations or moieties. α-glucosidases from different tissues showed different biochemical properties, with an optimum pH around 7.0–8.0 and KM between 0.37 and 4.7 mM, when MUαGlu was used as substrate. Using sucrose as substrate, the optimum pH was around 6.0, and KM ranges between 11 and 800 mM. Enzymes from the crop and midgut tissues showed inhibition in high substrate concentrations (sucrose), with KI ranging from 39 to 400 mM, which explains the high KM values found. Chromatographic profiles confirmed that different α-glucosidases are been produced in L. longipalpis in different physiological contexts, with the distinction of at least four α-glucosidases. The results suggest that some of these enzymes are involved in different metabolic processes, like digestion of plant sugars, digestion of blood glycoproteins or glycolipids, and mobilization of energetic storages during starvation

Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE−/−) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001). Conclusion: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted. Keywords: Non-alcoholic fatty liver disease, Glyoxalase, Methylglyoxal, Proteomics, iTRA