Structure and function of olfactory proteins in mosquitoes and aphids.

This thesis reports the experimental results of my research in the field of insect chemoreception. The focus is on biochemical events occurring at the periphery of the olfactory system, where odorants and pheromones are detected and discriminated. In particular, I have studied a class of soluble proteins that very recently have been shown to represent the key elements in insect chemosensing. As suggested by their name, Odorant-binding proteins (OBPs) bind chemicals present in the environment and convey the information to membrane-bound receptors, where the chemical messages are translaed into electric signals to be processed by the brain. For my experimental work I chose species very different in their biology, chemical ecology and phylogenetic position, aphids and mosquitoes. Both are of great economical importance, aphids represent one of the major pests in agriculture, not only for their direct damage to plants, but also as carriers of deadly microorganisms. Mosquitoes are vectors of serious diseases worldwide, causing enormous numbers of victims particularly among the populations of developing countries. In aphids, we have cloned and characterised for the first time 4 OBPs in different aphid species. Three of them have been expressed in bacterial system and functionally studied. Unlike OBPs in other insects, those of aphids are well conserved across even very distant species. Ligand-binding experiments have shown similar behaviour of the three proteins towards several organic compounds, but also some significant selectivities. In particular, (E)-β-farnesene, the aphids alarm pheromone and its related compound farnesol exhibited good affinity to OBP3, but did not bind the other two proteins. On the basis of such results we can suggest that OBP3 mediates the typical response of aphids to the alarm pheromone and propose a strategy for the design and synthesis of new repellents for aphids. In the malaria mosquito, Anopheles gambiae, seven OBPs have been expressed, purified and characterised for their ligand-binding properties. Binding experiments performed with mixtures of two OBPs of An. gambiae, suggested that OBP4 could form heterodimers with OBP1 and OBP3, thus generating new protein species with different properties. Such phenomenon could also occur in vivo, based on the observation that the genes encoding OBP1 and OBP4 are co-expressed in the same sensillum. Western blot has revealed that OBP47, an anomalous member of this class of proteins, being longer and more complex, is glycosylated in the mosquito. This is the first report of glycosylation in insect OBPs. The same protein was also successfully expressed both in bacterial and yeast systems. A highly purified samples has been crystallised by our collaborator, Christian Cambillau, Marseille, and its folding has been solved, being the first three-dimensional structure of a C-plus OBP. Moreover, the expression of OBPs has been mapped in the antennae of An. gambiae using a proteomic approach in collaboration with the group of CISM, University of Firenze, revealing that only two OBPs are expressed at relatively high levels in male antennae of An. gambiae, while at least a dozen are expressed in the female antennae. A parallel work investigated the expression of the most abundant OBP in the sex organs of An. gambiae, as well as of another mosquito species, Aedes albopictus. In both species this protein (OBP9 in An. gambiae, OBP22 in Ae. albopictus) is produced in the male sex organs and transferred to the female during mating. In the latter species, OBP22 is complexed with nonanal, a new putative male pheromone. This work supports the idea that, as in vertebrates, also in insects OBPs may play a double role in the detection and in the release of pheromones. The results reported in this thesis represent a major contribution to the characterisation of OBPs in insect and provide strong evidence that OBPs are directly and specifically involved in insect chemical communication. In particular, controlling the populations of dangerous insects can be best achieved after the fine and specific interactions between semiochemicals and their binding proteins are completely understood and clarified

Crystal structure of a novel type of odorant binding protein from Anopheles gambiae, belonging to the C+ class

International audienceAnopheles gambiae (Agam) relies on its olfactory system to target human prey, leading eventually to injection of Plasmodium falciparum, the malaria vector. Odorant-binding proteins (OBPs) are the first line of proteins involved in odorant recognition. They interact with olfactory receptors and thus constitute an interesting target for insect control. We undertook a large-scale study of proteins belonging to the olfactory system of Agam with the aim of preventing insect bites by designing strong olfactory repellents. We determined the 3D structures of several Agam OBPs alone or in complex with model compounds. Here, we report the first 3D structure of a member of the OBP C+ class, AgamOBP47, which has a longer sequence than classical OBPs and contains 6 disulphide bridges. AgamOBP47 possesses a core of six a-helices and three disulphide bridges, similar to the classical OBP fold. Two extra loops and the N- and C-terminal extra segments contain two additional a-helices and are maintained together by three disulphide bridges. They are embrace the classical OBP core domain. The binding site of OBP47 is located between the core and the additional domains. Two crevices are observed on opposite sides of OBP47, which are joined together by a shallow channel of sufficient size to accommodate a model of the best tested ligand. The binding sites of C+ class OBPs exhibit therefore different characteristics of their binding site, as compared to classical OBPs, which should leave to markedly diverse functional implications

Adopting a Theophylline-Responsive Riboswitch for Flexible Regulation and Understanding of Glycogen Metabolism in Synechococcus elongatus PCC7942

Cyanobacteria are supposed to be promising photosynthetic microbial platforms that recycle carbon dioxide driven into biomass and bioproducts by solar energy. Glycogen synthesis serves as an essential natural carbon sink mechanism, storing a large portion of energy and organic carbon source of photosynthesis. Engineering glycogen metabolism to harness and rewire carbon flow is an important strategy to optimize efficacy of cyanobacteria platforms. ADP-glucose pyrophosphorylase (GlgC) catalyzes the rate-limiting step for glycogen synthesis. However, knockout of glgC fails to promote cell growth or photosynthetic production in cyanobacteria, on the contrary, glgC deficiency impairs cellular fitness and robustness. In this work, we adopted a theophylline-responsive riboswitch to engineer and control glgC expression in Synechococcus elongatus PCC7942 and achieved flexible regulation of intracellular GlgC abundance and glycogen storage. With this approach, glycogen synthesis and glycogen contents in PCC7942 cells could be regulated in a range from about 40 to 300% of wild type levels. In addition, the results supported a positive role of glycogen metabolism in cyanobacteria cellular robustness. When glycogen storage was reduced, cellular physiology and growth under standard conditions was not impaired, while cellular tolerance toward environmental stresses was weakened. While when glycogen synthesis was enhanced, cells of PCC7942 displayed optimized cellular robustness. Our findings emphasize the significance of glycogen metabolism for cyanobacterial physiology and the importance of flexible approaches for engineering and understanding cellular physiology and metabolism

DTI-ALPS: An MR biomarker for motor dysfunction in patients with subacute ischemic stroke

PurposeBrain glymphatic dysfunction is involved in the pathologic process of acute ischemic stroke (IS). The relationship between brain glymphatic activity and dysfunction in subacute IS has not been fully elucidated. Diffusion tensor image analysis along the perivascular space (DTI-ALPS) index was used in this study to explore whether glymphatic activity was related to motor dysfunction in subacute IS patients.MethodsTwenty-six subacute IS patients with a single lesion in the left subcortical region and 32 healthy controls (HCs) were recruited in this study. The DTI-ALPS index and DTI metrics (fractional anisotropy, FA, and mean diffusivity, MD) were compared within and between groups. Spearman's and Pearson's partial correlation analyses were performed to analyze the relationships of the DTI-ALPS index with Fugl-Meyer assessment (FMA) scores and with corticospinal tract (CST) integrity in the IS group, respectively.ResultsSix IS patients and two HCs were excluded. The left DTI-ALPS index of the IS group was significantly lower than that of the HC group (t = −3.02, p = 0.004). In the IS group, a positive correlation between the left DTI-ALPS index and the simple Fugl-Meyer motor function score (ρ = 0.52, p = 0.019) and a significant negative correlation between the left DTI-ALPS index and the FA (R = −0.55, p = 0.023) and MD (R = −0.48, p = 0.032) values of the right CST were found.ConclusionsGlymphatic dysfunction is involved in subacute IS. DTI-ALPS could be a potential magnetic resonance (MR) biomarker of motor dysfunction in subacute IS patients. These findings contribute to a better understanding of the pathophysiological mechanisms of IS and provide a new target for alternative treatments for IS

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector. © 2013 Mastrobuoni et al

Rhizosphere Microenvironments of Eight Common Deciduous Fruit Trees Were Shaped by Microbes in Northern China

The rhizosphere microenvironment is the site of nutrient circulation and microbial community formation, and thus is an ongoing topic of research. Although research on this topic is extensive, studies into the rhizosphere microenvironment of fruit trees remain rare. To elucidate the mechanisms driving the fruit tree rhizosphere microenvironment, we assessed soil physicochemical properties, enzyme activities, the community-level physiological profile (CLPP) and microbial diversity in rhizospheric soils of eight common deciduous fruit trees in northern China. We found that the available minerals, pH, enzyme activities, microbial utilization of six types of carbon (C) substrates, and microbial diversity in the rhizosphere varied among tree species. Redundancy analysis (RDA) showed that rhizosphere microenvironmental parameters (ammonia nitrogen content, soil pH and invertase activity) were closely related to the soil microbial community. Further analysis revealed that the soil microbial utilization of six C sources, nitrate nitrogen content, and invertase activity were negatively correlated with Ambiguous species and Alternaria; however, these groups were positively correlated with pH. The ammonia nitrogen content was positively correlated with C source utilization and negatively correlated with Ambiguous, Lysobacter, Nitrospira, Alternaria, Fusarium, and Colletotrichum. Interestingly, invertase was closely linked to the microbial community, especially fungal diversity, and was positively correlated with plant-beneficial microbes such as Mortierella, Geomyces, Lysobacter, and Chaetomium, but negatively correlated with pathogenic microbes such as Alternaria, Fusarium, and Colletotrichum. Hence, rhizosphere soil physicochemical properties, enzyme activities and microbial community were significantly affected by tree species. Additionally, a variety of environmental factors were closely related to the microbial community in the rhizospheric soils of eight species of deciduous fruit trees

The association between stress hyperglycemia and unfavorable outcomes in patients with anterior circulation stroke after mechanical thrombectomy

Background and purposeStress hyperglycemia is common in critical and severe diseases. However, few studies have examined the association between stress hyperglycemia and the functional outcomes of patients with anterior circulation stroke, after mechanical thrombectomy (MT), in different diabetes status. This study therefore aimed to determine the relationship between stress hyperglycemia and the risk of adverse neurological functional outcomes in anterior circulation stroke patients with and without diabetes after MT.MethodsData of 408 patients with acute anterior circulation stroke treated with MT through the green-channel treatment system for emergency stroke at the First Affiliated Hospital of Jinan University between January 2016 and December 2020 were reviewed retrospectively. The stress hyperglycemia ratio (SHR) was calculated as fasting plasma glucose (mmol/L) divided by glycosylated hemoglobin (%). The patients were stratified into four groups by quartiles of SHR (Q1-Q4). The primary outcome was an excellent (nondisabled) functional outcome at 3 months after admission (modified Rankin Scale score of 0–1). The relationship between stress hyperglycemia and neurological outcome after stroke was assessed using multivariate logistic regression.ResultsAfter adjusting for potential confounders, compared with patients in Q1, those in Q4 were less likely to have an excellent outcome at 3 months (odds ratio [OR], 0.32, 95% confidence interval [CI], 0.14–0.66, p = 0.003), a good outcome at 3 months (OR, 0.41, 95% CI, 0.20–0.84, p = 0.020), and major neurological improvement (OR, 0.38, 95% CI, 0.19–0.73, p = 0.004). Severe stress hyperglycemia increased risks of 3-months all-cause mortality (OR, 2.82, 95% CI, 1.09–8.29, p = 0.041) and ICH (OR, 2.54, 95% CI, 1.21–5.50, p = 0.015).ConclusionStress hyperglycemia was associated with a reduced rate of excellent neurological outcomes, and increased mortality and ICH risks in patients with anterior circulation stroke after MT regardless of diabetes status

Striated muscle activator of Rho signalling (STARS) is reduced in ageing human skeletal muscle and targeted by miR-628-5p

Aim: The striated muscle activator of Rho signalling (STARS) is a muscle-specific actin-binding protein. The STARS signalling pathway is activated by resistance exercise and is anticipated to play a role in signal mechan-otransduction. Animal studies have reported a negative regulation of STARS signalling with age, but such regulation has not been investigated in humans. Methods: Ten young (18–30 years) and 10 older (60–75 years) subjects completed an acute bout of resistance exercise. Gene and protein expres-sion of members of the STARS signalling pathway and miRNA expression of a subset of miRNAs, predicted or known to target members of STARS signalling pathway, were measured in muscle biopsies collected pre-exer-cise and 2 h post-exercise. Results: For the first time, we report a significant downregulation of the STARS protein in older subjects. However, there was no effect of age on the magnitude of STARS activation in response to an acute bout of exer-cise. Finally, we established that miR-628-5p, a miRNA regulated by age and exercise, binds to the STARS 3’UTR to directly downregulate its tran-scription. Conclusion: This study describes for the first time the resistance exercise-induced regulation of STARS signalling in skeletal muscle from older humans and identifies a new miRNA involved in the transcriptional con-trol of STARS

Understanding the Local Sustainable Economic Development from New “3D” Perspective: Case of Hainan Island

This paper proposes a new T-3D measurement framework for calculating tourism economic space and as a tool able to assist in determining the sustainability of tourism development. The T-3D framework is described as a tourism-specific version of the Density, Distance, Division (3D) framework. Tourism economic concentration, level of integration and the degree of specialization are used to provide a touristic interpretation of density, distance and division. Taking Hainan Province as an example, this paper outlines the T-3D characteristics of tourism economic space. The results show that Hainan Province has large differences in the distribution of the tourism economy. In addition to the spatial division of the tourism economy, the spatial density and distance of the tourism economy are basically consistent in value. Further, the spatial density and division of the tourism economy exhibits a dual-core based on the cities of Sanya and Haikou, and the spatial distance of the tourism economy exhibits “core-peripheral” characteristics. The tourism economic space shows that the highest agglomeration based on T-3D characteristics occurs in the east followed by the west with the lowest agglomeration in the middle of the province. Using empirical analysis, the validity of the T-3D analysis system of the tourism economic space is verified and this is more conducive to improving the competitiveness of the tourism industry and promoting sustainable tourism development