Characterization of the caleosin gene family in the Triticeae

Background

The caleosin genes encode proteins with a single conserved EF hand calcium-binding domain and comprise small gene families found in a wide range of plant species. Some members of the gene family have been shown to be upregulated by environmental stresses including low water availability and high salinity. Caleosin 3 from wheat has been shown to interact with the α-subunit of the heterotrimeric G proteins, and to act as a GTPase activating protein (GAP). This study characterizes the size and diversity of the gene family in wheat and related species and characterizes the differential tissue-specific expression of members of the gene family.

Results

A total of 34 gene family members that belong to eleven paralogous groups of caleosins were identified in the hexaploid bread wheat, T. aestivum. Each group was represented by three homeologous copies of the gene located on corresponding homeologous chromosomes, except the caleosin 10, which has four gene copies. Ten gene family members were identified in diploid barley, Hordeum vulgare, and in rye, Secale cereale, seven in Brachypodium distachyon, and six in rice, Oryza sativa. The analysis of gene expression was assayed in triticale and rye by RNA-Seq analysis of 454 sequence sets and members of the gene family were found to have diverse patterns of gene expression in the different tissues that were sampled in rye and in triticale, the hybrid hexaploid species derived from wheat and rye. Expression of the gene family in wheat and barley was also previously determined by microarray analysis, and changes in expression during development and in response to environmental stresses are presented.

Conclusions

The caleosin gene family had a greater degree of expansion in the Triticeae than in the other monocot species, Brachypodium and rice. The prior implication of one member of the gene family in the stress response and heterotrimeric G protein signaling, points to the potential importance of the caleosin gene family. The complexity of the family and differential expression in various tissues and under conditions of abiotic stress suggests the possibility that caleosin family members may play diverse roles in signaling and development that warrants further investigation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-239) contains supplementary material, which is available to authorized users.     

Thermal performance of the Chagas disease vector,Triatoma infestans,under thermal variability

Vector-borne diseases (VBD) are particularly susceptible to climate change because most of the diseases’ vectors are ectotherms, which themselves are susceptible to thermal changes. The Chagas disease is one neglected tropical disease caused by the protozoan parasite, Trypanosoma cruzi. One of the main vectors of the Chagas disease in South America is Triatoma infestans, a species traditionally considered to be restricted to domestic or peridomestic habitats, but sylvatic foci have also been described along its distribution. The infestation of wild individuals, together with the projections of environmental changes due to global warming, urge the need to understand the relationship between temperature and the vector’s performance. Here, we evaluated the impact of temperature variability on the thermal response of T. infestans. We acclimated individuals to six thermal treatments for five weeks to then estimate their thermal performance curves (TPCs) by measuring the walking speed of the individuals. We found that the TPCs varied with thermal acclimation and body mass. Individuals acclimated to a low and variable ambient temperature (18°C ± 5°C) exhibited lower performances than those individuals acclimated to an optimal temperature (27°C ± 0°C); while those individuals acclimated to a low but constant temperature (18°C ± 0°C) did not differ in their maximal performance from those at an optimal temperature. Additionally, thermal variability (i.e., ± 5°C) at a high temperature (30°C) increased performance. These results evidenced the plastic response of T. infestans to thermal acclimation. This plastic response and the non-linear effect of thermal variability on the performance of T. infestans posit challenges when predicting changes in the vector’s distribution range under climate change.     

Differences in the inflammatory response induced by acute pancreatitis in different white adipose tissue sites in the rat

Background

There is increasing evidence of the role of adipose tissue on the systemic effects of acute pancreatitis. Patients with higher body mass index have increased risk of local and systemic complications and patients with android fat distribution and higher waist circumference are at greater risk for developing the severe form of the disease. Here we evaluated the changes on different areas of adipose tissue and its involvement on the inflammatory response in an experimental model of acute pancreatitis.

Methods

Pancreatitis was induced in male Wistar rats by intraductal administration of sodium taurocholate. Orlistat was administered to inhibit lipase activity. Activation of peritoneal macrophages was evaluated by measuring IL1β and TNFα expression. Inflammation was evaluated by measuring myeloperoxidase activity in mesenteric, epididymal and retroperitoneal areas of adipose tissue. Changes in the expression of inflammatory mediator in these areas of adipose tissue were also evaluated by RT-PCR.

Results

Pancreatitis induces the activation of peritoneal macrophages and a strong inflammatory response in mesenteric and epididymal sites of adipose tissue. By contrast, no changes were found in retroperitoneal adipose tissue. Inhibition of lipase prevented the activation of macrophages and the local inflammation in adipose tissue.

Conclusions

Our results confirm the involvement of adipose tissue on the progression of systemic inflammatory response during acute pancreatitis. However, there is a considerable diversity in different adipose tissue sites. These differences need to be taken into account in order to understand the progression from local pancreatic damage to systemic inflammation during acute pancreatitis.     

Protein expression from synthetic genes: selection of clones using GFP

Construction of synthetic genes is today the most elegant way to optimize the heterologous expression of a recombinant protein. However, the selection of positive clones that incorporate the correct synthetic DNA fragments is a bottleneck as current methods of gene synthesis introduce 3.5 nucleotide deletions per kb. Furthermore, even when all predictable optimizations for protein production have been introduced into the synthetic gene, production of the protein is often disappointing: protein is produced in too low amounts or end up in inclusion bodies. We propose a strategy to overcome these two problems simultaneously by cloning the synthetic gene upstream of a reporter gene. This permits the selection of clones devoid of frame-shift mutations. In addition, beside nucleotide deletion, an average of three non-neutral mutations per kb are introduced during gene synthesis. Using a reporter protein downstream of the synthetic gene, allows the selection of clones with random mutations improving the expression or the folding of the protein of interest. The problem of errors found in synthetic genes is then turned into an advantage since it provides polymorphism useful for molecular evolution. The use of synthetic genes appears as an alternative to the error-prone PCR strategy to generate the variations necessary in protein engineering experiments.     

Temporal and spatial variability in nearshore bacterioplankton communities of Lake Michigan

The spatial and temporal variability of bacterial communities were determined for the nearshore waters of Lake Michigan, an oligotrophic freshwater inland sea. A freshwater estuary and nearshore sites were compared six times during 2006 using denaturing gradient gel electrophoresis (DGGE). Bacterial composition clustered by individual site and date rather than by depth. Seven 16S rRNA gene clone libraries were constructed, yielding 2717 bacterial sequences. Spatial variability was detected among the DGGE banding patterns and supported by clone library composition. The clone libraries from deep waters and the estuary environment revealed highest overall bacterial diversity. Betaproteobacteria sequence types were the most dominant taxa, comprising 40.2–67.7% of the clone libraries. BAL 47 was the most abundant freshwater cluster of Betaproteobacteria , indicating widespread distribution of this cluster in the nearshore waters of Lake Michigan. Incertae sedis 5 and Oxalobacteraceae sequence types were prevalent in each clone library, displaying more diversity than previously described in other freshwater environments. Among the Oxalobacteraceae sequences, a globally distributed freshwater cluster was determined. The nearshore waters of Lake Michigan are a dynamic environment that experience forces similar to the coastal ocean environment and share common bacterial diversity with other freshwater habitats.     

Müller glial cell-provided cellular light guidance through the vital guinea-pig retina

In vertebrate eyes, images are projected onto an inverted retina where light passes all retinal layers on its way to the photoreceptor cells. Light scattering within this tissue should impair vision. We show that radial glial (Müller) cells in the living retina minimize intraretinal light scatter and conserve the diameter of a beam that hits a single Müller cell endfoot. Thus, light arrives at individual photoreceptors with high intensity. This leads to an optimized signal/noise ratio, which increases visual sensitivity and contrast. Moreover, we show that the ratio between Müller cells and cones-responsible for acute vision-is roughly 1. This suggests that high spatiotemporal resolution may be achieved by each cone receiving its part of the image via its individual Müller cell-light guide.     

Screening for engineered neomycin riboswitches that control translation initiation

Riboswitches are genetic control elements that regulate gene expression in a small molecule-dependent way. We developed a two-stage strategy of in vitro selection followed by a genetic screen and identified several artificial small molecule-binding riboswitches that respond to the aminoglycoside neomycin. Structure-function relationships and structural probing revealed that they adopt the general neomycin-binding motif. They display no sequence similarities to in vitro selected neomycin aptamers but contain parts of the decoding site that is the binding site for neomycin on the ribosomal RNA. We propose a model of a composed binding pocket of an internal loop as primary docking site and a terminal flaplike loop structure fixing neomycin in a sandwich-like manner. Such binding pockets characterized by multiple contacts between ligand and RNA are described for both natural and engineered riboswitches. We anticipate that combination of in vitro selection and in vivo screening is a useful strategy to identify RNA molecules with a desired functionality.     

Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

Colorectal cancer (CRC) is the most common digestive cancer in the Western world. Despite effective therapies, resistance and/or recurrence frequently occur. The present study investigated the impact of two survival pathways—neurotrophic factors (TrkB/BDNF) and autophagy—on cell fate and tumour evolution. In vitro studies were performed on two CRC cell lines, SW480 (primary tumour) and SW620 (lymph node invasion), which were also used for subcutaneous xenografts on a nude mouse model. In addition, the presence of neurotrophic factors (NTs) and autophagy markers were assessed in tissue samples representative of different stages. On the basis of our previous study (which demonstrated that TrkB overexpression is associated with prosurvival signaling in CRC cells), we pharmacologically inhibited NTs pathways with K252a. As expected, an inactivation of the PI3K/AKT pathway was observed and CRC cells initiated autophagy. Conversely, blocking the autophagic flux with chloroquine or with ATG5‐siRNA overactivated TrkB/BDNF signaling. In vitro, dual inhibition improved the effectiveness of single treatment by significantly reducing metabolic activity and enhancing apoptotic cell death. These findings were accentuated in vivo, in which dual inhibition induced a spectacular reduction in tumour volume following long‐term treatment (21 days for K252a and 12 days for CQ). Finally, significant amounts of phospho‐TrkB and LC3II were found in the patients’ tissues, highlighting their relevance in CRC tumour biology. Taken together, our results show that targeting NTs and autophagy pathways potentially constitutes a new therapeutic approach for CRC.     

Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis

The aim of this study was to analyze the type of immune response (Th1, Th2) and protein composition of bronchoalveolar lavage (BAL) of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Flow cytometry analysis of intracellular cytokines revealed different patterns: in IPF and SSc Th2 profiles were predominant, whereas in sarcoidosis Th1 prevailed. The proteomic analysis of BAL fluid (BALF) showed that there were quantitative differences between the three diseases. These were more evident between sarcoidosis and IPF, confirming our previous observations, whereas SSc had an intermediate profile between the two, however with some peculiarities. Comparison of BALF protein maps, constructed with the same quantity of total proteins, enabled us to identify the main profiles of the three diseases: an increase in plasma protein prevalent in sarcoidosis and also present in SSc, though for fewer proteins with respect to IPF and a greater abundance of low molecular weight proteins, mainly locally produced, in IPF. These findings are in line with the different pathogenesis of these diseases: IPF is considered a prevalently fibrotic disorder limited to the lung, with intense local production of functionally different proteins, whereas sarcoidosis and SSc are systemic immunoinflammatory diseases.