Expression Profiling of Rectal Tumors Defines Response to Neoadjuvant Treatment Related Genes

To date, no effective method exists that predicts the response to preoperative chemoradiation (CRT) in locally advanced rectal cancer (LARC). Nevertheless, identification of patients who have a higher likelihood of responding to preoperative CRT could be crucial in decreasing treatment morbidity and avoiding expensive and time-consuming treatments. The aim of this study was to identify signatures or molecular markers related to response to pre-operative CRT in LARC. We analyzed the gene expression profiles of 26 pre-treatment biopsies of LARC (10 responders and 16 non-responders) without metastasis using Human WG CodeLink microarray platform. Two hundred and fifty seven genes were differentially over-expressed in the responder patient subgroup. Ingenuity Pathway Analysis revealed a significant ratio of differentially expressed genes related to cancer, cellular growth and proliferation pathways, and c-Myc network. We demonstrated that high Gng4, c-Myc, Pola1, and Rrm1 mRNA expression levels was a significant prognostic factor for response to treatment in LARC patients (p<0.05). Using this gene set, we were able to establish a new model for predicting the response to CRT in rectal cancer with a sensitivity of 60% and 100% specificity. Our results reflect the value of gene expression profiling to gain insight about the molecular pathways involved in the response to treatment of LARC patients. These findings could be clinically relevant and support the use of mRNA levels when aiming to identify patients who respond to CRT therapy.     

Linking Cascading Effects of Fish Predation and Zooplankton Grazing to Reduced Cyanobacterial Biomass and Toxin Levels Following Biomanipulation

Eutrophication has been one of the largest environmental problems in aquatic ecosystems during the past decades, leading to dense, and often toxic, cyanobacterial blooms. In a way to counteract these problems many lakes have been subject to restoration through biomanipulation. Here we combine 13 years of monitoring data with experimental assessment of grazing efficiency of a naturally occurring zooplankton community and a, from a human perspective, desired community of large Daphnia to assess the effects of an altered trophic cascade associated with biomanipulation. Lake monitoring data show that the relative proportion of Daphnia spp. grazers in June has increased following years of biomanipulation and that this increase coincides with a drop in cyanobacterial biomass and lowered microcystin concentrations compared to before the biomanipulation. In June, the proportion of Daphnia spp. (on a biomass basis) went from around 3% in 2005 (the first year of biomanipulation) up to around 58% in 2012. During months when the proportion of Daphnia spp. remained unchanged (July and August) no effect on lower trophic levels was observed. Our field grazing experiment revealed that Daphnia were more efficient in controlling the standing biomass of cyanobacteria, as grazing by the natural zooplankton community never even compensated for the algal growth during the experiment and sometimes even promoted cyanobacterial growth. Furthermore, although the total cyanobacterial toxin levels remained unaffected by both grazer communities in the experimental study, the Daphnia dominated community promoted the transfer of toxins to the extracellular, dissolved phase, likely through feeding on cyanobacteria. Our results show that biomanipulation by fish removal is a useful tool for lake management, leading to a top-down mediated trophic cascade, through alterations in the grazer community, to reduced cyanobacterial biomass and lowered cyanobacterial toxin levels. This improved water quality enhances both the ecological and societal value of lakes as units for ecosystem services.     

Development of a modified transformation platform for apomixis candidate genes research in Paspalum notatum (bahiagrass)

The aim of this work was to improve existing transformation protocols and to transform specific genotypes of Paspalum notatum (bahiagrass) for functional analyses of candidate genes involved in reproduction. Three different explants were assayed for in vitro plant regeneration: mature seeds, mature embryos, and shoot meristems. Plant regeneration was achieved with all explant types, but mature seeds produced the optimal rate (78.0%) and were easiest to manipulate. A method based on serial re-induction of calli from meristems of the regenerated lines was also developed, which could be useful in plant breeding strategies pursuing somaclonal variation. Transient transformation experiments were performed on calli obtained from mature seeds using a compressed helium gene gun. Transient transformation constructs included anthocyanin-synthesis genes cloned under the CAMV 35S promoter and an enhanced green fluorescent protein gene (egfp) driven by the rice actin1 (act1) promoter. Selection curves for ammonium glufosinate were developed in order to determine the optimal selective pressure for stable transformation (1.0 mg/L). Stable co-transformation experiments were carried out with two different constructs containing: (1) the reporter egfp gene cloned under the rice act1 promoter and (2) the selector bar gene driven by the ubiquitin promoter. A total of 27 (64.2%) transgenic plants out of 42 resistant plants analyzed were obtained. The presence of the transgenes in regenerated plants was confirmed by polymerase chain reaction and DNA gel blot analysis. Gene expression was demonstrated by eGFP fluorescence detection and in vivo assays for ammonium glufosinate tolerance. This platform is being used to generate transgenic plants of P. notatum to analyze the function of apomixis-associated candidate genes.     

Use of Synthetic Genes for Cloning,Production and Functional Expression of the Bacteriocins Enterocin A and Bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.     

Bile acid supplementation improves established liver steatosis in obese mice independently of glucagon-like peptide-1 secretion

Bile acids or its derivatives may influence non-alcoholic fatty liver disease development through multiple mechanisms. Intestinal L-cells secrete glucagon-like peptide-1 (GLP-1) and can be activated by bile acids (BA) influencing insulin resistance and hepatic steatosis development and progression. The aim of the present study was to assess the effects of cholic acid (CA) or ursodeoxycholic acid (UDCA) administration on portal and systemic levels of GLP-1 in genetically obese mice with established hepatic steatosis. Eight-week-old ob/ob mice were fed CA or UDCA during 4 weeks. Systemic and portal GLP-1 levels were measured as well as glucose tolerance test, serum and biliary parameters, hepatic triglyceride content, liver histology, and hepatic gene expression of relevant genes related to bile secretion. Eight-week-old ob/ob mice exhibited marked obesity, hyperinsulinemia, and fasting hyperglycemia. Administration of both CA and UDCA was associated to decreased hepatic triglyceride content and complete reversion of histological steatosis. BA-fed animals did not exhibit significant differences in glucose tolerance. In addition, neither CA nor UDCA administration significantly influenced portal or systemic GLP-1 levels. CA and UDCA strongly ameliorated established fatty liver in ob/ob mice independently of the GLP-1 incretin pathway. Thus, the anti-steatotic action of these bile acids is likely related to direct hepatic effects.     

The validity of ecogeographical rules is context‐dependent: testing for Bergmann's and Allen's rules by latitude and elevation in a widespread Andean duck

Consistent responses by various organisms to common environmental pressures represent strong evidence of natural selection driving geographical variation. According to Bergmann's and Allen's rules, animals from colder habitats are larger and have smaller limbs than those from warmer habitats to minimize heat loss. Although evidence supporting both rules in different organisms exists, most studies have considered only elevational or latitudinal temperature gradients. We tested for the effects of temperature associated with both elevation and latitude on body and appendage size of torrent ducks (Merganetta armata), a widespread species in Andean rivers. We found a negative relationship between body size and temperature across latitude consistent with Bergmann's rule, whereas there was a positive relationship between these variables along replicate elevational gradients at different latitudes. Limb‐size variation did not support Allen's rule along latitude, nor along elevation. High‐elevation ducks were smaller and had longer wings than those inhabiting lower elevations within a river. We hypothesize that temperature is likely a major selective pressure acting on morphology across latitudes, although hypoxia or air density may be more important along elevational gradients. We conclude that the effect of temperature on morphology, and hence the likelihood of documenting ecogeographical ‘rules’, depends on the environmental context in which temperature variation is examined. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 850–862.     

Pannexin1 channels act downstream of P2X7 receptors in ATP-induced murine T-cell death

Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X₇ receptors (P2X₇Rs). However, a link between P2X₇Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4⁺) and cytotoxic (CD8⁺) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (¹⁰Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1^−/− mice. Moreover, electrophysiological measurements in wild-type CD4⁺ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1^−/− mice, in which levels of P2X₇Rs and ATP-induced intracellular free Ca²⁺ responses were enhanced suggesting that P2X₇Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP.     

Biological Control of Anastrepha spp. (Diptera: Tephritidae) in Mango Orchards through Augmentative Releases of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae)

Diachasmimorpha longicaudata (Ashmead) parasitoids were released by air on a weekly basis over 1600 ha of commercial mango orchards, backyard orchards, and patches of native vegetation, at a density of ca. 940 parasitoids/ha. Releases were made during 2 consecutive years, beginning at flower onset and lasting until the end of the production cycle. Two areas, 7 km apart, were compared. In one area parasitoids were released, whereas the other area was used as a control. During the 2nd year treatments were reversed. Fruit was sampled in commercial mango orchards and in backyard orchards to assess levels of parasitism in fruit fly larvae. Highly significant differences in percentage parasitism were found in release and control zones in backyard orchards. Furthermore, trapping results indicated that D. longicaudata releases were associated with ca. 2.7-fold suppression of Anastrepha spp. populations in backyard orchards. Results suggest that suppression might be affected by environmental conditions and by the parasitoid:fly ratio achieved. Anastrepha obliqua McQuart populations were suppressed more effectively by use of parasitoids than those of Anastrepha ludens Loew, perhaps due to the type of host fruits used by each species. Augmentative parasitoid releases in marginal areas surrounding commercial orchards (backyard orchards, wild vegetation) can substantially suppress fly populations. Through this approach, the number of flies that later move into commercial orchards can be significantly reduced. Such a strategy, when combined with sound orchard management schemes, can allow growers to produce clean fruit without the need to resort to the widespread use of insecticides.     

Spatial dynamics of Chinese Muntjac related to past and future climate fluctuations

Climate fluctuations in the past and in the future are likely to result in population expansions, shifts, or the contraction of the ecological niche of many species, and potentially leading to the changes in their geographical distributions. Prediction of suitable habitats has been developed as a useful tool for the assessment of habitat suitability and resource conservation to protect wildlife. Here, we model the ancestral demographic history of the extant modern Chinese Muntjac Muntiacus reevesi populations using approximate Bayesian computation (ABC) and used the maximum entropy model to simulate the past and predict the future spatial dynamics of the species under climate oscillations. Our results indicated that the suitable habitats for the M. reevesi shifted to the Southeast and contracted during the Last Glacial Maximum, whereas they covered a broader and more northern position in the Middle Holocene. The ABC analyses revealed that the modern M. reevesi populations diverged in the Middle Holocene coinciding with the significant contraction of the highly suitable habitat areas. Furthermore, our predictions suggest that the potentially suitable environment distribution for the species will expand under all future climate scenarios. These results indicated that the M. reevesi diverged in the recent time after the glacial period and simultaneously as its habitat’s expanded in the Middle Holocene. Furthermore, the past and future climate fluctuation triggered the change of Chinese muntjac spatial distribution, which has great influence on the Chinese muntjac’s population demographic history.