The res (restored cell structure by salinity) tomato mutant reveals the role of the DEAD-box RNA helicase SlDEAD39 in plant development and salt response

Increasing evidences highlight the importance of DEAD-box RNA helicases in plant development and stress responses. In a previous study, we characterized the tomato res mutant (restored cell structure by salinity), showing chlorosis and development alterations that reverted under salt-stress conditions. Map-based cloning demonstrates that RES gene encodes SlDEAD39, a chloroplast-targeted DEAD-box RNA helicase. Constitutive expression of SlDEAD39 complements the res mutation, while the silencing lines had a similar phenotype than res mutant, which is also reverted under salinity. Functional analysis of res mutant proved SlDEAD39 is involved in the in vivo processing of the chloroplast, 23S rRNA, at the hidden break-B site, a feature also supported by in vitro binding experiments of the protein. In addition, our results show that other genes coding for chloroplast-targeted DEAD-box proteins are induced by salt-stress, which might explain the rescue of the res mutant phenotype. Interestingly, salinity restored the phenotype of res adult plants by increasing their sugar content and fruit yield. Together, these results propose an unprecedented role of a DEAD-box RNA helicase in regulating plant development and stress response through the proper ribosome and chloroplast functioning, which, in turn, represents a potential target to improve salt tolerance in tomato crops.     

SIMEX for correction of dietary exposure effects with Box-Cox transformed data

Modelling dietary data, and especially 24-hr dietary recall (24HDR) data, is a challenge. Ignoring the inherent measurement error (ME) leads to biased effect estimates when the association between an exposure and an outcome is investigated. We propose an adapted simulation extrapolation (SIMEX) algorithm for modelling dietary exposures. For this purpose, we exploit the ME model of the NCI method where we assume the assumption of normally distributed errors of the reported intake on the Box-Cox transformed scale and of unbiased recalls on the original scale. According to the SIMEX algorithm, remeasurements of the observed data with additional ME are generated in order to estimate the association between the level of ME and the resulting effect estimate. Subsequently, this association is extrapolated to the case of zero ME to obtain the corrected estimate. We show that the proposed method fulfils the key property of the SIMEX approach, that is, that the MSE of the generated data will converge to zero if the ME variance converges to zero. Furthermore, the method is applied to real 24HDR data of the I.Family study to correct the effects of salt and alcohol intake on blood pressure. In a simulation study, the method is compared with the NCI method resulting in effect estimates with either smaller MSE or smaller bias in certain situations. In addition, we found our method to be more informative and easier to implement. Therefore, we conclude that the proposed method is useful to promote the dissemination of ME correction methods in nutritional epidemiology.     

Functional Integration and Individuality in Prokaryotic Collective Organisations

Acta Biotheoretica - Both physiological and evolutionary criteria of biological individuality are underpinned by the idea that an individual is a functionally integrated whole. However, a precise...     

Protozoa and human macrophages infection by Legionella pneumophila environmental strains belonging to different serogroups

Three Legionella pneumophila strains isolated from municipal hot tap water during a multicentric Italian survey and belonging to serogroups 1, 6, 9 and the reference strain Philadelphia-1 were studied to determine the intracellular replication capability and the cytopathogenicity in human monocyte cell line U937 and in an Acanthamoeba polyphaga strain. Our results show that both serogroups 1 and Philadelphia-1 were able to multiply into macrophages inducing cytopathogenicity, while serogroup 6 and ever more serogroup 9 were less efficient in leading to death of the infected macrophages. Both serogroups 1 and 6 displayed a quite good capability of intracellular replication in A. polyphaga, although serogroup 1 was less cytopathogenic than serogroup 6. Serogroup 9, like Philadelphia-1 strain, showed a reduced efficiency of infection and replication and a low cytopathogenicity towards the protozoan. Our study suggests that bacterial pathogenesis is linked to the difference in the virulence expression of L. pneumophila serogroups in both hosts, as demonstrated by the fact that only L. pneumophila serogroup 1 shows the contextual expression of the two virulence traits. Serogroup 6 proves to be a good candidate as pathogen since it shows a good capacity for intracellular replication in protozoan.     

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.     

ADAM9 silencing inhibits breast tumor cell invasion in vitro

ADAM9 (A Disintegrin And Metalloproteinase 9) is a member of the ADAM protein family which contains a disintegrin domain. This protein family plays key roles in many physiological processes, including fertilization, migration, and cell survival. The ADAM proteins have also been implicated in various diseases, including cancer. Specifically, ADAM9 has been suggested to be involved in metastasis. To address this question, we generated ADAM9 knockdown clones of MDA-MB-231 breast tumor cells using silencing RNAs that were tested for cell adhesion, proliferation, migration and invasion assays. In RNAi-mediated ADAM9 silenced MDA-MB-231 cells, the expression of ADAM9 was lower from the third to the sixth day after silencing and inhibited tumor cell invasion in matrigel by approximately 72% when compared to control cells, without affecting cell adhesion, proliferation or migration. In conclusion, the generation of MDA-MB-231 knockdown clones lacking ADAM9 expression inhibited tumor cell invasion in vitro, suggesting that ADAM9 is an important molecule in the processes of invasion and metastasis.     

Oxidative damage in relation to a female plumage badge: evidence for signalling costs

Badges of status may be controlled by costs derived from increased aggression from dominant individuals. This cost could be translated into elevated metabolic levels and a concomitant disruption of oxidative balance. Some females in Iberian pied flycatcher Ficedula hypoleuca populations exhibit a white forehead patch similar to that exhibited by all males in this species, functioning in aggressive interactions between females when competing for breeding sites. To test if social stress imposes costs on signalling, we painted white patches on females without natural patches (NP) and compared them with females with natural control (NU). We also over-painted the natural patch in other females (FP) and compared to females with control natural patches (FU). We obtained for the whole sample of females data on reproductive investment, morphology and oxidative damage measured by blood malondialdehydes (MDA), and in a subsample of females variables related to parental care during incubation and the early nestling stage. FP and FU did not differ significantly in any variable which negates an effect of paint itself. However, NP females showed significant higher levels of MDA than NU females when controlling for breeding success for the whole sample, and for female incubation attendance for the parental care subsample. When including the four treatments, there was a significant interaction between the paint treatment and the presence/absence of badges before the experiment when controlling for the significant negative effect of incubation attendance on MDA. Addition of a badge to females without one leads to increased oxidative damage possibly mediated by social control. Badges of status in female pied flycatchers may operate as badges of oxidative status.     

Structural,energetic, and dynamic responses of the native state ensemble of staphylococcal nuclease to cavity‐creating mutations

The effects of cavity‐creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C‐terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the β‐barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions. © 2012 Wiley Periodicals, Inc.     

In vitro evaluation of archaeosome vehicles for transdermal vaccine delivery

Abstract

Archaeosomes composed of archaeal total polar lipids (TPL) or semi-synthetic analog vesicles have been used as vaccine adjuvants and delivery systems in animal models for many years. Typically administered by intramuscular or subcutaneous injections, archaeosomes can induce robust, long-lasting humoral and cell-mediated immune responses against entrapped antigens and provide protection in murine models of infectious disease and cancer. Herein, we evaluated various archaeosomes for transdermal delivery, since this route may help eliminate needle-stick injuries and needle re-use, and therefore increase patient compliance. Archaeosomes composed of TPL from different archaea (Halobacterium salinarum, Methanobrevibacter smithii, Haloferax volcanii) and various semi-synthetic glycolipid combinations were evaluated for their ability to diffuse across the skin barrier using an ex vivo pig skin model and the results were compared to conventional synthetic ester liposomes. Physicochemical characteristics were determined for selected formulations including vesicle size, size distribution, zeta potential, fluidity, antigen (ovalbumin) incorporation efficiency and release. Archaeosomes, in particular those composed of M. smithii TPL or the synthetic glycolipid sulfated S-lactosylarchaeol (SLA) mixed with uncharged glycolipid lactosyl archaeol (LA), appeared to be effective carriers for ovalbumin, achieving much better antigen distribution and vesicle accumulation in the skin epidermis than conventional liposomes. The enhanced skin permeation of archaeosomes may be attributed to their chemical structure and physicochemical properties such as particle size, surface charge, stability, and fluidity of their lipid bilayer.     

Short Sleep Is Associated with Obesity among Truck Drivers

Recent studies suggest that short‐sleep duration is independently associated with obesity in the general population. The population of truck drivers is of particular interest, because they frequently work irregular shifts that in turn are associated with short‐sleep duration. In addition, truck drivers have a high prevalence of sedentary habits, poor diet, and obesity. The present study aimed at verifying the association between sleep patterns and factors associated with obesity in this population. The study sample consisted in 4,878 truck drivers who participated in a campaign promoted by a highway company in the State of São Paulo, Brazil. This campaign offered highway truck drivers a medical and laboratorial evaluation. The truck drivers completed a questionnaire concerning demographic data, sleep duration, consumption of medications, and medical problems, such as diabetes, cardiopathy, and hypertension; as well as the Berlin questionnaire, which is able to discriminate low and high risk for obstructive sleep apnea. Blood samples were collected to measure glucose and cholesterol levels. Also, body weight and height were registered to calculate the body mass index (BMI). The mean age (±SD) of the truck drivers studied was 40±10 years. Out of the truck drivers analyzed, 28.3% (n=1,379) had a BMI ≥30.0 Kg/m² (obesity). Among the 4,878 drivers included in the study, 1,199 (24.6%) were on medications and 334 (6.8%) were diabetic. Drivers (26.9%) with the greater BMI had a short sleep length. The independent factors associated with obesity were sleep duration <8 h/day (OR=1.24), age >40 years (OR=1.20), glucose levels >200 (OR=2.02), cholesterol levels >240 (OR=1.57), snoring (OR=1.74), and hypertension (OR=2.14). Smoking was not associated with obesity (OR=0.69), and diabetes was considered a control variable. In conclusion, this study supports the hypothesis that short sleep duration as well as age >40 years are independently associated with obesity. This particular combination (short‐sleep duration and obesity) is independently associated with several healthcare problems, including high levels of cholesterol, glucose, snoring, and hypertension. However, due to the cross‐sectional nature of this study, no cause–effect relationship can be drawn from these results.