The correlation between architecture and mRNA abundance in the genetic regulatory network of Escherichia coli

Background  

Two aspects of genetic regulatory networks are the static architecture that describes the overall connectivity between the genes and the dynamics that describes the sequence of genes active at any one time as deduced from mRNA abundances. The nature of the relationship between these two aspects of these networks is a fundamental question. To address it, we have used the static architecture of the connectivity of the regulatory proteins of Escherichia coli to analyse their relationship to the abundance of the mRNAs encoding these proteins. In this we build on previous work which uses Boolean network models, but impose biological constraints that cannot be deduced from the mRNA abundances alone.     

Male and female effects on sperm precedence in the giant sperm species <Emphasis Type="Italic">Drosophila bifurca</Emphasis>

Sperm competition is expected to be a driving force in sexual selection. In internally fertilized organisms, it occurs when ejaculates from more than one male are present simultaneously within the female’s reproductive tract. It has been suggested that greater sperm size may improve the competitive ability of sperm, but studies provide contradictory results depending on the species. More recently, the role of females in the evolution of sperm morphology has been pointed out. We investigate here the male and female effects that influence sperm precedence in the giant sperm species, Drosophila bifurca Patterson & Wheeler. Females were mated with two successive males, and the paternity outcomes for both males were analyzed after determining sperm transfer and storage. We found very high values of last male sperm precedence, suggesting a strong interaction between rival sperm. However, the data also indicate high frequencies of removal of the sperm of the first male from the female reproductive tract prior to any interaction with the second male. This implies that successful paternity depends mainly on successful sperm storage. Knowing what happens to the sperm within females appears to be a prerequisite for disentangling post-copulatory sexual interactions between males and females.     

Labeling of Bifidobacterium longum Cells with 13C-Substituted Leucine for Quantitative Proteomic Analyses

Stable isotope labeling of amino acids in cell culture was used for Bifidobacterium longum. A comprehensive proteomic strategy was developed and validated by designing an appropriate semidefined medium that allows stable replacement of natural leucine by [¹³C₆]leucine. Using this strategy, proteins having variations of at least 50% in their expression rates can be quantified with great confidence.     

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.     

Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells

Background

Preclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC.

Methods

Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models.

Results

Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours.

Conclusions

Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.     

High lipid order of Arabidopsis cell‐plate membranes mediated by sterol and DYNAMIN‐RELATED PROTEIN1A function

Membranes of eukaryotic cells contain high lipid‐order sterol‐rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher‐plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell‐plate formation in Arabidopsis relies on sterol‐ and DYNAMIN‐RELATED PROTEIN1A (DRP1A)‐dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order‐sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid‐order membrane domain. The cell‐plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co‐localize with DRP1A at the cell plate, and DRP1A accumulates in detergent‐resistant membrane fractions. Modifications of sterol concentration or composition reduce cell‐plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid‐order domain, and pave the way to explore potential feedback between lipid order and function of dynamin‐related proteins during cytokinesis.     

Assignment of PolyProline II conformation and analysis of sequence--structure relationship

Background

Secondary structures are elements of great importance in structural biology, biochemistry and bioinformatics. They are broadly composed of two repetitive structures namely α-helices and β-sheets, apart from turns, and the rest is associated to coil. These repetitive secondary structures have specific and conserved biophysical and geometric properties. PolyProline II (PPII) helix is yet another interesting repetitive structure which is less frequent and not usually associated with stabilizing interactions. Recent studies have shown that PPII frequency is higher than expected, and they could have an important role in protein – protein interactions.

Methodology/Principal Findings

A major factor that limits the study of PPII is that its assignment cannot be carried out with the most commonly used secondary structure assignment methods (SSAMs). The purpose of this work is to propose a PPII assignment methodology that can be defined in the frame of DSSP secondary structure assignment. Considering the ambiguity in PPII assignments by different methods, a consensus assignment strategy was utilized. To define the most consensual rule of PPII assignment, three SSAMs that can assign PPII, were compared and analyzed. The assignment rule was defined to have a maximum coverage of all assignments made by these SSAMs. Not many constraints were added to the assignment and only PPII helices of at least 2 residues length are defined.

Conclusions/Significance

The simple rules designed in this study for characterizing PPII conformation, lead to the assignment of 5% of all amino as PPII. Sequence – structure relationships associated with PPII, defined by the different SSAMs, underline few striking differences. A specific study of amino acid preferences in their N and C-cap regions was carried out as their solvent accessibility and contact patterns. Thus the assignment of PPII can be coupled with DSSP and thus opens a simple way for further analysis in this field.