Small‐angle scattering for structural biology—Expanding the frontier while avoiding the pitfalls

The last decade has seen a dramatic increase in the use of small‐angle scattering for the study of biological macromolecules in solution. The drive for more complete structural characterization of proteins and their interactions, coupled with the increasing availability of instrumentation and easy‐to‐use software for data analysis and interpretation, is expanding the utility of the technique beyond the domain of the biophysicist and into the realm of the protein scientist. However, the absence of publication standards and the ease with which 3D models can be calculated against the inherently 1D scattering data means that an understanding of sample quality, data quality, and modeling assumptions is essential to have confidence in the results. This review is intended to provide a road map through the small‐angle scattering experiment, while also providing a set of guidelines for the critical evaluation of scattering data. Examples of current best practice are given that also demonstrate the power of the technique to advance our understanding of protein structure and function.     

Glutathione and transpiration as key factors conditioning oxidative stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to uranium

Although oxidative stress has been previously described in plants exposed to uranium (U), some uncertainty remains about the role of glutathione and tocopherol availability in the different responsiveness of plants to photo-oxidative damage. Moreover, in most cases, little consideration is given to the role of water transport in shoot heavy metal accumulation. Here, we investigated the effect of uranyl nitrate exposure (50 μM) on PSII and parameters involved in water transport (leaf transpiration and aquaporin gene expression) of Arabidopsis wild type (WT) and mutant plants that are deficient in tocopherol (vte1: null α/γ-tocopherol and vte4: null α-tocopherol) and glutathione biosynthesis (high content: cad1.3 and low content: cad2.1). We show how U exposure induced photosynthetic inhibition that entailed an electron sink/source imbalance that caused PSII photoinhibition in the mutants. The WT was the only line where U did not damage PSII. The increase in energy thermal dissipation observed in all the plants exposed to U did not avoid photo-oxidative damage of mutants. The maintenance of control of glutathione and malondialdehyde contents probed to be target points for the overcoming of photoinhibition in the WT. The relationship between leaf U content and leaf transpiration confirmed the relevance of water transport in heavy metals partitioning and accumulation in leaves, with the consequent implication of susceptibility to oxidative stress.     

Minor salivary gland mesenchymal stromal cells derived from patients with Sjӧgren's syndrome deploy intact immune plasticity

Background aimsMesenchymal stromal cells (MSCs) provide minor salivary glands (MSGs) with support and niche cells for epithelial glandular tissue. Little is known about resident MSG-derived MSCs (MSG-MSCs) in primary Sj?gren's syndrome (PSS). The authors’ objective is to define the immunobiology of endogenous PSS MSG-MSCs.MethodsUsing culture-adapted MSG-MSCs isolated from consenting PSS subjects (n = 13), the authors performed in vitro interrogation of PSS MSG-MSC immunobiology and global gene expression compared with controls. To this end, the authors performed phenotypic and immune functional analysis of indoleamine 2,3-dioxygenase (IDO), programmed death ligand 1 (PD-L1) and intercellular adhesion marker 1 (ICAM-1) before and after interferon γ (IFNγ) licensing as well as the effect of MSG-MSCs on T-cell proliferation. Considering the female predominance of PSS, the authors also addressed the influence of 17-β-estradiol on estrogen receptor α-positive-related MSC function.ResultsThe authors found that MSG-MSCs deployed normal immune regulatory functionality after IFNγ stimulation, as demonstrated by increased protein-level expression of IDO, PD-L1 and ICAM-1. The authors also found that MSG-MSCs suppressed T-cell proliferation in a dose-dependent manner independent of 17-β-estradiol exposure. Gene ontology and pathway analysis highlighted extracellular matrix deposition as a possible difference between PSS and control MSG-MSCs. MSG-MSCs demonstrated increased α-smooth muscle actin expression in PSS, indicating a partial myofibroblast-like adaptation.ConclusionsThese findings establish similar immune regulatory function of MSG-MSCs in both PSS and control patients, precluding intrinsic MSC immune regulatory defects in PSS. PSS MSG-MSCs show a partial imprinted myofibroblast-like phenotype that may arise in the setting of chronic inflammation, providing a plausible etiology for PSS-related glandular fibrosis.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Variation in phenotypic plasticity and selection patterns in blue tit breeding time: between- and within-population comparisons

1.?Phenotypic plasticity, the response of individual phenotypes to their environment, can allow organisms to cope with spatio-temporal variation in environmental conditions. Recent studies have shown that variation exists among individuals in their capacity to adjust their traits to environmental changes and that this individual plasticity can be under strong selection. Yet, little is known on the extent and ultimate causes of variation between populations and individuals in plasticity patterns. 2.?In passerines, timing of breeding is a key life-history trait strongly related to fitness and is known to vary with the environment, but few studies have investigated the within-species variation in individual plasticity. 3.?Here, we studied between- and within-population variation in breeding time, phenotypic plasticity and selection patterns for this trait in four Mediterranean populations of blue tits (Cyanistes caeruleus) breeding in habitats varying in structure and quality. 4.?Although there was no significant warming over the course of the study, we found evidence for earlier onset of breeding in warmer years in all populations, with reduced plasticity in the less predictable environment. In two of four populations, there was significant inter-individual variation in plasticity for laying date. Interestingly, selection for earlier laying date was significant only in populations where there was no inter-individual differences in plasticity. 5.?Our results show that generalization of plasticity patterns among populations of the same species might be challenging even at a small spatial scale and that the amount of within-individual variation in phenotypic plasticity may be linked to selective pressures acting on these phenotypic traits.     

Antibacterial activity against β- lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: fractional Inhibitory Concentration Index (FICI) determination

Background

The present study reports the antibacterial capacity of alkaloid compounds in combination with Methicillin and Ampicillin-resistants bacteria isolated from clinical samples. The resistance of different bacteria strains to the current antibacterial agents, their toxicity and the cost of the treatment have led to the development of natural products against the bacteria resistant infections when applied in combination with conventional antimicrobial drugs.

Method

The antibacterial assays in this study were performed by using inhibition zone diameters, MIC, MBC methods, the time-kill assay and the Fractional Inhibitory Concentration Index (FICI) determination. On the whole, fifteen Gram-positive bacterial strains (MRSA/ARSA) were used. Negative control was prepared using discs impregnated with 10 % DMSO in water and commercially available Methicillin and Ampicillin from Alkom Laboratories LTD were used as positive reference standards for all bacterial strains.

Results

We noticed that the highest activities were founded with the combination of alkaloid compounds and conventional antibiotics against all bacteria strains. Then, results showed that after 7 h exposition there was no viable microorganism in the initial inoculums.

Conclusion

The results of this study showed that alkaloid compounds in combination with conventional antibiotics (Methicillin, Ampicillin) exhibited antimicrobial effects against microorganisms tested. These results validate the ethno-botanical use of Cienfuegosia digitata Cav. (Malvaceae) in Burkina Faso. Moreover, this study demonstrates the potential of this herbaceous as a source of antibacterial agent that could be effectively used for future health care purposes.     

Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-22 from Lycopersicon esculentum

In tomato, infections by tomato mosaic virus are controlled by durable Tm-2² resistance. In order to gain insight into the processes underlying disease resistance and its durability, we cloned and analysed the Tm-2² resistance gene and the susceptible allele, tm-2. The Tm-2² gene was isolated by transposon tagging using a screen in which plants with a destroyed Tm-2² gene survive. The Tm-2² locus consists of a single gene that encodes an 861 amino acid polypeptide, which belongs to the CC-NBS-LRR class of resistance proteins. The putative tm-2 allele was cloned from susceptible tomato lines via PCR with primers based on the Tm-2² sequence. Interestingly, the tm-2 gene has an open reading frame that is comparable to the Tm-2² allele. Between the tm-2 and the Tm-2² polypeptide 38 amino acid differences are present of which 26 are located in the second half of the LRR-domain. Susceptible tomato plants, which were transformed with the Tm-2² gene, displayed resistance against ToMV infection. In addition, virus specificity, displayed by the Tm-2² resistance was conserved in these transgenic lines. To explain the durability of this resistance, it is proposed that the Tm-2²-encoded resistance is aimed at the Achilles' heel of the virus.