Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues

To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations.     

Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.     

Slightly modifying pseudoproline dipeptides incorporation strategy enables solid phase synthesis of a 54 AA fragment of caveolin‐1 encompassing the intramembrane domain

This work contributes to highlight the benefits of pseudoproline dipeptides introduction in difficult SPPS. We show how a slight modification in the positioning choice conditioned the synthesis achievement of a 54 amino acid long caveolin‐1 peptide encompassing the intramembrane domain. Furthermore, we report a side reaction correlated with the coupling steps and generating truncated fragments with a mass deviation of + 42 Da. Considering the need of structural data for membrane proteins, most of which are considered as prevalent therapeutic targets, chemical synthesis provides an interesting alternative pathway to obtain hydrophobic domains by pushing back the frontiers of conventional RP methods of purification. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Absence of male reproductive skew,along with high frequency of polyandry and conspecific brood parasitism in the lekking Houbara bustard Chlamydotis undulata undulata

Behavioural studies have led to the perception that lekking species experience a high male reproductive skew as a consequence of females’ selective mate choice. In addition, observations suggest that females copulate only once and therefore polyandry seems unlikely as females are supposed to choose the best male available. In order to analyse the mating strategy of the Houbara bustard, an endangered lekking species under reinforcement in eastern Morocco, we used microsatellite data to perform paternity analyses. None of our observations followed common expectations under a lek mating system: we found no male reproductive skew suggesting no apparent selective female mate choice and no apparent male benefit from lekking. In contrast, a high level of polyandry (60 % of the nests) was recorded suggesting that sperm competition may operate. In addition, we present another case of conspecific brood parasitism in a lekking species and this was an unexpected alternative strategy for a species presenting high parental cost and low fecundity. The increasing number of studies contradicting common assumptions on lekking species suggests that alternative breeding strategies such as males pursuing an off‐lek mating strategy, female polyandry and even conspecific brood parasitism might be more widespread in lekking species than previously thought.     

Methods of NMR structure refinement: molecular dynamics simulations improve the agreement with measured NMR data of a C-terminal peptide of GCN4-p1

The C-terminal trigger sequence is essential in the coiled-coil formation of GCN4-p1; its conformational properties are thus of importance for understanding this process at the atomic level. A solution NMR model structure of a peptide, GCN4p16–31, encompassing the GCN4-p1 trigger sequence was proposed a few years ago. Derived using a standard single-structure refinement protocol based on 172 nuclear Overhauser effect (NOE) distance restraints, 14 hydrogen-bond and 11 ϕ torsional-angle restraints, the resulting set of 20 NMR model structures exhibits regular α-helical structure. However, the set slightly violates some measured NOE bounds and does not reproduce all 15 measured ³J(H_N-H_Cα)-coupling constants, indicating that different conformers of GCN4p16–31 might be present in solution. With the aim to resolve structures compatible with all NOE upper distance bounds and ³J-coupling constants, we executed several structure refinement protocols employing unrestrained and restrained molecular dynamics (MD) simulations with two force fields. We find that only configurational ensembles obtained by applying simultaneously time-averaged NOE distance and ³J-coupling constant restraining with either force field reproduce all the experimental data. Additionally, analyses of the simulated ensembles show that the conformational variability of GCN4p16–31 in solution admitted by the available set of 187 measured NMR data is larger than represented by the set of the NMR model structures. The conformations of GCN4p16–31 in solution differ in the orientation not only of the side-chains but also of the backbone. The inconsistencies between the NMR model structures and the measured NMR data are due to the neglect of averaging effects and the inclusion of hydrogen-bond and torsional-angle restraints that have little basis in the primary, i.e. measured NMR data.     

Causal relationships between frequency bands of extracellular signals in visual cortex revealed by an information theoretic analysis

Characterizing how different cortical rhythms interact and how their interaction changes with sensory stimulation is important to gather insights into how these rhythms are generated and what sensory function they may play. Concepts from information theory, such as Transfer Entropy (TE), offer principled ways to quantify the amount of causation between different frequency bands of the signal recorded from extracellular electrodes; yet these techniques are hard to apply to real data. To address the above issues, in this study we develop a method to compute fast and reliably the amount of TE from experimental time series of extracellular potentials. The method consisted in adapting efficiently the calculation of TE to analog signals and in providing appropriate sampling bias corrections. We then used this method to quantify the strength and significance of causal interaction between frequency bands of field potentials and spikes recorded from primary visual cortex of anaesthetized macaques, both during spontaneous activity and during binocular presentation of naturalistic color movies. Causal interactions between different frequency bands were prominent when considering the signals at a fine (ms) temporal resolution, and happened with a very short (ms-scale) delay. The interactions were much less prominent and significant at coarser temporal resolutions. At high temporal resolution, we found strong bidirectional causal interactions between gamma-band (40–100 Hz) and slower field potentials when considering signals recorded within a distance of 2 mm. The interactions involving gamma bands signals were stronger during movie presentation than in absence of stimuli, suggesting a strong role of the gamma cycle in processing naturalistic stimuli. Moreover, the phase of gamma oscillations was playing a stronger role than their amplitude in increasing causations with slower field potentials and spikes during stimulation. The dominant direction of causality was mainly found in the direction from MUA or gamma frequency band signals to lower frequency signals, suggesting that hierarchical correlations between lower and higher frequency cortical rhythms are originated by the faster rhythms.     

ILCD Handbook Public Consultation Workshop

Introduction

The European Commission is supporting the development of the International Reference Life Cycle Data System (ILCD). This consists primarily of the ILCD Handbook and the ILCD Data Network. This paper gives an insight into the scientific positions of business, governments, consultants, academics, and others that were expressed at this public consultation workshop.

Workshop focus

The workshop focused on four of the topics of the main guidance documents of the ILCD Handbook: (1) general guidance on life cycle assessment (LCA); (2) guidance for generic and average life cycle inventory (LCI) data sets; (3) requirements for environmental impact assessment methods, models and indicators for LCA; and (4) review schemes for LCA.

Workshop participation

This consultation workshop was attended by more than 120 participants during the 4 days of the workshop. Representatives came from 23 countries, from both within and outside the European Union.

Workshop structure

Approximately half of the participants were from business associations or individual companies. Another 20% were governmental representatives. Others came predominantly from consultancies and academia.

Results

This public consultation workshop provided valuable inputs into the overall ILCD Handbook developments as well as for further development. This paper focuses on some of the main scientific issues that were raised.