New insights on ChlD1 function in Photosystem II from site-directed mutants of D1/T179 in Thermosynechococcus elongatus

The monomeric chlorophyll, Chl_D1, which is located between the P_D1P_D2 chlorophyll pair and the pheophytin, Pheo_D1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg²⁺ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn₄CaO₅ cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *Chl_D1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of P_D1P_D2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the Chl_D1⁺Pheo_D1^? radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of ¹O₂ generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to Chl_D1 upon Q_A^? formation, indicates that in the T179H-PSII and in the WT*3-PSII, the Chl_D1 itself is the chlorophyll that is first damaged by ¹O₂, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, Chl_D1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of Chl_D1 very likely contribute to the well-known electrochromic shifts observed at ~430?nm during the S-state cycle.     

Life‐history innovation to climate change: can single‐brooded migrant birds become multiple breeders?

When climatic conditions change and become outside the range experienced in the past, species may show life‐history innovations allowing them to adapt in new ways. We report such an innovation for pied flycatchers Ficedula hypoleuca. Decades of breeding biological studies on pied flycatchers have rarely reported multiple breeding in this long‐distance migrant. In two populations, we found 12 recent incidents of females with second broods, all produced by extremely early laying females in warm springs. As such early first broods are a recent phenomenon, because laying dates have gradually advanced over time, this innovation now allows individual females to enhance their reproductive success considerably. If laying dates continue advancing, potentially more females may become multiple breeders and selection for early (and multiple) breeding phenotypes increases, which may accelerate adaptation to climatic change.     

Personalized pathology test for Cardio-vascular disease: Approximate Bayesian computation with discriminative summary statistics learning

Cardio/cerebrovascular diseases (CVD) have become one of the major health issue in our societies. But recent studies show that the present pathology tests to detect CVD are ineffectual as they do not consider different stages of platelet activation or the molecular dynamics involved in platelet interactions and are incapable to consider inter-individual variability. Here we propose a stochastic platelet deposition model and an inferential scheme to estimate the biologically meaningful model parameters using approximate Bayesian computation with a summary statistic that maximally discriminates between different types of patients. Inferred parameters from data collected on healthy volunteers and different patient types help us to identify specific biological parameters and hence biological reasoning behind the dysfunction for each type of patients. This work opens up an unprecedented opportunity of personalized pathology test for CVD detection and medical treatment.     

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.     

Trophallaxis Mediates Uniformity of Colony Odor in Cataglyphis iberica Ants (Hymenoptera, Formicidae)

We studied the effect of nestmate separation on trophallaxis in the polydomous ant Cataglyphis iberica. After dividing three colonies into two equivalent subgroups, one queenright and one queenless, we quantified the frequency of trophallaxis within each subgroup, between the workers from the two subgroups (mixed trophallaxis), and trophallaxis involving the queen. Observations of trophallaxis were conducted over four periods of time: for 2 weeks before the separation of the two subgroups, 8 weeks during separation, immediately after reunification, and 3 weeks following reunification. Subgroups were identically fed on the eve of each day of observation. Group separation induced an increase in mixed frequencies of trophallaxis just after reunification, after which trophallaxis returned to the initial level observed before separation. Previous results showed that group separation in C. iberica induces hydrocarbon profile divergence and that reunification restores this chemical modification. The current results seem to indicate that increased trophallaxis permits a uniform odor to be reestablished among previously separated ants. Trophallaxis involving the queen is infrequent and does not seem to be crucial in the process of odor exchange. Our data confirm that trophallaxis plays a key role in establishing the Gestalt colony odor, particularly among naturally separated satellite nests in a polydomous species like C. iberica.     

Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes

Cytoskeleton damage, particularly microtubule (MT) alterations, may play an important role in the pathogenesis of ischemia-induced myocardial injury. However, this disorganization has been scarcely confirmed in the cellular context. We evaluated MT network disassembly in myoblast cell line H9c2 and in neonatal rat cardiomyocytes in an in vitro substrate-free hypoxia model of simulated ischemia (SI). After different duration of SI from 30 up to 180 min, the cells were fixed and the microtubule network was revealed by immunocytochemistry. The microtubule alterations were quantified using a house-developed image analysis program. Additionally, the tubulin fraction were extracted and quantified by Western blotting. The cell respiration, the release of cellular LDH and the cell viability were evaluated at the same periods. An early MT disassembly was observed after 60 min of SI. The decrease in MT fluorescence intensity at 60 and 90 min was correlated with a microtubule disassembly. Conversely, SI-induced significant LDH release (35%) and decrease in cell viability (34%) occurred after 120 min only. These results suggest that the simulated ischemia-induced changes in MT network should not be considered as an ultrastructural hallmark of the cell injury and could rather be an early ultrastructural correlate of the cellular reaction to the metabolic challenge.     

Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation

Bone marrow mesenchymal stem cells (MSC) are multipotent cells. To explain their plasticity, we postulated that undifferentiated MSC may express proteins from other tissues such as neuronal tissues. MSC are obtained by two different approaches: plastic adhesion or negative depletion (RosetteSep and magnetic beads CD45/glycophorin A). MSC are evaluated through FACS analysis using a panel of antibodies (SH2, SH3, CD14, CD33, CD34, CD45, etc.). To confirm the multipotentiality in vitro, we have differentiated MSC into adipocytes, chondrocytes, osteocytes, and neuronal/glial cells using specific induction media. We have evaluated neuronal and glial proteins (Nestin, Tuj-I, betaIII Tubulin, tyrosine hydroxylase [TH], MAP-2, and GFAP) by using flow cytometry, Western blots, and RT-PCR. We found that MSC constituently express native immature neuronal proteins such as Nestin and Tuj-1. After only five passages, MSC can already express more mature neuronal or glial proteins, such as TH, MAP-2, and GFAP, without any specific induction. We noticed an increase in the expression of more mature neuronal/glial proteins (TH, MAP-2, and GFAP) after exposure to neural induction medium, thus confirming the differentiation of MSC into neurons and astrocytes. The constitutive expression of Nestin or Tuj-1 by MSC suggests that these cells are "multidifferentiated" cells and thus can retain the ability for neuronal differentiation, enhancing their potentiality to be employed in the treatment of neurological diseases.