Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics

Stratified sulfurous lakes are appropriate environments for studying the links between composition and functionality in microbial communities and are potentially modern analogs of anoxic conditions prevailing in the ancient ocean. We explored these aspects in the Lake Banyoles karstic area (NE Spain) through metagenomics and in silico reconstruction of carbon, nitrogen and sulfur metabolic pathways that were tightly coupled through a few bacterial groups. The potential for nitrogen fixation and denitrification was detected in both autotrophs and heterotrophs, with a major role for nitrogen and carbon fixations in Chlorobiaceae. Campylobacterales accounted for a large percentage of denitrification genes, while Gallionellales were putatively involved in denitrification, iron oxidation and carbon fixation and may have a major role in the biogeochemistry of the iron cycle. Bacteroidales were also abundant and showed potential for dissimilatory nitrate reduction to ammonium. The very low abundance of genes for nitrification, the minor presence of anammox genes, the high potential for nitrogen fixation and mineralization and the potential for chemotrophic CO₂ fixation and CO oxidation all provide potential clues on the anoxic zones functioning. We observed higher gene abundance of ammonia-oxidizing bacteria than ammonia-oxidizing archaea that may have a geochemical and evolutionary link related to the dominance of Fe in these environments. Overall, these results offer a more detailed perspective on the microbial ecology of anoxic environments and may help to develop new geochemical proxies to infer biology and chemistry interactions in ancient ecosystems.     

Gene network analysis of Arabidopsis thaliana flower development through dynamic gene perturbations

Understanding how flowers develop from undifferentiated stem cells has occupied developmental biologists for decades. Key to unraveling this process is a detailed knowledge of the global regulatory hierarchies that control developmental transitions, cell differentiation and organ growth. These hierarchies may be deduced from gene perturbation experiments, which determine the effects on gene expression after specific disruption of a regulatory gene. Here, we tested experimental strategies for gene perturbation experiments during Arabidopsis thaliana flower development. We used artificial miRNAs (amiRNAs) to disrupt the functions of key floral regulators, and expressed them under the control of various inducible promoter systems that are widely used in the plant research community. To be able to perform genome‐wide experiments with stage‐specific resolution using the various inducible promoter systems for gene perturbation experiments, we also generated a series of floral induction systems that allow collection of hundreds of synchronized floral buds from a single plant. Based on our results, we propose strategies for performing dynamic gene perturbation experiments in flowers, and outline how they may be combined with versions of the floral induction system to dissect the gene regulatory network underlying flower development.     

Almost 20 years of Neanderthal palaeogenetics: adaptation,admixture, diversity,demography and extinction

Nearly two decades since the first retrieval of Neanderthal DNA, recent advances in next-generation sequencing technologies have allowed the generation of high-coverage genomes from two archaic hominins, a Neanderthal and a Denisovan, as well as a complete mitochondrial genome from remains which probably represent early members of the Neanderthal lineage. This genomic information, coupled with diversity exome data from several Neanderthal specimens is shedding new light on evolutionary processes such as the genetic basis of Neanderthal and modern human-specific adaptations—including morphological and behavioural traits—as well as the extent and nature of the admixture events between them. An emerging picture is that Neanderthals had a long-term small population size, lived in small and isolated groups and probably practised inbreeding at times. Deleterious genetic effects associated with these demographic factors could have played a role in their extinction. The analysis of DNA from further remains making use of new large-scale hybridization-capture-based methods as well as of new approaches to discriminate contaminant DNA sequences will provide genetic information in spatial and temporal scales that could help clarify the Neanderthal''s—and our very own—evolutionary history.     

Protein partition between the different phases comprising poly(ethylene glycol)-salt aqueous two-phase systems, hydrophobic interaction chromatography and precipitation: a generic description in terms of salting-out effects

The solution behaviour of selected proteins has been studied under conditions promoting precipitation, binding to mildly hydrophobic adsorbents or partition. Solvophobic theory may be used to describe these forms of protein partition. The tendency of a protein to partition therein is dependent upon surface properties of the protein solute mediated by the concentration and nature of added salts. As applied to partitioning in poly(ethylene glycol) (PEG)-salt systems this implies that linear (Brönsted) relationships apply only to proteins partitioned close to the critical point. At longer tie-line lengths protein partitioning is increasingly influenced by salting-out forces. This is confirmed by the observed behaviour of the proteins. The point at which this behaviour changes has been unambiguously defined enabling the direct comparison of phase transition of proteins during partition in all systems. The results obtained show that phase transition during adsorption and partition occur at similar concentrations of salt. This is less than that required to promote precipitation. It appears, from these limited studies, that top-phase preferring proteins are partitioned at salt concentrations above those required to cause adsorption. Proteins preferring the lower phase are partitioned at salt concentrations close to or below those required for adsorption. This raises questions regarding the solvated molecular form of the partitioned proteins and the definition of the partition coefficient.     

Spermatogenesis of the spider crab Maja brachydactyla (Decapoda: Brachyura)

This study describes spermatogenesis in a majid crab (Maja brachydactyla) using electron microscopy and reports the origin of the different organelles present in the spermatozoa. Spermatogenesis in M. brachydactyla follows the general pattern observed in other brachyuran species but with several peculiarities. Annulate lamellae have been reported in brachyuran spermatogenesis during the diplotene stage of first spermatocytes, the early and mid‐spermatids. Unlike previous observations, a Golgi complex has been found in mid‐spermatids and is involved in the development of the acrosome. The Golgi complex produces two types of vesicles: light vesicles and electron‐dense vesicles. The light vesicles merge into the cytoplasm, giving rise to the proacrosomal vesicle. The electron‐dense vesicles are implicated in the formation of an electron‐dense granule, which later merges with the proacrosomal vesicle. In the late spermatid, the endoplasmic reticulum and the Golgi complex degenerate and form the structures–organelles complex found in the spermatozoa. At the end of spermatogenesis, the materials in the proacrosomal vesicle aggregate in a two‐step process, forming the characteristic concentric three‐layered structure of the spermatozoon acrosome. The newly formed spermatozoa from testis show the typical brachyuran morphology. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Sperm ultrastructure of the spider crab Maja brachydactyla (Decapoda: Brachyura)

This study describes the morphology of the sperm cell of Maja brachydactyla, with emphasis on localizing actin and tubulin. The spermatozoon of M. brachydactyla is similar in appearance and organization to other brachyuran spermatozoa. The spermatozoon is a globular cell composed of a central acrosome, which is surrounded by a thin layer of cytoplasm and a cup‐shaped nucleus with four radiating lateral arms. The acrosome is a subspheroidal vesicle composed of three concentric zones surrounded by a capsule. The acrosome is apically covered by an operculum. The perforatorium penetrates the center of the acrosome and has granular material partially composed of actin. The cytoplasm contains one centriole in the subacrosomal region. A cytoplasmic ring encircles the acrosome in the subapical region of the cell and contains the structures‐organelles complex (SO‐complex), which is composed of a membrane system, mitochondria with few cristae, and microtubules. In the nucleus, slightly condensed chromatin extends along the lateral arms, in which no microtubules have been observed. Chromatin fibers aggregate in certain areas and are often associated with the SO‐complex. During the acrosomal reaction, the acrosome could provide support for the penetration of the sperm nucleus, the SO‐complex could serve as an anchor point for chromatin, and the lateral arms could play an important role triggering the acrosomal reaction, while slightly decondensed chromatin may be necessary for the deformation of the nucleus. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm

Morphologic and morphometric sperm characteristics of mouse epididymal extracts from animals exposed to static magnetic fields were evaluated. For this purpose, animals were exposed for 35 days to a field of 0.7 T generated by a commercial permanent magnet for either 1 or 24 h per day. The values of morphometric parameters were obtained using the morphometric module of the Sperm Class Analyzer® computerized image analysis system, and percentages of abnormalities were calculated. The size of sperm heads was unaffected by exposure to static magnetic fields. Lack of hook was a sperm head abnormality found significantly more frequently in animals exposed continually than in nonexposed animals, showing a possible alteration to the spermatogenic process after exposure to static magnetic fields. The percentage of sperm with coiled tails or of sperm with abnormal midpiece or tail was not altered by exposure. Bioelectromagnetics 19:377–383, 1998. © 1998 Wiley-Liss, Inc.     

Ultrafast light harvesting dynamics in the cryptophyte phycocyanin 645.

Steady-state and femtosecond time-resolved optical methods have been used to study spectroscopic features and energy transfer dynamics in the soluble antenna protein phycocyanin 645 (PC645), isolated from a unicellular cryptophyte Chroomonas CCMP270. Absorption, emission and polarization measurements as well as one-colour pump-probe traces are reported in combination with complementary quantum chemical calculations of electronic transitions of the bilins. Estimation of bilin spectral positions and energy transfer rates aids in the development of a model for light harvesting by PC645. At higher photon energies light is absorbed by the centrally located dimer (DBV, beta50/beta61) and the excitation is subsequently funneled through a complex interference of pathways to four peripheral pigments (MBV alpha19, PCB beta158). Those chromophores transfer the excitation energy to the red-most bilins (PCB beta82). We suggest that the final resonance energy transfer step occurs between the PCB 82 bilins on a timescale estimated to be approximately 15 ps. Such a rapid final energy transfer step cannot be rationalized by calculations that combine experimental parameters and quantum chemical calculations, which predict the energy transfer time to be 40 ps.     

The role of residue stability in transient protein–protein interactions involved in enzymatic phosphate hydrolysis. A computational study

Finding why protein–protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Statistical surveys of so‐called transient complexes (like those relevant for signal transduction mechanisms) have shown a tendency of polar residues to participate in the interaction region. Following this scheme, residues in the unbound partners have to compete between interacting with water or interacting with other residues of the protein. On the other hand, several works have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions. This preorganization can be related to the instability of the residues important for catalysis. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. In this article the linear response approximation version of the semimacroscopic protein dipoles Langevin dipoles (PDLD/S‐LRA) model is used to evaluate the stability of several residues in two phosphate hydrolysis enzymes upon complexation with their activating partners. In particular, the residues relevant for PPI and for phosphate hydrolysis in the CDK2/Cyclin A and Ras/GAP complexes are analyzed. We find that the evaluation of the stability of residues in these systems can be used to identify not only active site regions but it can also be used as a guide to locate “hot spots” for PPIs. We also show that conformational changes play a major role in positioning interfacing residues in a proper “energetic” orientation, ready to interact with the residues in the partner protein surface. Thus, we extend the preorganization theory to PPIs, extrapolating the results we obtained from the above‐mentioned complexes to a more general case. We conclude that the correlation between stability of a residue in the surface and the likelihood that it participates in the interaction can be a general fact for transient PPIs. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Kinetics of the action of chymosin (rennin) on a peptide bond of bovine alpha s1-casein. Comparison of the behaviour of this substrate with that of beta- and kappa o-caseins

The action of chymosin on the Phe23-Phe24 bond of bovine alpha s1-casein, in citrate buffer (pH 6.2) at 30 degrees C, was followed by reversed-phase HPLC quantification of residual alpha s1-casein or fragment 24-199 after different time periods and at different substrate concentrations. This allowed determination of the Michaelian parameters for the reaction under study which were compared with those previously obtained for the action of chymosin on beta- and kappa o-casein under identical reaction conditions. The whole efficiency of the three reactions, as estimated by kcat/Km, was 1.8, 20.6 and 1405.0 for alpha s1-, beta- and kappa o-caseins, respectively. The specificity of chymosin is discussed in the light of these results and of the known sequences of the 3 caseins.