Syntenic Relationships between the U and M Genomes of Aegilops,Wheat and the Model Species Brachypodium and Rice as Revealed by COS Markers

Diploid Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata are important wild gene sources for wheat. With the aim of assisting in alien gene transfer, this study provides gene-based conserved orthologous set (COS) markers for the U and M genome chromosomes. Out of the 140 markers tested on a series of wheat-Aegilops chromosome introgression lines and flow-sorted subgenomic chromosome fractions, 100 were assigned to Aegilops chromosomes and six and seven duplications were identified in the U and M genomes, respectively. The marker-specific EST sequences were BLAST-ed to Brachypodium and rice genomic sequences to investigate macrosyntenic relationships between the U and M genomes of Aegilops, wheat and the model species. Five syntenic regions of Brachypodium identified genome rearrangements differentiating the U genome from the M genome and from the D genome of wheat. All of them seem to have evolved at the diploid level and to have been modified differentially in the polyploid species Ae. biuncialis and Ae. geniculata. A certain level of wheat–Aegilops homology was detected for group 1, 2, 3 and 5 chromosomes, while a clearly rearranged structure was showed for the group 4, 6 and 7 Aegilops chromosomes relative to wheat. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species, wheat and model species will facilitate the targeted development of new markers specific for U and M genomic regions and will contribute to the understanding of molecular processes related to allopolyploidization.     

Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.     

Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is the most common cause of bacterial, seafood‐related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro‐ and anti‐inflammatory cytokines by V. parahaemolyticus‐infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL‐1α, IL‐6, TNF‐α and IL‐10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T‐cell co‐stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.     

DFT modelling of hydrogen on Cu(110)- and (111)-type clusters

Density Functional Theory (DFT) calculations using gaussian 98 have been performed on hydrogen adsorbed on clusters representing the (110) and (111) surfaces of Cu. Clusters were constructed to model different adsorption sites, and at least two different size clusters were used for each site. On the (111) surface, hydrogen prefers to adsorb in a hollow site, though with the hcp variant being favoured by the adsorption energy, and the fcc alternative by the vibrational frequencies. On the (110) surface, the "fcc" site on a (1 2 2) reconstructed surface is preferred.     

Re-cycling Paradigms: Cell Cycle Regulation in Adult Hippocampal Neurogenesis and Implications for Depression

Since adult neurogenesis became a widely accepted phenomenon, much effort has been put in trying to understand the mechanisms involved in its regulation. In addition, the pathophysiology of several neuropsychiatric disorders, such as depression, has been associated with imbalances in adult hippocampal neurogenesis. These imbalances may ultimately reflect alterations at the cell cycle level, as a common mechanism through which intrinsic and extrinsic stimuli interact with the neurogenic niche properties. Thus, the comprehension of these regulatory mechanisms has become of major importance to disclose novel therapeutic targets. In this review, we first present a comprehensive view on the cell cycle components and mechanisms that were identified in the context of the homeostatic adult hippocampal neurogenic niche. Then, we focus on recent work regarding the cell cycle changes and signaling pathways that are responsible for the neurogenesis imbalances observed in neuropathological conditions, with a particular emphasis on depression.     

Variations in the Rhythms of Respiration and Nitrogen Fixation in Members of the Unicellular Diazotrophic Cyanobacterial Genus Cyanothece

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.Nitrogen fixation is an important global phenomenon by which molecular nitrogen, one of the most abundant components of the earth’s atmosphere, is converted into a more reduced form suitable for incorporation into living systems. The majority of this nitrogen fixation is achieved by biological means through the activity of microorganisms (Burris and Roberts, 1993; Raymond et al., 2004; Rubio and Ludden, 2008). This process is energy intensive, and nitrogenase, the enzyme complex involved in the biological nitrogen fixation reaction, is generally known to be extremely sensitive to oxygen (Robson and Postgate, 1980; Hill et al., 1981; Berman-Frank et al., 2005). Thus, most microbes participating in this process fix nitrogen only when suitable anaerobic or microaerobic conditions are established in an otherwise oxygen-rich environment. However, some nitrogen-fixing (diazotrophic) microbes have the advantage of being able to fix nitrogen in aerobic environments. Outstanding among these are the photosynthetic prokaryotes called cyanobacteria, an extremely successful group of microbes with plant-like traits. These microbes are considered to be the progenitors of plant chloroplasts. Cyanobacteria perform both oxygen-evolving photosynthesis and oxygen-sensitive nitrogen fixation, thereby providing a platform to power the most metabolically expensive biological process (Simpson and Burris, 1984) with solar energy.Among the nitrogen-fixing cyanobacteria, filamentous strains have been extensively studied for their contribution to the nitrogen cycle in marine and terrestrial ecosystems (Mulligan and Haselkorn, 1989; Kaneko et al., 2001; Meeks et al., 2001; Sañudo-Wilhelmy et al., 2001; Wong and Meeks, 2001; Gomez et al., 2005). Some of these filamentous strains develop specialized cells called heterocysts that allow the spatial segregation of photosynthesis and nitrogen fixation. These heterocysts also have higher rates of respiratory oxygen consumption, which results in a virtually anoxic environment conducive for the nitrogenase enzyme (Bergman et al., 1997). All heterocystous strains are known to fix nitrogen aerobically. In contrast, nonheterocystous cyanobacteria lack any specialized oxygen-free compartments and often require incubation under microoxic or anaerobic conditions for nitrogen fixation (Rippka and Waterbury, 1977; Rippka et al., 1979; Brass et al., 1992). However, some nonheterocystous cyanobacterial strains can fix nitrogen under aerobic conditions. These include some filamentous genera like Trichodesmium spp., Lyngbya spp., and Oscillatoria spp. (Jones, 1990; Janson et al., 1994; Finzi-Hart et al., 2009) as well as unicellular genera like Gloeothece spp. and Cyanothece spp. (Wyatt and Silvey, 1969; Rippka and Waterbury, 1977; Huang and Chow, 1988; Van Ni et al., 1988; Schütz et al., 2004).In comparison with filamentous cyanobacteria, which have long been recognized for their nitrogen-fixing ability, the importance of unicellular cyanobacteria as key components of the environmental nitrogen cycle has only been recently uncovered. Studies over the last decade have established unicellular strains like Crocosphaera spp., Cyanothece spp., and UCYN-A as important players in the marine nitrogen cycle (Zehr et al., 2001; Montoya et al., 2004; Zehr, 2011). Since unicellular diazotrophic cyanobacteria utilize the same cellular platform for photosynthesis and nitrogen fixation, they are required to adjust their cellular metabolism to accommodate these two antagonistic processes. Systems-level studies in the unicellular genus Cyanothece have revealed a temporal separation of the two processes, photosynthesis occurring during the day and nitrogen fixation occurring at night (Stöckel et al., 2008; Toepel et al., 2008; Welsh et al., 2008). Cellular respiration plays a critical role during the transition from one phase to the next, rapidly freeing the intracellular environment of the photosynthetically generated oxygen and rendering it conducive for the induction of nitrogenase activity. In addition, respiration also sustains the process of nitrogen fixation, not only by maintaining a low-oxygen environment required for the functioning of the nitrogenase enzyme but also by mobilizing the stored solar energy to fuel this energy-intensive process.Unicellular diazotrophs exhibit great diversity in the efficiency of nitrogen fixation as well as in the physiological regulation of the process. For instance, members of the genus Gloeothece fix nitrogen aerobically during the day, but at 0% dissolved oxygen concentration, nitrogen fixation is shifted entirely to the dark period (Ortega-Calvo and Stal, 1991; Taniuchi et al., 2008). In contrast, some Synechococcus spp. strains can fix nitrogen only when incubated under anoxic conditions (Steunou et al., 2006). Members of the genus Cyanothece have been reported to engage in both aerobic and anaerobic nitrogen fixation, with nitrogenase activity peaking during the night (Reddy et al., 1993; Bergman et al., 1997; Turner et al., 2001). This suggests that, in addition to the regulations imposed by the diurnal cycle, strain-specific intracellular cues govern the process of nitrogen fixation in unicellular cyanobacteria, which may vary according to the genotype or the ecotype of the strains.Members of the unicellular cyanobacterial genus Cyanothece are diazotrophs that thrive in marine as well as terrestrial environments. This genus was originally grouped together with Synechococcus spp. but was later separated on the basis of distinct morphological and biochemical differences between the two genera (Komárek, 1976; Rippka and Cohen-Bazire, 1983). Some of the features that define the largely heterogeneous genus Cyanothece are oval to cylindrical cells, larger than 3 µm in size (they can be as large as 24 µm in diameter), radially arranged thylakoids, and a mucilaginous layer surrounding the cells (Komárek and Cepák, 1998; Porta et al., 2000; Liberton et al., 2011).It was recently demonstrated that Cyanothece sp. ATCC 51142, a member of the genus Cyanothece, has the unique ability to produce molecular hydrogen at exceptionally high rates under aerobic conditions (Bandyopadhyay et al., 2010). This striking observation was attributed to the nitrogenase enzyme system of Cyanothece sp. ATCC 51142. Our study also indicated that high rates of respiration in this strain might contribute to its nitrogenase-mediated aerobic hydrogen production. Glycerol was found to be an efficient source of reductants and energy for this process. In an effort to investigate if this atypical cyanobacterial trait was a characteristic of the genus Cyanothece, five additional Cyanothece spp. strains from different ecological habitats were sequenced to completion. The six strains display more than 90% identity in their 16S ribosomal RNA sequence but exhibit striking variability with respect to their genome sizes (with the largest genome being 7.8 Mb and the smallest being 4.4 Mb), the number of plasmids, and the percentage of pseudogenes (Bandyopadhyay et al., 2011). In addition, two of the strains possess linear chromosomal elements, features not known to occur in any other photosynthetic bacteria sequenced to date, which may impart niche-specific advantages to these strains. Analysis of the genome sequence of the Cyanothece spp. strains showed the presence of a nitrogenase gene cluster in all five strains, and preliminary analysis showed that four of the five strains were capable of aerobic nitrogen fixation and hydrogen production (Bandyopadhyay et al., 2011). In this study, we have focused on the patterns of nitrogen fixation and respiration in six different Cyanothece spp. strains in an effort to elucidate the underlying differences and similarities in these processes in unicellular diazotrophic strains with similar genotypic but varied ecological backgrounds. Our study reveals inherent differences in the regulation of these processes, which are likely controlled by strain-specific cellular signals. However, despite the differences in the patterns of nitrogenase activity, aerobic nitrogen fixation and hydrogen production was found to be a characteristic of this genus, with most members exhibiting nitrogenase-mediated hydrogen production at rates higher than any other wild-type cyanobacterial strain.     

The plant Selaginella moellendorffii possesses enzymes for synthesis and hydrolysis of the compatible solutes mannosylglycerate and glucosylglycerate

A mannosylglycerate synthase (MgS) gene detected in the genome of Selaginella moellendorffii was expressed in E. coli and the recombinant enzyme was purified and characterized. A remarkable and unprecedented feature of this enzyme was the ability to efficiently synthesize mannosylglycerate (MG) and glucosylglycerate (GG) alike, with maximal activity at 50 °C, pH 8.0 and with Mg²⁺ as reaction enhancer. We have also identified a novel glycoside hydrolase gene in this plant’s genome, which was functionally confirmed to be highly specific for the hydrolysis of MG and GG and named MG hydrolase (MgH), due to its homology with bacterial MgHs. The recombinant enzyme was maximally active at 40 °C and at pH 6.0–6.5. The activity was independent of cations, but Mn²⁺ was a strong stimulator. Regardless of these efficient enzymatic resources we could not detect MG or GG in S. moellendorffii or in the extracts of five additional Selaginella species. Herein, we describe the properties of the first eukaryotic enzymes for the synthesis and hydrolysis of the compatible solutes, MG and GG.