Temporal,regional and geochemical drivers of microbial community variation in the melt ponds of the Ross Sea region,Antarctica

To expand the understanding of the poorly described planktonic bacterial communities inhabiting Antarctic meltwater ponds, this study characterized the community composition and identified environmental drivers influencing community structure from a total of 41 meltwater ponds: 37 from the McMurdo Ice Shelf (Bratina Island) and four from a terrestrial locale (Miers Valley) during three austral summers. DNA fingerprinting coupled with in situ pH and conductivity was utilized to select ponds for in-depth nutrient and chemical analysis and high-throughput sequencing of the bacterial 16S rRNA gene V5–V6 hypervariable region. Conductivity was the strongest driver of community structure across all ponds and for all time points; however, other influential factors (pH, climatological, Hg, Fe, and PO₄) were also identified. Unique members of communities (sequences absent in at least one pond) represented a small percentage of total reads but also represented a large proportion of pond biodiversity that was strongly driven by differing environmental variables (Si, B and S). Significant temporal variation in community structure was also identified within the same ponds although major taxa remained present. Miers Valley ponds exhibit greater similarity to Bratina Island ponds rather than between each other, thereby suggesting regional movement of microorganisms. In summary, these data provide the first in-depth investigation of the intra-seasonal and regional variation of the microbial communities inhabiting these ponds and proved that a total of ten cosmopolitan OTUs were the dominant components of ponds throughout all sampling times and locations, their variable relative abundances driving the major dissimilarities in community structure.     

Implications of molecular diversity of chitin and its derivatives

Chitin is a long unbranched polysaccharide, made up of β-1,4-linked N-acetylglucosamine which forms crystalline fiber-like structure. It is present in the fungal cell walls, insect and crustacean cuticles, nematode eggshells, and protozoa cyst. We provide a critical appraisal on the chemical modifications of chitin and its derivatives in the context of their improved efficacy in medical applications without any side effect. Recent advancement in nanobiotechnology has helped to synthesize several chitin derivatives having significant biological applications. Here, we discuss the molecular diversity of chitin and its applications in enzyme immobilization, wound healing, packaging material, controlled drug release, biomedical imaging, gene therapy, agriculture, biosensor, and cosmetics. Also, we highlighted chitin and its derivatives as an antioxidant, antimicrobial agent, anticoagulant material, food additive, and hypocholesterolemic agent. We envisage that chitin and chitosan-based nanomaterials with their potential applications would augment nanobiotechnology and biomedical industries.

     

Copepod foraging and predation risk within the surface layer during night-time feeding forays

Vertical distribution data seem to indicate that certain speciesof diel vertical migrating copepods avoid the surface high chlorophyll(Chl) region within coastal and estuarine environments, evenduring the night. Copepods may avoid this layer to reduce predationmortality, avoid advective loss or to avoid consuming too muchtoxic algae. We hypothesize that copepods make several intermittentfeeding ‘forays’ into shallow surface layers duringthe night, returning to intermediate depths between forays.Using an individual-based model (IBM) of Calanus pacificus,we examined the implications of this behavior on feeding successand mortality risk, and tested whether a practical field-samplingscheme would be able to detect foray-like behavior. In somecases, mortality of the foray-foraging copepods was up to 50%less than that of randomly behaving controls, for a given amountof food ingested. The trapping scheme devised should be ableto detect the occurrence of foray behavior (FB) in the fieldand should show differences in the gut contents of copepodsentering and leaving the uppermost food-rich layer. The presenceor absence of foray-like behavior significantly altered therelative concentration of copepods within various surface strataand thus could influence the temporal availability of copepodsas prey for the larvae and juveniles of several important managedfish species. This paper is one of six on the subject of the role of zooplanktonpredator–prey interactions in structuring plankton communities.     

Metastasis-associated C4.4A, a GPI-anchored protein cleaved by ADAM10 and ADAM17

Metalloproteases play a complex role in tumor progression. While the activity of some ADAM, ADAMTS and matrix metalloproteases (MMPs) seems to be protumorigenic, the activity of others seems to prevent tumor progression. The identification of the array of substrates of a given metalloprotease (degradome) seems an adequate approach to predict the effect of the inhibition of a metalloprotease in tumors. Here, we present the proteomic identification of a novel substrate for ADAM10 and -17. We used SILAC (Stable Isotope Labeling by Amino acids in Cell culture), a proteomic technique based on the differential metabolic labeling of cells in different conditions. This was applied to MCF7 cells derived from an invasive mammary tumor, and the same cells expressing shRNAs that knock down ADAM10 or -17. Following this approach, we have identified C4.4A as a substrate to both metalloproteases. Since C4.4A is likely involved in tumor invasion, these results indicate that the cleavage of C4.4A by ADAM10 and ADAM17 contributes to tumor progression.     

Dynamics of cell proliferation and apoptosis reflect different life strategies in hydrothermal vent and cold seep vestimentiferan tubeworms

Deep-sea vestimentiferan tubeworms, which live in symbiosis with bacteria, exhibit different life strategies according to their habitat. At unstable and relatively short-lived hydrothermal vents, they grow extremely fast, whereas their close relatives at stable and long-persisting cold seeps grow slowly and live up to 300 years. Growth and age differences are thought to occur because of ecological and physiological adaptations. However, the underlying mechanisms of cell proliferation and death, which are closely linked to homeostasis, growth, and longevity, are unknown. Here, we show by immunohistochemical and ultrastructural cell cycle analyses that cell proliferation activities of the two species studied are higher than in any other characterized invertebrate, being only comparable with tumor and wound-healing processes. The slow growth in Lamellibrachia luymesi from cold seeps results from balanced activities of proliferation and apoptosis in the epidermis. In contrast, Riftia pachyptila from hydrothermal vents grows fast because apoptosis is down-regulated in this tissue. The symbiont-housing organ, the trophosome, exhibits a complex cell cycle and terminal differentiation pattern in both species, and growth is regulated by proliferation. These mechanisms have similarities to the up- and down-regulation of proliferation or apoptosis in various types of tumor, although they occur in healthy animals in this study, thus providing significant insights into the underlying mechanisms of growth and longevity. Financial support was provided by the Austrian Science Foundation (FWF; grants to M.B.), a DOC scholarship from the Austrian Academy of Science (to B.P.), and grants from the US National Science Foundation (to C.R. Fisher).     

Structural and Biophysical Characterization of the Proteins Interacting with the Herpes Simplex Virus 1 Origin of Replication

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA_15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (K_d ∼ 6 nm) followed by a second binding event (K_d ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA_15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.The initiation of DNA replication for most double-stranded DNA (dsDNA)⁶ viral genomes begins with the recognition of the origin by specific origin-binding proteins. The herpes simplex virus type 1 (HSV-1) genome encodes seven proteins required for origin-dependent DNA replication. These are the DNA polymerase (UL30) and its accessory protein (UL42), a heterotrimeric helicase-primase complex (UL5, UL8, and UL52), the single-stranded DNA-binding protein (ICP8 or UL29), and the origin-binding protein (UL9) (reviewed in Ref. 1). HSV-1 contains three functional origins, oriL and two copies of oriS. OriS, which is about 80 bp in length, consists of three UL9 recognition sites, in Boxes I, II, and III, which are arranged in two overlapping palindromes (2). Box I and Box III are part of an evolutionarily conserved palindrome that forms a stable hairpin in single-stranded DNA, which may be important in the origin rearrangement (3) during initiation of replication. Box I and II are separated by an AT-rich spacer sequence, which varies in length and nucleotide composition between the different members of the α-herpesvirus subfamily (2, 4–6).UL9 is a homodimer in solution, and EM studies, with UL9 bound to oriS, indicate the existence of a dimer or pair of dimers assembled on oriS (7). Several reports indicate that UL9 can physically interact not only with ICP8 (8) but also with other members of the HSV-1 replication complex, including UL8 (9) and UL42 (10). Thus UL9 functions as a docking protein to recruit these essential replication proteins to the viral origins. ICP8 stimulates the helicase activity of UL9 (11, 12) and binds to its C-terminal 27-aa residues (13). In the presence of ICP8, UL9 will open dsDNA containing Box I, leading to a conformational change in the origin, thus facilitating unwinding (14–16). As stated above, the changes in DNA conformation in the complete oriS may be more complex (3). Recently, it has been suggested that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by UL9. oriS* contains a hairpin formed by complementary base pairing between Box I and Box III in oriS (17). UL9, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-bp double-stranded minimal oriS fragment to oriS* and form a UL9-oriS* complex. The formation of a UL9-oriS* complex requires ATP hydrolysis (18). Therefore, the UL9-oriS* complex may serve as an assembly site for the herpesvirus replisome. Macao et al. (3) proposed a model in which full-length UL9 would be required to adopt a different conformation when binding to oriS or oriS*. The implication is that UL9 partially unwinds and introduces a hairpin into the origin of replication and that the formation of oriS* is aided, in some way, by ICP8 and requires ATP hydrolysis. Macao et al. (3) suggest that the length of the single-stranded tail of the probe DNA determines the stoichiometry of the UL9-DNA complex. oriS may bind two molecules of UL9, whereas oriS* may only bind one because the hairpin formation prevents the second interaction.Photo-cross-linking studies have shown that, although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I, suggesting that the C terminus of UL9 undergoes a conformational change upon binding to Box I (19). The results reported here are consistent with this observation. To date there is no three-dimensional structural information available on the full-length UL9 or either of the functionally characterized (helicase and DNA binding) domains. The ability to adopt different conformations and a tendency to proteolytic degradation may be responsible for this. It has been shown that UL9 binds with very high specificity to the Box I through its DNA-binding domain, consisting of the C-terminal 317 aa (UL9ct) (20, 21). Although the importance of the binding between UL9ct and oriS for the viral life cycle is well established, the mechanism behind this interaction still remains unclear. Even though UL9ct exists as a monomer in solution, uncertainty remains as to whether one or two molecules bind to a single Box I recognition sequence. Some reports have suggested that one UL9ct molecule binds to a single copy of the sequence (22–24), whereas others have proposed that UL9ct forms a dimer when bound to DNA (25, 26). This apparent difference may well result from the different protein concentrations used in different assays/experiments, which in turn highlights the difficulty of translating in vitro equilibrium experiments into cellular nonequilibrium situations.A few years ago, the crystal structure of a 60-residue C-terminal deletion mutant of ICP8 (ICP8ΔC) was determined to 3 Å resolution (Protein Data Bank code 1URJ (27)). The structure of ICP8ΔC consists of a large N-terminal domain (aa 9–1038) and a smaller entirely helical C-terminal domain (aa 1049–1120) connected to the N-terminal domain by a disordered linker (aa 1038–1049) spanning around 18 Å in the crystal structure. ICP8 preferentially binds ssDNA over dsDNA in a nonsequence-specific and cooperative manner (28). ICP8 is a zinc metalloprotein containing one zinc atom per molecule, which is coordinated by three cysteines (Cys-499, Cys-502, and Cys-510) and a histidine (His-512) (27).In this study, we show that the 60-amino acid C-terminal deletion of ICP8 (ICP8ΔC) binds strongly to UL9ct. We present three low resolution structures in solution using small angle x-ray scattering as follows: that of the UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer dsDNA (dsDNA_15-mer) containing the Box I sequence. Using these data and a variety of biophysical techniques, we demonstrate that the stoichiometries of the UL9ct-dsDNA_15-mer and UL9ct-ICP8ΔC-dsDNA_15-mer complexes are 2:1 and 2:1:1, respectively, at the micromolar protein concentrations used in this study. Using EM we visualize the assembly of the ICP8ΔC-UL9ct complex on oriS and estimate the size of the complex.     

The phylogeography of Adelie penguin faecal flora

The gut of animals changes quickly from a totally sterile environment before birth to a numerous and highly diverse microbial community shortly after birth. However, few studies have examined the source of the bacteria colonizing the gut. We used a genetic based approach to investigate the distribution and acquisition of faecal microbial communities by Adelie penguins, Pygoscelis adeliae , breeding in the Ross Sea region of Antarctica by cloning a portion of the 16S rRNA gene and by automated ribosomal intergenic spacer analysis (ARISA). We hypothesized that bacteria were either acquired from the penguins' neighbours or inherited from their ancestors. Samples were collected from Adelie penguin breeding colonies at the north-western edge of the Ross Sea coast through to the southernmost Adelie penguin colonies on Ross Island. Most of the bacterial sequences we obtained were only distantly homologous with previously published sequences. Bacterial taxa appear to have a restricted distribution as the majority of 16S rRNA clones were isolated from only one or two hosts. Faecal bacterial community similarity was strongly negatively correlated with the genetic distance between hosts suggesting that bacterial communities are inherited. There was little support for a correlation between distance between collection sites and community similarity.     

The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.