Duration of Hydrogen Formation by Anabaena cylindrica B629 in Atmospheres of Argon, Air, and Nitrogen

The time course of hydrogen formation by Anabaena cylindrica was followed beneath an argon atmosphere alone and also beneath atmospheres of argon, nitrogen, and air in the presence of carbon monoxide (0.2%) and acetylene (5%). Hydrogen production beneath argon alone was comparable in rate and duration (7 to 12 days) to that which occurred beneath air in the presence of carbon monoxide (0.2%) and acetylene (5%). However, much greater longevity (16 to 26 days) and improved rates of hydrogen formation were obtained when algae were incubated beneath argon and particularly nitrogen, each supplemented with carbon monoxide and acetylene. The total hydrogen produced by these cultures was up to three times as much as that released by cultures incubated beneath argon alone. Hydrogen-oxygen ratios for argon cultures either with or without carbon monoxide and acetylene were initially 1:5 but approximated 1:2 when measured over the entire incubation period. In each case oxygen production and nitrogenase activity (acetylene reduction) continued at reduced rates after hydrogen evolution had ceased. The effects of methionine sulfoximine (2 μM), ammonium ions (0.5 mM), or both on oxygen production were generally negligible, while effects on hydrogen production were variable depending on the atmosphere used; in most cases, eventual destabilization of the system occurred. A brief comparison was made of the time courses of anaerobic and aerobic hydrogen formation by the marine cyanobacterium Calothrix membranacea. It was found that shaking of cultures was beneficial for hydrogen production but not strictly necessary. It is concluded that hydrogen production by A. cylindrica in air and particularly nitrogen in the presence of carbon monoxide and acetylene offers the best potential of the atmospheres considered on the basis of four criteria: rates and longevity of hydrogen formation, practicality of the atmosphere used, and tolerance of hydrogen evolution to slight changes in composition of the atmosphere.     

Design,synthesis and evaluation of potent and selective inhibitors of mono-(ADP-ribosyl)transferases PARP10 and PARP14

A series of diaryl ethers were designed and synthesized to discern the structure activity relationships against the two closely related mono-(ADP-ribosyl)transferases PARP10 and PARP14. Structure activity studies identified 8b as a sub-micromolar inhibitor of PARP10 with?～15-fold selectivity over PARP14. In addition, 8k and 8m were discovered to have sub-micromolar potency against PARP14 and demonstrated moderate selectivity over PARP10. A crystal structure of the complex of PARP14 and 8b shows binding of the compound in a novel hydrophobic pocket and explains both potency and selectivity over other PARP family members. In addition, 8b, 8k and 8m also demonstrate selectivity over PARP1. Together, this study identified novel, potent and metabolically stable derivatives to use as chemical probes for these biologically interesting therapeutic targets.     

Cattle grazing does not reduce fire severity in eucalypt forests and woodlands of the Australian Alps

Large grazing herbivores can change fire regimes by altering fuel types and abundance, particularly in savanna biomes where the dominant fuel is grass. The use of herbivores as a fire management tool is receiving increasing consideration globally, but this intervention has a limited evidence‐base and is controversial because of potential deleterious ecological effects. These issues are well illustrated by the political and scientific debate about the capacity of cattle grazing to reduce fire hazard in the Victorian Alps of Australia; there have been remarkably few scientific studies to illuminate this issue. Here we use remote sensing and geographic information system analysis to determine the effect of active grazing licences on fire severity (crown scorch) in eucalypt forests and woodlands following large fires in the Alps during the summers of 2002/2003 and 2006/2007. Our statistical analyses, which controlled for spatial autocorrelation, found crown scorch was strongly related to vegetation type but there was no evidence that cattle grazing reduced fire severity. There was some evidence that grazing could increase fire severity by possibly changing fuel arrays. Such landscape analyses are a critical approach given that large‐scale grazing × fire trials are prohibitively expensive and impractical to conduct.     

Ordered,Random, Monotonic and Non-Monotonic Digital Nanodot Gradients

Cell navigation is directed by inhomogeneous distributions of extracellular cues. It is well known that noise plays a key role in biology and is present in naturally occurring gradients at the micro- and nanoscale, yet it has not been studied with gradients in vitro. Here, we introduce novel algorithms to produce ordered and random gradients of discrete nanodots – called digital nanodot gradients (DNGs) – according to monotonic and non-monotonic density functions. The algorithms generate continuous DNGs, with dot spacing changing in two dimensions along the gradient direction according to arbitrary mathematical functions, with densities ranging from 0.02% to 44.44%. The random gradient algorithm compensates for random nanodot overlap, and the randomness and spatial homogeneity of the DNGs were confirmed with Ripley''s K function. An array of 100 DNGs, each 400×400 µm², comprising a total of 57 million 200×200 nm² dots was designed and patterned into silicon using electron-beam lithography, then patterned as fluorescently labeled IgGs on glass using lift-off nanocontact printing. DNGs will facilitate the study of the effects of noise and randomness at the micro- and nanoscales on cell migration and growth.     

NLRP3/Cryopyrin Is Necessary for Interleukin-1�� (IL-1��) Release in Response to Hyaluronan, an Endogenous Trigger of Inflammation in Response to Injury

Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1β release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1β release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin → IL-1β pathway. These findings support the hypothesis that hyaluronan works through IL-1β and the cryopyrin system to signal sterile inflammation.Inflammation, as defined by changes in vascular permeability and leukocyte recruitment, is an essential step for the control of microbial invasion. Specific microbial products trigger this process through a diverse array of innate immune pattern recognition receptors. However, an inflammatory response independent of infection is also an important process for maintenance of biological homeostasis. For example, normal wound healing requires a controlled inflammatory response to enable the recruitment of monocytes and the release of growth factors required for repair. This response can occur in the absence of microbial stimuli. Furthermore, inflammation and the release of proinflammatory mediators is also associated with many diseases such as rheumatoid arthritis and Crohn disease (1). These diseases are not well understood in terms of their triggers but rather are described by the subsequent release of proinflammatory mediators. Identification of the triggers of sterile inflammation represents an important goal with immediate diagnostic and therapeutic significance.Recent work has begun to elucidate pathways of inflammation that occur in the absence of microbial stimuli. Stress signals such as heat-shock proteins, intracellular components of necrotic cells not normally seen by immune cells, and components of the extracellular matrix have all been implicated as endogenous triggers of injury (2–4). Among this group is the glycosaminoglycan hyaluronan (HA),⁶ an important structural component of the extracellular matrix that is also a common component of bacterial surfaces. HA is synthesized at the cell surface and typically exists as a high molecular mass polymer greater than 10⁶ Da and composed of repeating disaccharide units of N-acetylglucosamine and glucuronic acid (5, 6). Unlike other glycosaminoglycans such as heparan sulfate or chondroitin sulfates that encode specific activity by use of a diverse disaccharide sequence, HA is not sulfated or epimerized, and only changes in HA size, concentration, and location affect function.We have previously developed murine models of sterile injury to identify the innate elements that recognize and mediate sterile inflammation (7). Our results demonstrated that (a) the initiation of a sterile intrinsic inflammatory process is dependent on TLR4 activation, (b) sterile injury induces HA accumulation at the injured site, and (c) sterile intrinsic inflammation resembles signaling events that are activated by HA. Furthermore, we have defined a novel alternative recognition complex for HA that involves TLR4, MD-2, and CD44 (7). Taken together with other work associating HA and innate pattern recognition (4, 8–10), these observations have provided new insight into mechanisms responsible for sterile inflammation.Recently, the NLR (nucleotide-binding domain and leucine rich repeat-containing) family has been extensively analyzed as a group of intracellular pattern recognition receptors (11). NLRs have a leucine-rich repeat that recognizes pathogen-associated molecular patterns including bacterial cell wall components and viral nucleic acids. NOD2 and NLR family, pyrin containing 3 (NLRP3)/cryopyrin are two of the best characterized NLRs. NOD2 recognizes the bacterial peptidoglycan-derived molecule muramyl dipeptide and activates the NF-κB pathway to induce inflammatory responses (12). Mutations of the NOD2 gene were identified in individuals with chronic inflammatory disorders such as Crohn disease (13, 14) and Blau syndrome (15). Mouse knockin mutants of NOD2, which have the same mutation in NOD2 as human patients with Crohn disease, showed elevated proinflammatory cytokines following muramyl dipeptide challenge or dextran sodium sulfate-induced bowel inflammation (16). NLRP3, also known as cyropyrin, CIAS1, NALP3, PYPAF1, forms an “inflammasome” with ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 to convert pro-IL-1β to active IL-1β (17). Mutations in NLRP3 were identified in individuals with familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease (18–20). These individuals have recurrent or chronic inflammatory symptoms, including fever, arthritis, and a urticaria-like eruption characterized by neutrophilic infiltration. In FCAS, symptoms can be elicited by cold provocation by a mechanism that appears to be mediated through the skin (15, 21).Because disorders associated with mutations in NLRP3 are examples of inflammation under sterile conditions and HA has been shown to be a trigger of sterile inflammation, we sought to further understand the mechanism of the response to HA by examining the role of cryopyrin during injury and after exposure to HA. Our results show that cryopyrin and IL-1β are integral to sterile inflammation and the response to HA. These observations provide new insight into the function of HA as a “danger signal” of injury.     

The Bio3D packages for structural bioinformatics

Bio3D is a family of R packages for the analysis of biomolecular sequence, structure, and dynamics. Major functionality includes biomolecular database searching and retrieval, sequence and structure conservation analysis, ensemble normal mode analysis, protein structure and correlation network analysis, principal component, and related multivariate analysis methods. Here, we review recent package developments, including a new underlying segregation into separate packages for distinct analysis, and introduce a new method for structure analysis named ensemble difference distance matrix analysis (eDDM). The eDDM approach calculates and compares atomic distance matrices across large sets of homologous atomic structures to help identify the residue wise determinants underlying specific functional processes. An eDDM workflow is detailed along with an example application to a large protein family. As a new member of the Bio3D family, the Bio3D‐eddm package supports both experimental and theoretical simulation‐generated structures, is integrated with other methods for dissecting sequence‐structure–function relationships, and can be used in a highly automated and reproducible manner. Bio3D is distributed as an integrated set of platform independent open source R packages available from: http://thegrantlab.org/bio3d/ .     

Xenopus as a model organism in developmental chemical genetic screens

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.