Lake Fryxell is a perennially ice-covered lake in the McMurdo Dry Valleys, Antarctica, with a sharp oxycline in a water column that is density stabilized by a gradient in salt concentration. Dissolved oxygen falls from 20 mg liter ؊1 to undetectable over one vertical meter from 8.9-to 9.9-m depth. We provide the first description of the benthic mat community that falls within this oxygen gradient on the sloping floor of the lake, using a combination of micro-and macroscopic morphological descriptions, pigment analysis, and 16S rRNA gene bacterial community analysis. Our work focused on three macroscopic mat morphologies that were associated with different parts of the oxygen gradient: (i) "cuspate pinnacles" in the upper hyperoxic zone, which displayed complex topography and were dominated by phycoerythrin-rich cyanobacteria attributable to the genus Leptolyngbya and a diverse but sparse assemblage of pennate diatoms; (ii) a less topographically complex "ridge-pit" mat located immediately above the oxic-anoxic transition containing Leptolyngbya and an increasing abundance of diatoms; and (iii) flat prostrate mats in the upper anoxic zone, dominated by a green cyanobacterium phylogenetically identified as Phormidium pseudopriestleyi and a single diatom, Diadesmis contenta. Zonation of bacteria was by lake depth and by depth into individual mats. Deeper mats had higher abundances of bacteriochlorophylls and anoxygenic phototrophs, including Chlorobi and Chloroflexi. This suggests that microbial communities form assemblages specific to niche-like locations. Mat morphologies, underpinned by cyanobacterial and diatom composition, are the result of local habitat conditions likely defined by irradiance and oxygen and sulfide concentrations.

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Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin-depolymerizing factor homology (ADF-H) domains. ADF-H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best-characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF-H domain of human HIP-55-drebrin-like protein, the first published structure of a drebrin-like domain (mammalian), and the first published structure of GMF b (mouse). We also determined the structures of mouse GMF c, the mouse coactosin-like domain and the C-terminal ADF-H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F-actin) and globular actin (G-actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F-actin binding sites of mouse GMF b and GMF c contain two additional b-strands not seen in other ADF-H structures. The G-actin binding site of the ADF-H domain of human HIP-55-drebrin-like protein is absent and distorted in mouse GMF b and GMF c.

Hydrogels formed via free radical-mediated thiol-ene step-growth photopolymerization have been developed for a broad range of tissue engineering and regenerative medicine applications. While the crosslinking mechanism of thiol-ene hydrogels has been well-described, there has been only limited work exploring the physical differences among gels arising from variations in crosslinker properties. Here, we show that the character of linear polyethylene glycol (PEG) dithiols used to crosslink multi-arm polyethylene glycol norbornene (PEGNB) can be used as a facile strategy to tune hydrogel formation kinetics, and therefore the equilibrium hydrogel network architecture. Specifically, we report the dramatic effect of crosslinker length on PEGNB hydrogel formation kinetics and the formed hydrogel properties. It is shown that the hydrogel formation kinetics and formed hydrogel properties can be tuned by solely varying the crosslinker length. It was hypothesized that under identical reaction conditions, a more accessible 3.5 k PEG dithiol crosslinker would improve network ideality relative to a shorter 1.5 k crosslinker. Longer linkers consequently promote significantly more rapid macromer crosslinking and therefore gelation. Accelerated gel formation satisfies an urgent unmet need for rapid polymerization in droplet microfluidics. Using long linkers, we demonstrate the ability to photopolymerize PEGNB microgels under flow on a microfluidic chip, with reliable control over microgel size and shape in a high-throughput manner. To further validate the potential of this platform to produce novel, microstructured cell carrier vehicles, 3T3 fibroblasts were successfully encapsulated and cultured over 14 days with excellent cell viability. This study demonstrates that PEGNB hydrogel dynamics could be readily customized to fulfill a variety of needs in tissue engineering, controlled cell delivery, or drug release applications.

The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMPbound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.Glucose metabolism is central to the biochemistry of all living systems with the enzymatic phosphorylation of glucose playing a key role in cellular energy metabolism by ensuring that this energy-rich substrate is available to the cell. A relatively recently discovered ADP-dependent glucokinase (ADPGK) 3 (EC 2.7.1.147) catalyzes the phosphorylation of D-glucose to glucose-6-phosphate using MgADP as phosphoryl donor in contrast to the more typical ATP-utilizing hexokinases and glucokinases. ADPGK was first identified in Archaea, being involved in a modified Embden-Meyerhof glycolytic pathway (1), in which Archaea can also use an ADP-dependent phosphofructokinase (ADPPFK; EC 2.7.1.146). Bioinformatic analysis led to the identification of ADPGK in metazoa and the subsequent cloning and initial characterization of mammalian ADPGKs (2, 3). Mammalian ADPGKs show modest sequence similarity to archaeal orthologues (ϳ20% amino acid identity). ADPGK is highly expressed in a wide variety of both normal and tumor mammalian tissues (3) and has been found to be localized to the endoplasmic reticulum in T cells (4) consistent with the sequence-based annotation of an N-terminal signal peptide. Furthermore, ADPGK has been identified as a cholesterol binding protein in a proteomics screen (5). Despite the role of archaeal ADPGK in glycolysis, overexpression of ADPGK in H460 and HC116 human tumor cells showed no cell proliferative or glycolytic effects (3). ADPGK knock-out in t...