We investigated the ecological physiology and behavior of free-living wood frogs [Lithobates (Rana) sylvaticus] overwintering in Interior Alaska by tracking animals into natural hibernacula, recording microclimate, and determining frog survival in spring. We measured cryoprotectant (glucose) concentrations and identified the presence of antifreeze glycolipids in tissues from subsamples of naturally freezing frogs. We also recorded the behavior of wood frogs preparing to freeze in artificial hibernacula, and tissue glucose concentrations in captive wood frogs frozen in the laboratory to −2.5°C. Wood frogs in natural hibernacula remained frozen for 193±11 consecutive days and experienced average (October-May) temperatures of −6.3°C and average minimum temperatures of -14.6±2.8°C (range −8.9 to −18.1°C) with 100% survival (N=18). Mean glucose concentrations were 13-fold higher in muscle, 10-fold higher in heart and 3.3-fold higher in liver in naturally freezing compared with laboratory frozen frogs. Antifreeze glycolipid was present in extracts from muscle and internal organs, but not skin, of frozen frogs. Wood frogs in Interior Alaska survive freezing to extreme limits and durations compared with those described in animals collected in southern Canada or the Midwestern United States. We hypothesize that this enhancement of freeze tolerance in Alaskan wood frogs is due to higher cryoprotectant levels that are produced by repeated freezing and thawing cycles experienced under natural conditions during early autumn.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.

Methyl β-D-xylopyranosyl-(1→4)-β-D-mannopyranoside, C12H22O10, crystallized as colorless block-like needles from methanol-water solvent. Comparisons to the internal linkage conformations in the two crystallographic forms of the structurally related disaccharide methyl β-D-mannopyranosyl-(1→4)-β-D-xylopyranoside are discussed. Intramolecular inter-residue hydrogen bonding is observed between one mannopyranosyl hydroxy O atom and the ring O atom of the xylopyranosyl residue. Intermolecular hydrogen bonding yields a bilayered two-dimensional sheet of molecules that are located parallel to the bc plane.

Invasive outcomes of Group A Streptococcus (GAS) infections that involve damage to skin and other tissues are initiated when these bacteria colonize and disseminate via an open wound to gain access to blood and deeper tissues. Two critical GAS virulence factors, Plasminogen-Associated M-Protein (PAM) and streptokinase (SK), work in concert to bind and activate host human plasminogen (hPg) in order to create a localized proteolytic environment that alters wound-site architecture. Using a wound scratch assay with immortalized epithelial cells, real-time live imaging (RTLI) was used to examine dynamic effects of hPg activation by a PAM-containing skin-trophic GAS isolate (AP53R+S−) during the course of infection. RTLI of these wound models revealed that retraction of the epithelial wound required both GAS and hPg. Isogenic AP53R+S− mutants lacking SK or PAM highly attenuated the time course of retraction of the keratinocyte wound. We also found that relocalization of integrin β1 from the membrane to the cytoplasm occurred during the wound retraction event. We devised a combined in situ-based cellular model of fibrin clot-in epithelial wound to visualize the progress of GAS pathogenesis by RTLI. Our findings showed GAS AP53R+S− hierarchically dissolved the fibrin clot prior to the retraction of keratinocyte monolayers at the leading edge of the wound. Overall, our studies reveal that localized activation of hPg by AP53R+S−via SK and PAM during infection plays a critical role in dissemination of bacteria at the wound site through both rapid dissolution of the fibrin clot and retraction of the keratinocyte wound layer.

Methyl β-D-mannopyranosyl-(1→4)-β-D-xylopyranoside, C(12)H(22)O(10), (I), crystallizes as colorless needles from water, with two crystallographically independent molecules, (IA) and (IB), comprising the asymmetric unit. The internal glycosidic linkage conformation in molecule (IA) is characterized by a ϕ' torsion angle (O5'(Man)-C1'(Man)-O1'(Man)-C4(Xyl); Man is mannose and Xyl is xylose) of -88.38 (17)° and a ψ' torsion angle (C1'(Man)-O1'(Man)-C4(Xyl)-C5(Xyl)) of -149.22 (15)°, whereas the corresponding torsion angles in molecule (IB) are -89.82 (17) and -159.98 (14)°, respectively. Ring atom numbering conforms to the convention in which C1 denotes the anomeric C atom, and C5 and C6 denote the hydroxymethyl (-CH(2)OH) C atom in the β-Xylp and β-Manp residues, respectively. By comparison, the internal glycosidic linkage in the major disorder component of the structurally related disaccharide, methyl β-D-galactopyranosyl-(1→4)-β-D-xylopyranoside), (II) [Zhang, Oliver & Serriani (2012). Acta Cryst. C68, o7-o11], is characterized by ϕ' = -85.7 (6)° and ψ' = -141.6 (8)°. Inter-residue hydrogen bonding is observed between atoms O3(Xyl) and O5'(Man) in both (IA) and (IB) [O3(Xyl)...O5'(Man) internuclear distances = 2.7268 (16) and 2.6920 (17) Å, respectively], analogous to the inter-residue hydrogen bond detected between atoms O3(Xyl) and O5'(Gal) in (II). Exocyclic hydroxymethyl group conformation in the β-Manp residue of (IA) is gauche-gauche, whereas that in the β-Manp residue of (IB) is gauche-trans.