Upper lethal temperatures in three cold-tolerant insects are higher in winter than in summer.

Upper lethal temperatures (ULTs) of cold-adapted insect species in winter have not been previously examined. We anticipated that as the lower lethal temperatures (LLTs) decreased (by 20-30°C) with the onset of winter, the ULTs would also decrease accordingly. Consequently, given the recent increases in winter freeze-thaw cycles and warmer winters due to climate change, it became of interest to determine whether ambient temperatures during thaws were approaching ULTs during the cold seasons. However, beetle Dendroides canadensis (Coleoptera: Pyrochroidae) larvae had higher 24 and 48 h ULT50 (the temperature at which 50% mortality occurred) in winter than in summer. The 24 and 48 h ULT50 for D. canadensis in winter were 40.9 and 38.7°C, respectively. For D. canadensis in summer, the 24 and 48 h ULT50 were 36.7 and 36.4°C. During the transition periods of spring and autumn, the 24 h ULT50 was 37.3 and 38.5°C, respectively. While D. canadensis in winter had a 24 h LT50 range between LLT and ULT of 64°C, the summer range was only 41°C. Additionally, larvae of the beetle Cucujus clavipes clavipes (Coleoptera: Cucujidae) and the cranefly Tipula trivittata (Diptera: Tipulidae) also had higher ULTs in winter than in summer. This unexpected phenomenon of increased temperature survivorship at both lower and higher temperatures in the winter compared with that in the summer has not been previously documented. With the decreased high temperature tolerance as the season progresses from winter to summer, it was observed that environmental temperatures are closest to upper lethal temperatures in spring.

Methyl 4-O-ß-D-xylopyranosyl ß-D-mannopyranoside, a core disaccharide of an antifreeze glycolipid.

Methyl ß-D-xylopyranosyl-(1→4)-ß-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 ß-D-mannopyranosyl-(1→4)-ß-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.

Methyl 4-O-ß-D-mannopyranosyl ß-D-xylopyranoside.

Methyl ß-D-mannopyranosyl-(1→4)-ß-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 ß-Xylp and ß-Manp residues, respectively. By comparison, the internal glycosidic linkage in the major disorder component of the structurally related disaccharide, methyl ß-D-galactopyranosyl-(1→4)-ß-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 ß-Manp residue of (IA) is gauche-gauche, whereas that in the ß-Manp residue of (IB) is gauche-trans.

Group A Streptococcus-Induced Activation of Human Plasminogen Is Required for Keratinocyte Wound Retraction and Rapid Clot Dissolution.

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 ß1 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.

Synthetic Antimicrobial Peptide Tuning Permits Membrane Disruption and Interpeptide Synergy.

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.

Beetle, Dendroides canadensis, antifreeze proteins increased high temperature survivorship in transgenic fruit flies, Drosophila melanogaster.

Paradoxically, some insects have an increased capacity to survive higher temperatures in winter than summer. Possible contributors to this increased heat tolerance in winter could be their sub-zero adaptations (high polyol concentrations, antifreeze proteins, antifreeze glycolipids, etc.). To investigate if a sub-zero adaptation can increase organismal high temperature survivorship, we tested transgenic fruit flies, Drosophila melanogaster, with antifreeze proteins from the fire-colored beetle, Dendroides canadensis (DAFPs). Transgenic Drosophila melanogaster with individual DAFPs-1 and -4 had increased survivorship compared to control flies after 24 h when placed at 35-36.5 °C. The 24 h ULT50 (Upper Lethal Temperature at which 50% mortality occurred) was calculated to be 36.3 °C for DAFP-1 flies, 36.2 °C for DAFP-4 flies, 35.4 °C for wild-type controls, and 34.9 °C for GAL4 controls. The results indicate that DAFPs may have an alternative function in insects and be a contributor in the unexpected phenomenon of increased higher temperature survivorship in winter.