Equilibrium shapes of erythrocytes in rouleau formation

A well known physiological property of erythrocytes is that they can aggregate and form a rouleau. We present a theoretical analysis of erythrocyte shapes in a long rouleau composed of cells with identical sizes. The study is based on the area difference elasticity model of lipid membranes, and takes into consideration the adhesion of curved axisymmetric membranes. The analysis predicts that the erythrocytes in the rouleau can have either a discoid or a cup-like shape. These shapes are analogous to the discoid and stomatocyte shapes of free erythrocytes. The transitions between the discoid and cup-like shapes in the rouleau are characterized. The occurrence of these transitions depends on three model parameters: the cell relative volume, the preferred difference between the areas of the membrane bilayer leaflets, and the strength of the adhesion between the membranes. The cup-like shapes are favored at small relative volumes and small preferred area differences, and the discoid shapes are favored at large values of these parameters. Increased adhesion strength enlarges the contact area between the cells, flattens the cells, and consequently promotes the discoid shapes.     

Anatomical variations of the cystic artery

Thorough knowledge about the origin of the cystic artery is surgically important, especially when intraoperative or post-operative bleeding occurs in the gallbladder fossa. The arterial supply of the gallbladder was studied in 81 livers. The gallbladder was supplied by one cystic artery in 86% and by two arteries in 14% of cases. When a single artery was present, it originated from the right hepatic artery in 53% of livers. Other origins included the anterior or the posterior sectional hepatic artery, the replacing right hepatic artery, and in 5% of cases, segmental arteries for segments 4, 5, 6 and 8. When two cystic arteries supplied the gallbladder, both most commonly originated from the right hepatic artery (7% incidence). In 1% of cases, a subsegmental branch for segment 6 and a subsegmental branch for segment 5 respectively, originated from the cystic artery.     

TRAF2 recruitment via T61 in CD30 drives NFκB activation and enhances hESC survival and proliferation

CD30 (TNFRSF8), a tumor necrosis factor receptor family protein, and CD30 variant (CD30v), a ligand-independent form encoding only the cytoplasmic signaling domain, are concurrently overexpressed in transformed human embryonic stem cells (hESCs) or hESCs cultured in the presence of ascorbate. CD30 and CD30v are believed to increase hESC survival and proliferation through NFκB activation, but how this occurs is largely unknown. Here we demonstrate that hESCs that endogenously express CD30v and hESCs that artificially overexpress CD30v exhibit increased ERK phosphorylation levels, activation of the canonical NFκB pathway, down-regulation of the noncanonical NFκB pathway, and reduced expression of the full-length CD30 protein. We further find that CD30v, surprisingly, resides predominantly in the nucleus of hESC. We demonstrate that alanine substitution of a single threonine residue at position 61 (T61) in CD30v abrogates CD30v-mediated NFκB activation, CD30v-mediated resistance to apoptosis, and CD30v-enhanced proliferation, as well as restores normal G₂/M-checkpoint arrest upon H₂O₂ treatment while maintaining its unexpected subcellular distribution. Using an affinity purification strategy and LC-MS, we identified TRAF2 as the predominant protein that interacts with WT CD30v but not the T61A-mutant form in hESCs. The identification of Thr-61 as a critical residue for TRAF2 recruitment and canonical NFκB signaling by CD30v reveals the substantial contribution that this molecule makes to overall NFκB activity, cell cycle changes, and survival in hESCs.     

Structural and Biochemical Analysis of a Single Amino-Acid Mutant of WzzBSF That Alters Lipopolysaccharide O-Antigen Chain Length in Shigella flexneri

Lipopolysaccharide (LPS), a surface polymer of Gram-negative bacteria, helps bacteria survive in different environments and acts as a virulence determinant of host infection. The O-antigen (Oag) component of LPS exhibits a modal chain-length distribution that is controlled by polysaccharide co-polymerases (PCPs). The molecular basis of the regulation of Oag chain-lengths remains unclear, despite extensive mutagenesis and structural studies of PCPs from Escherichia coli and Shigella. Here, we identified a single mutation (A107P) of the Shigella flexneri WzzB_SF, by a random mutagenesis approach, that causes a shortened Oag chain-length distribution in bacteria. We determined the crystal structures of the periplasmic domains of wild-type WzzB_SF and the A107P mutant. Both structures form a highly similar open trimeric assembly in the crystals, and show a similar tendency to self-associate in solution. Binding studies by bio-layer interferometry reveal cooperative binding of very short (VS)-core-plus-O-antigen polysaccharide (COPS) to the periplasmic domains of both proteins, but with decreased affinity for the A107P mutant. Our studies reveal that subtle and localized structural differences in PCPs can have dramatic effects on LPS chain-length distribution in bacteria, for example by altering the affinity for the substrate, which supports the role of the structure of the growing Oag polymer in this process.     

The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs

Innate immune signaling by Toll-like receptors (TLRs) involves receptor phosphorylation, which helps to shape and drive key inflammatory outputs, yet our understanding of the kinases and mechanisms that mediate TLR phosphorylation is incomplete. Spleen tyrosine kinase (Syk) is a nonreceptor protein tyrosine kinase, which is known to relay adaptive and innate immune signaling, including from TLRs. However, TLRs do not contain the conserved dual immunoreceptor tyrosine-based activation motifs that typically recruit Syk to many other receptors. One possibility is that the Syk-TLR association is indirect, relying on an intermediary scaffolding protein. We previously identified a role for the palmitoylated transmembrane adapter protein SCIMP in scaffolding the Src tyrosine kinase Lyn, for TLR phosphorylation, but the role of SCIMP in mediating the interaction between Syk and TLRs has not yet been investigated. Here, we show that SCIMP recruits Syk in response to lipopolysaccharide-mediated TLR4 activation. We also show that Syk contributes to the phosphorylation of SCIMP and TLR4 to enhance their binding. Further evidence pinpoints two specific phosphorylation sites in SCIMP critical for its interaction with Syk-SH2 domains in the absence of immunoreceptor tyrosine-based activation motifs. Finally, using inhibitors and primary macrophages from SCIMP^-/- mice, we confirm a functional role for SCIMP-mediated Syk interaction in modulating TLR4 phosphorylation, signaling, and cytokine outputs. In conclusion, we identify SCIMP as a novel, immune-specific Syk scaffold, which can contribute to inflammation through selective TLR-driven inflammatory responses.     

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.     

Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models

The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato‐labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non‐perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.     

Prevalence of mandibular osteomyelitis in roe deer (Capreolus capreolus) in Slovenia

Mandibular osteomyelitis in free-ranging cervids is a rare, but eventually fatal, disease. We examined 41,895 defleshed mandibles of roe deer collected throughout Slovenia in 2007. Mandibles from 14,679 fawns had no signs of osteomyelitis, and were excluded from further analysis. Of the remaining 27,216 specimens, chronic osteomyelitis ("lumpy jaw") was found in 113 mandibles (4.2%; 7.0% of adults). The majority of cases were observed from the Mediterranean and subalpine regions, near larger cities and thermal power plants. There was no statistically significant correlation between severity of the mandibular osteomyelitis and body weight. Females were more frequently affected than males. Coarse and abrasive food, and to some extent dental fluorosis, are the most probable triggers for development of lesions.     

Ligand-induced conformational changes within a hexameric Acyl-CoA thioesterase

Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyze coenzyme A from acyl-CoA esters to mediate a range of cellular functions including β-oxidation, lipid biosynthesis, and signal transduction. Here, we present the crystal structure of a hexameric hot-dog domain-containing acyl-CoA thioesterase from Bacillus halodurans in the apo-form and provide structural and comparative analyses to the coenzyme A-bound form to identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and the trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% and decrease to about half the size in the ligand-bound state. We also assessed the specificity of the enzyme against a range of fatty acyl-CoA substrates and have identified a preference for short-chain fatty acyl-CoAs. Coenzyme A was shown both to negatively regulate enzyme activity, representing a direct inhibitory feedback, and consistent with the structural data, to destabilize the quaternary structure of the enzyme. Coenzyme A-induced conformational changes in the C-terminal helices of enzyme were assessed through mutational analysis and shown to play a role in regulating enzyme activity. The conformational changes are likely to be conserved from bacteria through to humans and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.