Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis

Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error. Furthermore, considerable disagreement and speculation exists on which specific damage events underlie observed miscoding lesions. The root of the problem is that it has previously been difficult to assemble sufficient data to test the hypotheses, and near-impossible to accurately determine the specific strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin, and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390 965 bp of modern chloroplast and 131 474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved specimen, Type 2 (cytosine→thymine/guanine→adenine) miscoding lesions represent the overwhelming majority of damage-derived miscoding lesions. Additionally, we show that an as yet unidentified guanine→adenine analogue modification, not the conventionally argued cytosine→uracil deamination, underpins a significant proportion of Type 2 damage. How widespread these implications are for aDNA will become apparent as future studies analyse data recovered from a wider range of substrates.     

Small molecule ago-allosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor

Following our previous publication describing the biological profiles, we herein describe the structure-activity relationships of a core set of quinoxalines as the hGLP-1 receptor agonists. The most potent and efficacious compounds are 6,7-dichloroquinoxalines bearing an alkyl sulfonyl group at the C-2 position and a secondary alkyl amino group at the C-3 position. These findings serve as a valuable starting point for the discovery of more drug-like small molecule agonists for the hGLP-1 receptor.     

Novel tetrahydroisoquinolines are histamine H3 antagonists and serotonin reuptake inhibitors

A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.     

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.     

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.     

Phylogenetic analysis of freshwater sponges provide evidence for endemism and radiation in ancient lakes

Morphologic and phylogenetic analysis of freshwater sponges endemic to lakes in Central Sulawesi, Siberia and South-East Europe is presented. We also analyzed several cosmopolitan sponge species from Eurasia and North America and included sponge sequences from public databases. In agreement with previous reports [Addis, J.S., Peterson, K.J., 2005. Phylogenetic relationships of freshwater sponges (Porifera, Spongillina) inferred from analyses of 18S rDNA, COI mtDNA, and ITS2 rDNA sequences. Zool. Scr. 34, 549-557], the metaniid sponge Corvomeyenia sp. was the most deeply branching species within a monophyletic lineage of the suborder Spongillina. Pachydictyum globosum (Malawispongiidae) and Nudospongilla vasta (Spongillidae), two morphologically quite distinct species from Sulawesi were found in a joint clade with Trochospongilla (Spongillidae) rendering Trochospongilla paraphyletic. Furthermore, Ochridaspongia sp., another Malawispongiidae, clustered far away from that clade, together with Ephydatia fluviatilis, making the latter family polyphyletic. The Lubomirskiidae endemic to Lake Baikal, Lubomirskia abietina, Baikalospongia bacillifera, B. intermedia, and Swartschewskia papyracea formed a well-supported clade that was most closely linked to the genus Ephydatia (99.9% identity over a total length of 2169 concatenated nucleotide positions). Our study indicates the frequent and independent origin of sponge species endemic to different freshwater ecosystems from a few cosmopolitan founder species. The highly specific primer sets newly developed here facilitate work on the molecular phylogeny and DNA barcoding of sponges.     

Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO(3)(-) exchanger AE1

Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl^– removal, and energy depletion activate Ca²⁺-permeable cation channels with Ca²⁺-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1^–/– mice) than in their wild-type littermates (AE1^+/+ mice) despite increased percentages of reticulocytes (AE1^–/–: 49%, AE1^+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1^–/–erythrocytes/reticulocytes (10%) than in AE1^+/+ erythrocytes (1%). Osmotic shock (addition of 400 mM sucrose), Cl^– removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1^–/– erythrocytes/reticulocytes than in AE1^+/+ erythrocytes. The increase of annexin binding following exposure to the Ca²⁺ ionophore ionomycin (1 µM) was, however, similar in AE1^–/– and in AE1^+/+ erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca²⁺ permeability in AE1^–/– erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1^–/– mice than in AE1^+/+ mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca²⁺ entry and subsequent scrambling of cell membrane phospholipids. annexin; cell volume; osmolarity; phosphatidylserine; energy depletion     

Toxoplasma gondii inhibits Fas/CD95-triggered cell death by inducing aberrant processing and degradation of caspase 8

Ligation of the death receptor Fas/CD95 activates an apoptotic cascade and plays critical roles during infectious diseases. Previous work has established that infection with the intracellular parasite Toxoplasma gondii renders cells resistant to multiple inducers of apoptosis. However, the effect of T. gondii on the death receptor pathway is poorly characterized. Here we have determined the impact of the parasite on apoptosis in type I cells that transduce Fas/CD95 engagement via the death receptor pathway without the need of a mitochondrial amplification loop. The results have shown that T. gondii significantly reduced Fas/CD95-triggered apoptosis by impairing activation of the initiator caspase 8. Parasitic infection diminished the cellular amount of procaspase 8, resulting in its decreased recruitment to the death-inducing signalling complex and the impaired activation of effector caspases. Remarkably, downregulation of caspase 8 protein in T. gondii-infected cells also occurred in the absence of Fas/CD95 engagement and was associated with the appearance of non-canonical caspase 8 cleavage fragments. Distinct parasite proteins were associated with caspase 8 and its proteolytic fragments. These findings indicate that T. gondii aberrantly processes and finally degrades the initiator caspase 8, thereby, blocking Fas/CD95-mediated apoptosis which signals independently of the apoptogenic function of host cell mitochondria.     

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.