PARP inhibition impedes the maturation of nascent DNA strands during DNA replication

Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1−/− cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds

Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

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The enzymatic de-epithelialization technique determines denuded amniotic membrane integrity and viability of harvested epithelial cells.

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture

Bordetella Adenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells

Macrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agent Bordetella pertussis. The adenylate cyclase toxin (CyaA) mediates immune evasion of B. pertussis by suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells. Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producing B. pertussis bacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression on in vitro differentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiated in vitro. The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens

Reply to the Letter to the Editor by Paul O. Sheridan, Sylvia H. Duncan, Alan W. Walker, Karen P. Scott, Petra Louis and Harry J. Flint, referring to our paper ‘Reclassification of Eubacterium rectale (Prévot et al. 1967) in a new genus Agathobacter gen. nov., as Agathobacter rectalis comb. nov., within the family Lachnospiraceae, and description of Agathobacter ruminis sp. nov., from the rumen’, Int J Syst Evol Microbiol, DOI 10.1099/ijsem.0.000788

No abstract available

Reply to the Letter to the Editor by Paul O. Sheridan, Sylvia H. Duncan, Alan W. Walker, Karen P. Scott, Petra Louis and Harry J. Flint, referring to our paper ‘Reclassification of Eubacterium rectale (Prévot et al. 1967) in a new genus Agathobacter gen. nov., as Agathobacter rectalis comb. nov., within the family Lachnospiraceae, and description of Agathobacter ruminis sp. nov., from the rumen’, Int J Syst Evol Microbiol, DOI 10.1099/ijsem.0.000788

No abstract available

KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy