Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation

Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.     

Biophysical Investigation of the Membrane-Disrupting Mechanism of the Antimicrobial and Amyloid-Like Peptide Dermaseptin S9

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with ²H- and ³¹P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors.     

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.     

Adverse responses of Cabera pusaria caterpillars to high dietary manganese concentration

The adaptation of insects to environmental changes can constitute a crucial factor in their development and activity. The response of Cabera pusaria L. (Lepidoptera: Geometridae) caterpillars to high manganese (Mn) concentrations in the diet was studied. Birch leaves were treated by dipping in MnCl₂·4H₂O solutions, thereby achieving Mn contents of 370 (T0), 695 (T1), 3 198 (T2), and 6 302 mg kg⁻¹ (T3). The reactions were determined by observing caterpillar mortality, development time, food consumption, and pupal weight. Manganese concentrations in larval excrement, pupae, and food were determined. Manganese in the diet at unnaturally increased concentrations caused great stress for caterpillars. All individuals in the treatment with the highest Mn concentration (T3) died during rearing and successful pupation occurred in only four individuals in T2. Even in the case of caterpillars from T1 (twofold higher than T0) a negative reaction (increased food consumption and prolonged development) was recorded. We also determined significantly increased Mn concentration in pupae from T1 (T2 and T3 were not included in this evaluation due to mortality) and excrement (T1‒T3) compared with T0 having a natural Mn concentration. Caterpillars were seen to eliminate negatively acting dietary Mn by its translocation to excrement. However, the highest mortality rate in T2 and T3 and negative reactions of individuals in T1 very likely demonstrate energy insufficiency and the high energy requirements of Mn elimination mechanisms.     

Errors in egg-laying by female Common Cuckoo Cuculus canorus in nests of its common host

Dozens of studies have documented that brood parasites are well adapted to a brood parasitic lifestyle but not all parasitism events are successful. Co-evolution between brood parasites and their hosts is a dynamic process so it is reasonable to expect that a female brood parasite may commit errors during egg deposition by laying her eggs outside the laying period of the host, with consequent impacts on her fitness. Using an extensive dataset from a long-term study, we evaluated egg-laying patterns and errors related to the timing of egg-laying in the Common Cuckoo Cuculus canorus (hereafter ‘Cuckoo’). Specifically, we tested whether the Cuckoo avoids laying before or on the day of host clutch initiation to reduce the risk of rejection of parasitic eggs, whether laying errors will be more frequent in periods with a lack of active host nests, and whether the laying errors will be more frequent in periods with intense Cuckoo parasitism and a consequent lack of suitable host nests. We found that about one-third of Cuckoo eggs were laid on the host clutch initiation day or 1 day before, and the percentage of Cuckoo eggs laid decreased thereafter. Surprisingly, the probability of Cuckoo egg acceptance by the hosts was not affected by the egg-laying stage of the host clutch. Errors in the timing of egg-laying with fatal consequences (i.e. those precluding Cuckoo hatching because of laying in incubated or deserted clutches) were recorded in about 5% of cases. Only laying date of a Cuckoo egg had a significant effect on the probability of errors, which increased during the breeding season. This may be related to the higher number of deserted and incubated host nests at the site at the end of the breeding season. Errors in egg-laying may be attributed to young and inexperienced females but also impaired body condition or intraspecific competition may cause this behaviour. Future studies, which will test these possible explanations, will help to understand better the mechanism of co-evolutionary arms races and differences between host specialist and generalist brood parasites in various host–parasite systems.     

Human immune response against salivary antigens of Simulium damnosum s.l.: A new epidemiological marker for exposure to blackfly bites in onchocerciasis endemic areas

BackgroundSimulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated.Methodology/Principal findingsBlood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC–MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC–MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase.Conclusions/SignificanceThis study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.     

Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5–188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560 kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70 kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610 kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. In vitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.