Drug treatment in the development of mismatch repair defective acute leukemia and myelodysplastic syndrome

DNA from therapy-related acute leukemia/myelodysplastic syndrome cases (tAL/MDS) from the GIMEMA [Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto] Archive was examined for the microsatellite instability (MSI(+)) phenotype that is diagnostic for defective DNA mismatch repair. More than 60% (16/25) of tAL/MDS cases were MSI(+) in contrast to <4% (0/28) of de novo cases. hMLH1 gene silencing was rare and evidence of promoter methylation was found in less than one-third of the MSI(+) cases. Among the GIMEMA patients who had been treated for breast cancer there was an apparent trend towards early onset primary breast disease. This suggests that there might be common predisposing factors for breast cancer and tAL/MDS. There were also three examples of mutations in the MRE11 gene among the 25 tAL/MDS cases suggesting that defective recombinational DNA repair may promote the development of secondary malignancy. MSI(+) tAL/MDS was significantly associated with previous chemotherapy and the frequency of MSI(+) among radiotherapy patients was considerably lower. In view of the established relationship between drug resistance and mismatch repair defects, we suggest that selection for therapeutic drug resistance may contribute to the incidence of MSI(+) tAL/MDS.     

The 37 kDa/67 kDa laminin receptor is required for PrP(Sc) propagation in scrapie-infected neuronal cells

The accumulation of PrP^Sc in scrapie-infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA-expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti-LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrP^Sc in these cells. The treatments also reduced PrP^c levels. The anti-LRP/LR antibody, W3, abolished PrP^Sc accumulation and reduced PrP^c levels after seven days of incubation. Cells remained free of PrP^Sc after being cultured for 14 additional days without the antibody, whereas the PrP^c level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrP^Sc propagation in cultured cells and suggest that LRP/LR-specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.     

Inter-familial relationships of the shorebirds (Aves: Charadriiformes) based on nuclear DNA sequence data

Background  

Phylogenetic hypotheses of higher-level relationships in the order Charadriiformes based on morphological data, partly disagree with those based on DNA-DNA hybridisation data. So far, these relationships have not been tested by analysis of DNA sequence data. Herein we utilize 1692 bp of aligned, nuclear DNA sequences obtained from 23 charadriiform species, representing 15 families. We also test earlier suggestions that bustards and sandgrouses may be nested with the charadriiforms. The data is analysed with methods based on the parsimony and maximum-likelihood criteria.     

Diurnal and circadian rhythm in compound eye of cricket (Gryllus bimaculatus): changes in structure and photon capture efficiency

Day-night changes in rhabdom size of compound eyes were investigated in three groups of crickets (Gryllus bimaculatus): nymphs and adult males and females. In both adults and nymphs, the rhabdoms were larger at night than during a day. In adults, the mean rhabdom occupation ratios (RORs) of ommatidial retinulae at midnight were about two times greater than the values at midday. This change contributes to control of the photon capture efficiency (PCE) of the eye according to photic environment. The RORs of adult males at midnight were higher than those of both adult females and nymphs. This suggests that the PCE of the compound eye of adult males is the greatest of all groups. Under constant darkness, day-night changes in ROR were detected only in adult males, but neither in adult females nor in nymphs. On the other hand, no day-night changes were detected in any experimental group under constant light. These results suggest that the change in rhabdom size between day and night is an adaptation to the photic environment that is controlled mainly by the light-dark (day-night) cycle. However, the change in male adults is induced by an endogenous circadian clock.     

Survival and energy metabolism in an oxygen deficient environment. Field and laboratory studies on the bottom fauna from the profundal zone of Lake Esrom,Denmark

The three macroinvertebrate taxa, Potamothrix hammoniensis, Chironomus anthracinus and Pisidium spp. are permanent inhabitants of the regularly microxic/anoxic profundal zone in Lake Esrom. In situ and laboratory studies (10 °C) of metabolism (aerobic and anaerobic) and anaerobic survival in P. hammoniensis and Pisidium spp. are compared with previous results from C. anthracinus. The late summer microxic conditions in the lake lasts 2–2 months, during which the three taxa display metabolic and behavioral strategies in order to survive. All three are respiratory oxy-regulators with critical oxygen levels at 1 mg O₂ l^–1 (P. hammoniensis and Pisidium spp.) or 2–3 mg O₂ l^–1 (C. anthracinus). The lethal time (LD₅₀) in experimental anoxia follows a similar trend, with 150–170 days of survival in P. hammoniensis and Pisidium spp., compared to 2–5 weeks in C. anthracinus. The glycogen stores are almost (C. anthracinus) or fully exploited (P. hammoniensis and Pisidium spp.) during anaerobis and the animals finally enter a state of quiescence or dormancy. During the late phase of anoxia, their metabolism is down at (C. anthracinus) or below (P. hammoniensis and Pisidium spp.) 1% of normoxic metabolism. The populations in the lake behave rather similar in so far that the energy gain from anaerobic degradation of glycogen maximizes 1% of normoxic conditions regardless of species. Also, in Pisidium this appears to be the only energy source during dormancy. However, as previously presented in case of C. anthracinus, P. hammoniensis maintain a partly aerobic metabolism constituting 44% of normoxia during the microxic period, compared to the 12–19% obtained by C. anthracinus. It is thus demonstrated that P. hammoniensis and Pisidium spp. possess a remarkable ability to survive in situ severe oxygen depletion. P. hammoniensis can benefit from the presence of merely traces of oxygen, whereas C. anthracinus with poorer anaerobic survival is strongly dependent on minute oxygen supplies.     

Flax (Linum usitatissimum L.) depends on arbuscular mycorrhizal fungi for growth and P uptake at intermediate but not high soil P levels in the field

The contribution of indigenous arbuscular mycorrhizal fungi (AMF) to growth and phosphorus (P) uptake by oilseed flax (Linum usitatissimum L.) was examined in two field experiments covering soil P levels from 20–86 mg kg-1 NaHCO3-extractable P. The fumigant dazomet was applied to the soil in half of the plots to obtain control plants with reduced mycorrhiza formation. An extensive AMF colonization of up to 48% of the root length was established in untreated soil of both experiments, although P fertilization reduced colonization to 28–39% at the latest harvests. Fumigation markedly decreased or totally prevented AMF colonization throughout the experiments. Root growth responded to fumigation by increased total and specific root length. Shoot P uptake was decreased by fumigation at soil P levels lower than ca. 50 mg kg-1 whereas shoot growth was reduced by fumigation at soil P levels lower than ca. 40 mg kg-1. The effects of fumigation were ascribed to the suppression of mycorrhiza formation. The effect of the AMF increased with decreasing soil P levels. Phosphorus inflow through roots (based on shoot P uptake) was reduced more strongly by fumigation than total P uptake. The P inflow through fungal tissue in roots was estimated to 4 × 10-14 mol P cm-1 s-1. We conclude that AMF are essential to flax growth at soil P levels below ca. 40 mg P kg-1, which is representative of the conditions under which most flax is grown.     

The Arabidopsis Athb-8, -9 and genes are members of a small gene family coding for highly related HD-ZIP proteins

We report the isolation and characterization of two Arabidopsis homeobox genes highly related to the Athb-8 gene. The full-length cDNAs encode proteins of 841 and 852 amino acids which we have designated Athb-9 and -14, respectively. Athb-8, -9 and -14 are members of a small family of HD-Zip proteins (HD-ZIP III) characterized by a HD-Zip motif confined to the N-terminus of the polypeptide. The spatial organization of the HD-Zip domain of Athb-8, -9 and -14 is different from that of the Athb-1 (a member of the HD-ZIP I family) and Athb-2 (a member of the HD-ZIP II family) HD-Zip domains. DNA binding analysis performed with random-sequence DNA templates showed that the Athb-9 HD-Zip (HD-Zip-9) domain, but not the Athb-9 HD alone, binds to DNA. The HD-Zip-9 domain recognizes a 11 bp pseudopalindromic sequence (GTAAT(G/C)ATTAC), as determined by selecting high-affinity binding sites from random-sequence DNA. Moreover, gel retardation assays demonstrated that the HD-Zip-9 domain binds to DNA as a dimer. These data support the notion that the HD-ZIP III domain interacts with DNA recognition elements in a fashion similar to the HD-ZIP I and II domains.     

Adaptive Potential of Hybridization among Malaria Vectors: Introgression at the Immune Locus TEP1 between Anopheles coluzzii and A. gambiae in ‘Far-West’ Africa

“Far-West” Africa is known to be a secondary contact zone between the two major malaria vectors Anopheles coluzzii and A. gambiae. We investigated gene-flow and potentially adaptive introgression between these species along a west-to-east transect in Guinea Bissau, the putative core of this hybrid zone. To evaluate the extent and direction of gene flow, we genotyped site 702 in Intron-1 of the para Voltage-Gated Sodium Channel gene, a species-diagnostic nucleotide position throughout most of A. coluzzii and A. gambiae sympatric range. We also analyzed polymorphism in the thioester-binding domain (TED) of the innate immunity-linked thioester-containing protein 1 (TEP1) to investigate whether elevated hybridization might facilitate the exchange of variants linked to adaptive immunity and Plasmodium refractoriness. Our results confirm asymmetric introgression of genetic material from A. coluzzii to A. gambiae and disruption of linkage between the centromeric "genomic islands" of inter-specific divergence. We report that A. gambiae from the Guinean hybrid zone possesses an introgressed TEP1 resistant allelic class, found exclusively in A. coluzzii elsewhere and apparently swept to fixation in West Africa (i.e. Mali and Burkina Faso). However, no detectable fixation of this allele was found in Guinea Bissau, which may suggest that ecological pressures driving segregation between the two species in larval habitats in this region may be different from those experienced in northern and more arid parts of the species’ range. Finally, our results also suggest a genetic subdivision between coastal and inland A. gambiae Guinean populations and provide clues on the importance of ecological factors in intra-specific differentiation processes.     

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8⁺ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8⁺ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8⁺ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8⁺ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8⁺ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.