Environmental exposure, endogenous metabolism and cancer chemotherapy can give rise to alkylation of DNA, and the resulting alkylated thymidine (alkyldT) lesions were found to be poorly repaired and persistent in mammalian tissues. Unrepaired DNA lesions may compromise genomic integrity by inhibiting DNA replication and inducing mutations in these processes. In this study, we explored how eight O4-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, (R)-sBu and (S)-sBu, are recognized by DNA replication machinery in HEK293T human embryonic kidney cells. We found that the O4-alkyldT lesions are moderately blocking to DNA replication, with the bypass efficiencies ranging from 20 to 33% in HEK293T cells, and these lesions induced substantial frequencies T→C transition mutation. We also conducted the replication experiments in the isogenic cells where individual translesion synthesis (TLS) DNA polymerases were depleted by the CRISPR/Cas9 genome editing method. Our results showed that deficiency in Pol η or Pol ζ, but not Pol κ or Pol ι, led to pronounced drops in bypass efficiencies for all the O4-alkyldT lesions except O4-MedT. In addition, depletion of Pol ζ resulted in significant decreases in T→C mutation frequencies for all the O4-alkyldT lesions except O4-MedT and O4-nBudT. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O4-alkyldT lesions and defined the roles of TLS polymerases in bypassing these lesions in human cells.

Mangrove ecosystems are in serious decline around the world and various initiatives are underway to assess their current coverage and loss in cover. These ecosystems occur as thin strips along coastlines or rivers and, due to the strong environmental gradients present, mangroves show high spatial variability along short transects. Remote sensing tools that offer high spatial resolution mapping and high information content are needed to provide good differentiation of the various mangrove zones and types. The added complexities of tropical atmospheric conditions provide further challenges in terms of the selection of sensors and image analysis methodologies. This paper explores the possibility of combining a high spatial/spectral resolution scanner, 'Compact Airborne Spectrographic Imager' (CASI), with the airborne National Aeronautics & Space Administration's polarimetric radar, 'AIRSAR', for mapping and monitoring of mangrove estuaries. The Daintree River estuary in far North Queensland, Australia was chosen for this study due to its diversity of mangrove species. Imagery acquired by both the CASI airborne scanner (14 bands, 2.5 m pixel) and the AIRSAR (L-and P-band polarimetry, C-band interferometry, 10 m pixel) has been used to produce detailed maps of the mangrove zones in the estuary.The advantages and difficulties associated with multi-source data integration are investigated in this paper. While radar provides general structural information in relation to mangrove zonation, high-resolution hyperspectral scanners allow for finer-detailed analysis and green-biomass information. Classifications (maximum likelihood) of both the individual and integrated datasets are performed, with the latter producing more accurate results. Application of a hierarchical neural network classification is also explored, where the more general mangrove zones are separated first based on structural information, then species or specie-complexes are extracted in subsequent levels using spectral differences.

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O4-alkylthymidine (O4-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O4-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O4-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O4-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O4-iPrdT, O4-iBudT, and O4-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O4-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT and O4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N6-CMdA nor N4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O4-CMdT and O6-CMdG moderately blocked DNA replication and induced substantial frequencies of T→A (81%), T→C (68%) and G→A (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol η resulted in significant drops in bypass efficiencies of N4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N6-CMdA and O6-CMdG upon depletion of Pol κ, and for O6-CMdG upon removal of Pol ζ. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O6-CMdG, N3-CMdT and O4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.

Sleep deficiencies may play a role in depressing immune parameters. Little is known about the impact of exercise after sleep deprivation on mucosal immunity. The purpose of this study was to quantify salivary antimicrobial proteins (AMPs) in response to sleep loss before and after exercise. Four men and 4 women (age: 22.8 ± 2; : 49.1 ± 7.1 ml · kg(-1) · min(-1)) completed 2 exercise trials consisting of 45 minutes of running at 75% VO2peak after a normal night of sleep (CON) and after a night without sleep (WS). Exercise trials were separated by 10 ± 3 days. Saliva was collected before, immediately after, and 1 hour after exercise. LL-37, HNP1-3, Lactoferrin (Lac), and Lysozyme (Lys) were measured. Sleep loss did not affect the concentration or secretion rate of AMPs before or in response to exercise. However, exercise increased the concentration from pre- to post-exercise of LL-37 (pre: 15.5 ± 8.7; post: 22.3 ± 16.2 ng · ml(-1)), HNP1-3 (pre: 2.2 ± 2.3; post: 3.3 ± 2.5 µg · ml(-1)), Lac (pre: 5,234 ± 4,202; post: 12,283 ± 10,995 ng · ml(-1)), and Lys (pre: 5,831 ± 4,465; post: 12,542 ± 10,755 ng · ml(-1)), p <= 0.05. The secretion rates were higher immediately after and 1 hour after exercise compared with before exercise for LL-37 (pre: 3.1 ± 2.1; post: 5.1 ± 3.7; +1: 6.9 ± 8.4 ng · min(-1)), HNP1-3 (pre: 0.38 ± 0.38; post: 0.80 ± 0.75; +1: 0.84 ± 0.67 µg · min(-1)), Lac (pre: 1,096 ± 829; post: 2,948 ± 2,923; +1: 2,464 ± 3,785 ng · min(-1)), and Lys (pre: 1,534 ± 1,790; post: 3,042 ± 2,773; +1: 1,916 ± 1,682 ng · min-(1)), p<= 0.05. These data suggest that the major constituents of the mucosal immune system are unaffected by acute sleep loss and by exercise after acute sleep loss. Exercise increased the concentration and secretion rate of each AMP suggesting enhanced immunity and control of inflammation, despite limited sleep.