Quantitative and qualitative determination of microplastics in oyster, seawater and sediment from the coastal areas in Zhuhai, China.

Microplastics as a new class of environmental contaminants have become the hot issue of global concern. We conducted quantitative and qualitative experiments to investigate microplastics in oyster, seawater and sediment along the Zhuhai coastline. The soft tissues of oysters were digested with potassium hydroxide (10%) and hydrogen peroxide (30%), seawaters and sediments with hydrogen peroxide (30%) to degrade organic matter, and analyzed using a digital camera, optical microscopy and micro-ATR-FTIR. The abundance of microplastics were in the range of 0.14-7.90 n/g in oysters (wet weight), 10.00-27.50 n/L in seawaters and 0.053-0.26 n/g in sediments. The fiber and fragment shape, black color, 101-500 µm of size and polyethylene composition were all classified as the major constituents of microplastics. The level of contaminants in oysters was correlated to those in their surrounding environments. Therefore, oysters may serve as a promising sentinel species for the indication of microplastic pollution in the coastal zone of Zhuhai.

Puffer Fish Gut Microbiota Studies Revealed Unique Bacterial Co-Occurrence Patterns and New Insights on Tetrodotoxin Producers.

Tetrodotoxin (TTX) is a potent neurotoxin isolated mainly from toxic puffer fish. To date, the TTX biosynthetic mechanism inside its hosts remains unresolved. Here, we hypothesize the TTX synthesis relies on the host gut microbiota, including the neglected non-culturable bacteria. In these studies, we collected the gut contents from 5 puffer fish species of the genus Takifugu including one suspected hybrid species for gut microbiota study by 16S rRNA amplicon metagenomics approach. Their gut samples were divided into toxic and non-toxic groups based on the TTX concentrations in the livers detected by LC-MS/MS. Bacterial diversity studies showed that gut microbiota structures were significantly different between toxic and non-toxic species. Vibrio and Cyanobacteria centered at the gut bacterial co-occurrence network, suggesting their importance in TTX biosynthesis. The results of PICRUSt2 metagenomic prediction and gene set enrichment analysis provided new support of arginine-precursor required in TTX biosynthesis. This is the first study to profile the gut microbiota in toxic and non-toxic puffer fish species by 16S rRNA amplicon metagenomic approach, defining significant microbial co-occurrence patterns in their gut environment. Our data supported the proposed biosynthesis of TTX inside the hosts by their gut bacterial symbionts using arginine as a precursor.

Homology modeling of four Y-family, lesion-bypass DNA polymerases: the case that E. coli Pol IV and human Pol kappa are orthologs, and E. coli Pol V and human Pol eta are orthologs.

Y-family DNA polymerases (DNAPs) are a superfamily of evolutionarily related proteins that exist in cells to bypass DNA damage caused by both radiation and chemicals. Cells have multiple Y-family DNAPs, presumably to conduct translesion synthesis (TLS) on DNA lesions of varying structure and conformation. The potent, ubiquitous environmental mutagen/carcinogen benzo[a]pyrene (B[a]P) induces all classes of mutations with G-->T base substitutions predominating. We recently showed that a G-->T mutagenesis pathway for the major adduct of B[a]P ([+ta]-B[a]P-N2-dG) in Escherichia coli depends on Y-family member DNAP V. Since no X-ray crystal study for DNAP V has been reported, no structure is available to help in understanding the structural basis for dATP insertion associated with G-->T mutations from [+ta]-B[a]P-N2-dG. Herein, we do homology modeling to construct a model for UmuC, which is the polymerase subunit of DNAP V. The sequences of eight Y-family DNAPs were aligned based on the positioning of conserved amino acids and an analysis of conserved predicted secondary structure, as well as insights gained from published X-ray structures of five Y-family members. Starting coordinates for UmuC were generated from the backbone coordinates for the Y-family polymerase Dpo4 for reasons discussed, and were refined using molecular dynamics with CHARMM 27. A survey of the literature revealed that E. coli DNAP V and human DNAP eta show a similar pattern of dNTP insertion opposite a variety of DNA lesions. Furthermore, E. coli DNAP IV and human DNAP kappa show a similar dNTP insertional pattern with these same DNA lesions, although the insertional pattern for DNAP IV/kappa differs from the pattern for DNAPs V/eta. These comparisons prompted us to construct and refine models for E. coli DNAP IV and human DNAPs eta and kappa as well. The dNTP/template binding pocket of all four DNAPs was inspected, focusing on the array of seven amino acids that contact the base of the incoming dNTP, as well as the template base. DNAPs V and eta show similarities in this array, and DNAPs IV and kappa also show similarities, although the arrays are different for the two pairs of DNAPs. Thus, there is a correlation between structural similarities and insertional similarities for the pairs DNAPs V/eta and DNAPs IV/kappa. Although the significance of this correlation remains to be elucidated, these observations point the way for future experimental studies.

Free-energy perturbation methods to study structure and energetics of DNA adducts: results for the major N2-dG adduct of benzo[a]pyrene in two conformations and different sequence contexts.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is activated to (+)-anti-B[a]PDE, which induces a variety of mutations (e.g., G --> T, G --> A, etc.) via its major adduct [+ta]-B[a]P-N2-dG. One hypothesis is that adducts (such as [+ta]-B[a]P-N2-dG) induce different mutations via different conformations, probably when replicated by different lesion-bypass DNA polymerases (DNAPs). We showed that Escherichia coli DNAP V was responsible for G --> T mutations with [+ta]-B[a]P-N2-dG in a 5'-TGT sequence (Yin et al., (2004) DNA Repair 3, 323), so we wish to study conformations of this adduct/sequence context by molecular modeling. The development of a CHARMM-based molecular dynamics (MD) simulations protocol with free-energy calculations in the presence of solvent and counterions is described. A representative base-pairing and base-displaced conformation of [+ta]-B[a]P-N2-dG in the 5'-TGT sequence are used: (1) BPmi5, which has the B[a]P moiety in the minor groove pointing toward the base on the 5'-side of the adduct, and (2) Gma5, which has the B[a]P moiety stacked with the surrounding base pairs and the dG moiety displaced into the major groove. The MD output structures are reasonable when compared to known NMR structures. Changes in DNA sequence context dramatically affect the biological consequences (e.g., mutagenesis) of [+ta]-B[a]P-N2-dG. Consequently, we also developed a MD-based free-energy perturbation (FEP) protocol to study DNA sequence changes. FEP involves the gradual "fading-out" of atoms in a starting structure (A) and "fading-in" of atoms in a final structure (B), which allows a realistic assessment of the energetic and structural changes when two structures A and B are closely related. Two DNA sequence changes are described: (1) 5'-TGT --> 5'-TGG, which involves two steps [T:A --> T:C --> G:C], and (2) 5'-TGT --> 5'-TGC, which involves three steps [T:A --> T:2AP --> C:2AP --> C:G], where 2AP (2-aminopurine) is included, because T:2AP and C:2AP retain more-or-less normal pairing orientations between complementary bases. FEP is also used to evaluate the impact that a 5'-TGT to 5'-UGT sequence change might have on mutagenesis with [+ta]-B[a]P-N2-dG. In summary, we developed (1) a CHARMM-based molecular dynamics (MD) simulations protocol with free-energy calculations in the presence of solvent and counterions to study B[a]P-N2-dG adducts in DNA duplexes, and (2) a MD-based free-energy perturbation (FEP) protocol to study DNA sequence context changes around B[a]P-N2-dG adducts.

Molecular modeling of the major benzo[a]pyrene N2-dG adduct in cases where mutagenesis results are known in double stranded DNA.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC-->TA, GC-->AT, etc.). One hypothesis for this complexity is that different mutations are induced by different conformations of its major adduct [+ta]-B[a]P-N2-dG when bypassed during DNA replication (probably by different DNA polymerases). Previous molecular modeling studies suggested that B[a]P-N2-dG adducts can in principle adopt at least 16 potential conformational classes in ds-DNA. Herein we report on molecular modeling studies with the eight conformations most likely to be relevant to base substitution mutagenesis in 10 cases where mutagenesis has been studied in ds-DNA plasmids in E. coli with B[a]P-N2-dG adducts of differing stereoisomers and DNA sequence contexts, as well as in five cases where the conformation is known by NMR. Of the approximately 11,000 structures generated in this study, the computed lowest energy structures are reported for 120 cases (i.e. eight conformations and 15 examples), and their conformations compared. Of the eight conformations, four are virtually always computed to be high in energy. The remaining four lower energy conformations include two with the BP moiety in the minor groove (designated: BPmi5 and BPmi3), and two base-displaced conformations, one with the dG moiety in the major groove (designated: Gma5) and one with the dG in the minor groove (designated: Gmi3). Interestingly, these four are the only conformations that have been observed for B[a]P-N2-dG adducts in NMR studies. Independent of sequence contexts and adduct stereochemistry, BPmi5 structures tend to look reasonably similar, as do BPmi3 structures, while the base-displaced structures Gma5 and BPmi3 tend to show greater variability in structure. A correlation was sought between modeling and mutagenesis results in the case of the low energy conformations BPmi5, BPmi3, Gma5 and Gma3. Plots of log[(G-->T)/(G-->A)] versus energy[(conformation X)-(conformation Y)] were constructed for all six pairwise combinations of these four conformations, and the only plot giving a straight line involved Gma5 and Gmi3. While this finding is striking, its significance is unclear (as discussed).

Molecular modeling of four stereoisomers of the major B[a]PDE adduct (at N(2)-dG) in five cases where the structure is known from NMR studies: molecular modeling is consistent with NMR results.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which is known to induce a variety of mutations (e.g., GC --> TA, GC --> AT, etc.). One hypothesis for this complexity is that different mutations are induced by different conformations of its major adduct [+ta]-B[a]P-N(2)-dG when bypassed during DNA replication (perhaps by different DNA polymerases). Our previous molecular modeling studies have suggested that conformational complexity might be extensive in that B[a]P-N(2)-dG adducts appeared capable of adopting at least sixteen potential conformational classes in ds-DNA [e.g., Kozack and Loechler (1999) Carcinogenesis 21, 1953], although only eight seemed likely to be relevant to base substitution mutagenesis. Such molecular modeling studies are only likely to be valuable for the interpretation of mutagenesis results if global minimum energy conformations for adducts are found and if the differences in the energies of these different conformations can be computed reasonably accurately. One approach to assessing the reliability of our molecular modeling techniques is considered herein. Using a five-step molecular modeling protocol, which importantly included a molecular dynamics version of simulated annealing, eight conformations are studied in each of five cases. (The five cases are listed below, and were chosen because in each case the preferred solution conformation is known from a NMR study.) Of the eight conformations studied, the one computed to be lowest in energy is the same conformation as the one observed by NMR in four of the five cases: 5'-CGC sequence with [+ta]-, [-ta]-, and [+ca]-B[a]P-N(2)-dG, and 5'-TGC sequence with [+ta]-B[a]P-N(2)-dG. In the fifth case (5'-CGC sequence with [-ca]-B[a]P-N(2)-dG), the known NMR conformation is computed to be second lowest in energy, but it is within approximately 1.7 kcal of the computed lowest energy conformation. These results suggest that molecular modeling is surprisingly accurate in computing lowest energy conformations and that it should be useful in assessing the relative energies of different conformations. This is especially important given that currently molecular modeling is the only means available to study the energetics of minor conformations of DNA adducts.