Urinary 1-hydroxypyrene and mutagenicity in bus drivers and mail carriers exposed to urban air pollution in Denmark

BACKGROUND: Previous studies in Denmark have shown that bus drivers and tramway employees were at an increased risk for developing several types of cancer and that bus drives from central Copenhagen have high levels of biomarkers of DNA damage.AIMS: The present study evaluates 1-hydroxypyrene concentrations and mutagenic activity in urine as biomarkers of exposure in non-smoking bus drivers in city and rural areas on a work day and a day off and in non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). METHODS: Twenty-four hour urine samples were collected on a working day and a day off from 60 non-smoking bus drivers in city and rural areas and from 88 non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). The concentration of 1-hydroxypyrene was measured by means of HPLC and the mutagenic activity was assessed by the Ames assay with Salmonella tester strain YG1021 and S9 mix. The N-acetyltransferase (NAT2) phenotype was used as a biomarker for susceptibility to mutagenic/carcinogenic compounds. RESULTS: Bus drivers excreted more 1-hydroxypyrene in urine than did mail carriers. The differences were slightly smaller when NAT2 phenotype, cooking at home, exposure to vehicle exhaust, and performing physical exercise after work were included. The NAT2 slow acetylators had 29% (1.29 [CI: 1.15-1.98]) higher 1-hydroxypyrene concentrations in urine than the fast acetylators. Male bus drivers had 0.92 revertants/mol creatinine [CI: 0.37-1.47] and female bus drivers 1.90 revertants/mol creatinine [CI: 1.01-2.79] higher mutagenic activity in urine than mail carriers. CONCLUSION: The present study indicates that bus drivers are more exposed to polycyclic aromatic hydrocarbons (PAH) and mutagens than mail carriers. Mail carriers who worked outdoors had higher urinary concentration of 1-hydroxypyrene, a marker of exposure to PAH, than those working indoors. The individual levels of urinary mutagenic activity were not correlated to excretion of 1-hydroxypyrene. This might be due to the fact that the most potent mutagenic compounds in diesel exhaust are not PAH but dinitro-pyrenes. Among bus drivers, fast NAT2 acetylators had higher mutagenic activity in urine than slow NAT2 acetylators and female bus drivers had higher mutagenic activity than male bus drivers.     

Sucrose and IQ induced mutations in rat colon by independent mechanism

Sucrose-rich diets have repeatedly been observed to have co-carcinogenic actions in colon and liver of rats and to increase the number of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induced aberrant crypt foci in rat colon. To investigate a possible interaction between sucrose and IQ on the genotoxicity in rat liver and colon, we gave Big Blue rats a diet containing sucrose (0%, 3.45% or 13.4% w/w) and/or IQ (70 ppm) for a period of 3 weeks. Sucrose and IQ increased the mutation frequency in the colon. The effect of combined treatments with IQ and sucrose on the mutation frequencies was additive indicating that sucrose and IQ act independently. This was supported by the mutation spectra where sucrose expands the background mutations in the colon, whereas IQ, in other studies, more specifically has induced G:C --> T:A transversions. In the liver IQ increased the mutation frequency, whereas addition of sucrose reduced the effect of IQ in a dose-dependent manner. The level of bulky DNA adducts in liver and colon was increased in animals exposed to either sucrose or IQ. In animals exposed to IQ, addition of sucrose had marginal effects on the level of bulky DNA adducts. Markers of oxidative damage and DNA repair were generally unaffected by the treatments. In conclusion, sucrose and IQ in the diet induced mutations in the colon by independent mechanisms, whereas an interaction was observed in liver leading to a decrease in mutations by the combined treatment.     

CyDisCo production of functional recombinant SARS‐CoV‐2 spike receptor binding domain

The COVID‐19 pandemic caused by SARS‐CoV‐2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS‐CoV‐2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre‐expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin‐converting enzyme) receptor as well as antibodies in COVID‐19 patient sera.     

Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores

The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.     

Effects of Mesolithic hunter-gatherers on local vegetation in a non-uniform glacio-isostatic land uplift area, northern Sweden

The oldest early Mesolithic settlements found so far (i.e. 8600 b.p.) in the interior of northern Sweden, in the province of Norrbotten, have been discovered through the application of a model simulating glacio-isostatic land uplift. The objective of this study was to investigate, through pollen and charred particle analyses, vegetation composition and the influence of man on vegetation and fire pattern in the vicinity of two of these early settlements. Early Holocene vegetation was characterised by Betula, Hippophaë and Salix-species, but no initial impact by hunter-gatherers on vegetation was detected. Subsequently Betula and Pinus became dominant but abrupt changes in the tree layer followed, Poaceae, Hippophaë and Salix-species increased, and Humulus occurred. These changes, which were synchronous with dates of archaeological findings at the settlements, indicated local vegetation changes caused by man. The relationship between these changes and fire was, however, subtle. Eventually Pinus forests became dominant and human impact on vegetation became less apparent. It is concluded that the impact of early Mesolithic hunter-gatherers on vegetation was detectable, but that their effect on fire pattern was difficult to evaluate. Further, we show that the glacio-isostatic land uplift must be considered in the search for Mesolithic settlements in areas previously covered by the Weichselian Ice, and that the process has influenced the biological archives through re-deposition of material.     

ERCC1, XPD and RAI mRNA levels in lymphocytes are not associated with lung cancer risk in a prospective study of Danes

Low DNA-repair capacity has been associated with increased risk of several types of cancer. mRNA levels of the nucleotide excision repair genes ERCC1 and XPD have been shown to correlate with the DNA-repair capacity. Likewise, mRNA levels of several DNA-repair genes including ERCC1 have been shown to be lower in lymphocytes from patients with lung cancer and head and neck cancer compared with healthy persons. In these studies, the low DNA-repair gene expression levels could be either a risk factor for disease or a consequence of the same. In this nested case-cohort study, which to our knowledge, is the first prospective study of DNA-repair gene mRNA levels as predictors of lung cancer, we have investigated the occurrence of lung cancer in relation to the mRNA level of the two DNA-repair genes ERCC1 and XPD and the NF kappaB inhibitor RAI in blood samples prior to disease. Among 54,220 members of a Danish prospective cohort study, 265 lung cancer cases were identified and a sub-cohort comprising 272 individuals was used for comparison. The expression levels of the three adjacent genes were found to be highly inter-correlated, to be higher in women compared to men and to be lower in older individuals. The incidence rate ratios for lung cancer in association with one log-unit increase (natural logarithm) in mRNA levels were 1.12 (CI=0.89-1.41) for ERCC1, 1.00 (CI=0.83-1.21) for XPD and 1.25 (0.89-1.74) for RAI. In conclusion, this study indicated no association between mRNA expression of the DNA-repair genes ERCC1 and XPD and risk of subsequent development of lung cancer.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

Active PI3K pathway causes an invasive phenotype which can be reversed or promoted by blocking the pathway at divergent nodes

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP(3) production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.     

Binding of Two Intrinsically Disordered Peptides to a Multi-Specific Protein: A Combined Monte Carlo and Molecular Dynamics Study

The unique ability of intrinsically disordered proteins (IDPs) to fold upon binding to partner molecules makes them functionally well-suited for cellular communication networks. For example, the folding-binding of different IDP sequences onto the same surface of an ordered protein provides a mechanism for signaling in a many-to-one manner. Here, we study the molecular details of this signaling mechanism by applying both Molecular Dynamics and Monte Carlo methods to S100B, a calcium-modulated homodimeric protein, and two of its IDP targets, p53 and TRTK-12. Despite adopting somewhat different conformations in complex with S100B and showing no apparent sequence similarity, the two IDP targets associate in virtually the same manner. As free chains, both target sequences remain flexible and sample their respective bound, natively -helical states to a small extent. Association occurs through an intermediate state in the periphery of the S100B binding pocket, stabilized by nonnative interactions which are either hydrophobic or electrostatic in nature. Our results highlight the importance of overall physical properties of IDP segments, such as net charge or presence of strongly hydrophobic amino acids, for molecular recognition via coupled folding-binding.     

Realist synthesis: illustrating the method for implementation research

ABSTRACT: BACKGROUND: Realist synthesis is an increasingly popular approach to the review and synthesis of evidence, which focuses on understanding the mechanisms by which an intervention works (or not). There are few published examples of realist synthesis. This paper therefore fills a gap by describing, in detail, the process used for a realist review and synthesis to answer the question 'what interventions and strategies are effective in enabling evidence-informed healthcare?' The strengths and challenges of conducting realist review are also considered. METHODS: The realist approach involves identifying underlying causal mechanisms and exploring how they work under what conditions. The stages of this review included: defining the scope of the review (concept mining and framework formulation); searching for and scrutinising the evidence; extracting and synthesising the evidence; and developing the narrative, including hypotheses. RESULTS: Based on key terms and concepts related to various interventions to promote evidenceinformed healthcare, we developed an outcome-focused theoretical framework. Questions were tailored for each of four theory/intervention areas within the theoretical framework and were used to guide development of a review and data extraction process. The search for literature within our first theory area, change agency, was executed and the screening procedure resulted in inclusion of 52 papers. Using the questions relevant to this theory area, data were extracted by one reviewer and validated by a second reviewer. Synthesis involved organisation of extracted data into evidence tables, theming and formulation of chains of inference, linking between the chains of inference, and hypothesis formulation. The narrative was developed around the hypotheses generated within the change agency theory area. CONCLUSIONS: Realist synthesis lends itself to the review of complex interventions because it accounts for context as well as outcomes in the process of systematically and transparently synthesising relevant literature. While realist synthesis demands flexible thinking and the ability to deal with complexity, the rewards include the potential for more pragmatic conclusions than alternative approaches to systematic reviewing. A separate publication will report the findings of the review.