Photo-repair effect of a bacterial Antarctic CPD-photolyase on UVC-induced DNA lesions in human keratinocytes.

Exposure to ultraviolet radiation from sunlight induces oxidative DNA lesions and bipyrimidine photoproducts that can lead to photo-aging and skin carcinogenesis. CPD-photolyases are flavoproteins that repair cyclobutane pyrimidine dimers using blue light as an energy source. In the present work, we evaluated the photo-repair effect of the recombinant CPD-photolyase PhrAHym from the Antarctic bacterium Hymenobacter sp. UV11 on DNA lesions in human keratinocytes induced by UVC light. By performing immunochemistry assays we observed that PhrAHym repairs in a highly efficient way the CPD-photoproducts and reduces the γH2AX formation. Since this enzyme is non-cytotoxic and repairs UVC-induced DNA lesions in human keratinocytes, we propose that PhrAHym could be used as a biotherapeutic agent against UV-induced skin cancer, photoaging, and related diseases.

Lab-made 3D printed stoppers as high-throughput cell migration screening tool.

Cell migration is a process that underlies the development and maintenance of multicellular organisms, with profound implications in various pathologies. The study of cell migration is fundamental in various fields of basic biology and pharmaceutical development. Wound healing assay is an indirect way to assess cell migration. Conventional methods, such as the scratch test, are inexpensive and easy to execute but have the disadvantages of being poorly reproducible and difficult to perform on a high-throughput scale. Meanwhile, commercial strategies are expensive. In the present work, we developed a lab-made wound healing assay device that is inexpensive, easy to handle, and reproducible. We designed 3D-printed stoppers compatible with cell culture in 96-well plates. These stoppers did not affect HaCaT cells viability. The stopper-produced initial wound size was reproducible on a high-throughput scale. Also, stoppers demonstrated their effectiveness to evaluate cell migration and allowed differentiating treatments with and without fetal bovine serum. Finally, proliferation assay was determined in this wound healing model. In conclusion, our lab-made 3D-printed stopper-based assay is a more economical alternative to currently available strategies for developing reproducible, high-throughput assays to assess cell migration and proliferation.

A natural occurring bifunctional CPD/(6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9.

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme. KEY POINTS: • We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.

Furoxans and tocopherol analogs-furoxan hybrids as anticancer agents.

We determined the antiproliferative and nitric oxide (NO)-releasing activity of furoxans and tocopherol analogs-furoxan hybrids by tandem Griess/resazurin/sulforhodamin B assays in HeLa, 253J, T24, and HepG2 cancer cells. In addition, to investigate the NO implications in the inhibition of cell growth, cells were pretreated with the NO scavenger hemoglobin and the genotoxic damage was determined. The compounds 1 and 3 emerged as good anticancer agents for bladder cancer treatment. The NO-releasing activity of these compounds appears to be necessary to obtain the antiproliferative effect. Although compound 1 exerted a DNA damage mechanism of action, compound 3 seemed to act in a different way. The low toxicity levels against normal cell line HaCaT point them out as a very promising scaffold for the further design of new anticancer agents.