The cytotoxic effects of topoisomerase I inhibitors such as camptothecin can be modulated by replication fork reversal, in a process that requires Poly(ADP-ribose) polymerase (PARP) activity. Here human RECQ1 helicase is shown to restore such regressed replication forks, whereas PARP1 activity restrains this RECQ1 function. Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

Here we present a new bonding protocol for SU-8 negative tone photoresist that exploits the chemical modifications induced in the resin by exposure to 254 nm (UVC) light. Fourier Transform Infrared microspectroscopy (μ-FTIR) was used to carry out a thorough study on the chemical processes and modifications occurring within the epoxy resin by exposure to 365 nm and 254 nm light. In particular, we established that UVC light promotes the opening of the epoxy rings bypassing the post-exposure bake. The possibility to promote a further activation of the resin, already patterned with standard UV lithography, was exploited to produce closed microfluidic devices. Specifically, we were able to fabricate fluidic chips, characterized by broadband transparency from mid-IR to UV and long term stability in continuous flow conditions. CaF₂ was used as substrate, coated by sputtering with a nanometric silicon film, in order to make surface properties of this material more suitable for standard fabrication processes with respect to the original substrate. The fabricated microfluidic chips were used to study by μ-FTIR the biochemical response of live breast cancer MCF-7 cells to osmotic stress and their subsequent lysis induced by the injection of deionized water in the device. μ-FTIR analyses detected fast changes in protein, lipid and nucleic acid content as well as cytosol acidification.