The COVID-19 pandemic focused attention on a pressing need for fast, accurate, and low-cost diagnostic tests. This work presents an electrochemical capillary driven immunoassay (eCaDI) developed to detect SARS-CoV-2 nucleocapsid (N) protein. The low-cost flow device is made of polyethylene terephthalate (PET) and adhesive films. Upon addition of a sample, reagents and washes are sequentially delivered to an integrated screen-printed carbon electrode for detection, thus automating a full sandwich immunoassay with a single end-user step. The modified electrodes are sensitive and selective for SARS-CoV-2 N protein and stable for over 7 weeks. The eCaDI was tested with influenza A and Sindbis virus and proved to be selective. The eCaDI was also successfully applied to detect nine different SARS-CoV-2 variants, including Omicron.

Canine malignant melanoma (CMM) is a locally and systemically aggressive cancer that shares many biological and clinical characteristics with human mucosal melanoma. Hypofractionated radiation protocols have been used to treat CMM but little is known about its radiation biology. This pilot study is designed to investigate response of CMM cell lines to various ionizing radiations and cytotoxic agents to better understand this canine cancer. Four CMM cell lines were evaluated by clonogenic survival assay under aerobic and hypoxic conditions and parameters such as alpha beta (α/β) ratio, oxygen enhancement ratio (OER), and relative biological effectiveness (RBE) were calculated after 137Cs, 6 megavoltage (MV) photon, or carbon ion irradiation. Six cytotoxic agents (cisplatin, camptothecin, mitomycin C, bleomycin, methtyl methanesulfonate and etoposide) were also assessed for their efficacy. Under aerobic condition with 6 MV photon, the α/β ratio of the four cell lines ranged from 0.3 to >100, indicating a wide variation of cellular sensitivity. The ratio increased under hypoxic condition compared to aerobic condition and this was more dramatic in 137Cs and 6 MV photon treatments. OER of carbon was lower than 137Cs at D10 in 3 of the 4 cell lines. The RBE values generally increased with the increase of LET. Different cell lines showed sensitivity/resistance to different cytotoxic agents. This study revealed that CMM has a wide range of radiosensitivity and that hypoxia can reduce it, indicating that widely used hypofractionated protocols may not be optimal for all CMM patients. Several cytotoxic agents that have never been clinically assessed can improve treatment outcome.

Quercetin has been demonstrated to produce DNA damage in the presence of metal ions. In the present study, 7 natural and 5 semi‑synthetic glycosylated flavonoids were utilized to investigate the cupric ion (Cu2+)‑dependent DNA damage in vitro. The reaction mixture, containing single‑stranded DNA, different concentrations of flavonoids and cupric ion in the buffer, was incubated at three different temperatures. DNA damage was then assessed by gel electrophoresis followed by densitometric analysis. The reaction mixture with quercetin at 4, 20 and 54˚C induced DNA damage in a concentration‑ and temperature‑dependent manner. Furthermore, only the reaction at 54˚C resulted in DNA damage in flavonoids with glucosyl substitution of the hydroxyl group at the 3‑position on the C ring in quercetin. By contrast, loss of the hydroxyl group at the 3‑position on the C ring, or at the 3'‑ or 4'‑position on the B ring of quercetin, did not portray DNA damage formation at the investigated experimental temperatures. In addition, the experimental results suggested that the hydroxyl group at the 3‑position on the C ring produced the strongest capability to induce DNA damage in the presence of cupric ions. Furthermore, hydroxyl groups at the 3'‑ or 4'‑position on the B ring were only able to induce DNA damage at higher temperatures, and were less efficient in comparison with the hydroxyl group at the 3‑position on the C ring. Cupric ion chelating capacity was also assessed with spectroscopic analysis, and quercetin presented the largest chelating capacity among the tested flavonoids. Hydroxyl radical formation was assessed with a luminol reaction, and quercetin presented faster consumption of luminol. These results suggest that the 3‑position hydroxyl group of the C ring is required to induce DNA damage at low temperatures. Furthermore, the results of the present study also indicated that the presence of cupric ions will decrease the activity of the glycosylated quercetins, in terms of their ability to induce DNA damage.

Tea polyphenols are known antioxidants presenting health benefits due to their observed cellular activities. In this study, two tea polyphenols, epigallocatechin gallate, which is common in green tea, and theaflavin, which is common in black tea, were investigated for their PARP inhibitory activity and selective cytotoxicity to BRCA2 mutated cells. The observed cytotoxicity of these polyphenols to BRCA2 deficient cells is believed to be a result of PARP inhibition induced synthetic lethality. Chinese hamster V79 cells and their BRCA2 deficient mutant V-C8, and V-C8 with gene complemented cells were tested against epigallocatechin gallate and theaflavin. In addition, Chinese hamster ovary (CHO) wild-type cells and rad51D mutant 51D1 cells were used to further investigate the synthetic lethality of these molecules. The suspected PARP inhibitory activity of epigallocatechin and theaflavin was confirmed through in vitro and in vivo experiments. Epigallocatechin gallate showed a two-fold increase of cytotoxicity to V-C8 cells compared to V79 and gene complimented cells. Compared to CHO wild type cells, 51D1 cells also showed elevated cytotoxicity following treatment with epigallocatechin gallate. Theaflavin, however, showed a similar increase of cytotoxicity to VC8 compared to V79 and gene corrected cells, but did not show elevation of cytotoxicity towards rad51D mutant cells compared to CHO cells. Elevation of sister chromatid exchange formation was observed in both tea polyphenol treatments. Polyphenol treatment induced more micronuclei formation in BRCA2 deficient cells and rad51D deficient cells when compared against the respective wild type cells. In conclusion, tea polyphenols, epigallocatechin gallate, and theaflavin may present selective cytotoxicity to BRCA2 deficient cells through synthetic lethality induced by PARP inhibition.

Cu2+ and Co2+ are metals known to increase DNA damage in the presence of hydrogen peroxide through a Fenton type reaction. We hypothesized that these metals could increase DNA damage following irradiations of increasing LET values as hydrogen peroxide is a product of the radiolysis of water. The reaction mixtures contain either double- or single-stranded DNA in solution with Cu2+ or Co2+ and was irradiated either with X-ray, carbon-ion or iron-ion beams or was treated with hydrogen peroxide or bleomycin at increasing radiation dosages or chemical concentrations. DNA damage was then assessed by gel electrophoresis followed by band intensity analysis. DNA in solution with metals demonstrated the most DNA damage when treated with hydrogen peroxide followed by irradiation with low-LET (X-Ray), high-LET (carbon-ion and iron-ion), respectively, and demonstrated the least damage with treatment of bleomycin. Cu2+ portrayed greater DNA damage than Co2+ following all experimental conditions. The metals effect caused more DNA damage and was observed to be LET dependent for single-strand break formation but inversely dependent for double-strand break formation. These results suggest that Cu2+ is more efficient than Co2+ at inducing both DNA single-strand and double-strand breaks following all irradiations and chemical treatments.