Antioxidant Strategies to Modulate NETosis and the Release of Neutrophil Extracellular Traps during Chronic Inflammation.

Extracellular traps are released by neutrophils and other immune cells as part of the innate immune response to combat pathogens. Neutrophil extracellular traps (NETs) consist of a mesh of DNA and histone proteins decorated with various anti-microbial granule proteins, such as elastase and myeloperoxidase (MPO). In addition to their role in innate immunity, NETs are also strongly linked with numerous pathological conditions, including atherosclerosis, sepsis and COVID-19. This has led to significant interest in developing strategies to inhibit NET release. In this study, we have examined the efficacy of different antioxidant approaches to selectively modulate the inflammatory release of NETs. PLB-985 neutrophil-like cells were shown to release NETs on exposure to phorbol myristate acetate (PMA), hypochlorous acid or nigericin, a bacterial peptide derived from Streptomyces hygroscopicus. Studies with the probe R19-S indicated that treatment of the PLB-985 cells with PMA, but not nigericin, resulted in the production of HOCl. Therefore, studies were extended to examine the efficacy of a range of antioxidant compounds that modulate HOCl production by MPO to prevent NETosis. It was shown that thiocyanate, selenocyanate and various nitroxides could prevent NETosis in PLB-985 neutrophils exposed to PMA and HOCl, but not nigericin. These results were confirmed in analogous experiments with freshly isolated primary human neutrophils. Taken together, these data provide new information regarding the utility of supplementation with MPO inhibitors and/or HOCl scavengers to prevent NET release, which could be important to more specifically target pathological NETosis in vivo.

DNA extraction of bacterial cells using a semi-automated filtration system.

The COVID-19 pandemic lockdown created problems with importing of commercial kits resulting in extended turnaround times for consumable deliveries. One way to circumvent this was to use an inexpensive optimized in-house method for DNA extraction from water. • The DNA extraction methods were optimized on a 96-well plate using a semi-automated filtration system to increase the number of samples from 24 to 96 at a time in 2 h. The DNA extraction method optimizations included: (a) Guanidium thiocyanate method plus dilution series of celite to determine DNA binding capacity; (b) QIamp 96 Qiacube HT kit (Qiagen®); (c) Guanidium thiocyanate with the celite replaced with a binding buffer. • The in-house DNA extraction methods and adapted in-house DNA extraction method were compared to QIamp 96 Qiacube HT kit (Qiagen®), which is used on a 96-well semi-automated filtration system. The results showed maximum capacity of the 96-well filter plates was 400 µâ„“ broth (OD600 = 0.45 = 3.6 × 108 cells/mℓ) before the 96-well filters blocked. • When the methods were compared, there was no significant difference between the in-house DNA extraction method with 1:420 celite dilution (P-value = 0.126) and the adapted in-house method with binding buffer (P-value = 0.298) DNA yield or amplification of PCR products.

The Association Between Environmental Perchlorate, Nitrate, and Thiocyanate Exposure and Oral Pain in NHANES.

Aim: To examine the human exposure to perchlorate, nitrate, and thiocyanate, and their associations with oral pain (OP) in the general population from the U.S. Methods: A total of 13,554 participants were enrolled in the National Health and Nutrition Examination Survey. The urinary perchlorate, nitrate, and thiocyanate were measured using ion chromatography coupled with an electrospray tandem mass spectrometry. The multivariable linear and logistic regressions were performed to explore the associations of the urinary perchlorate, nitrate, and thiocyanate, with the prevalence of oral pain. Restricted cubic splines were used to explore the non-linearity. Results: There are 3,129 OP cases. There was a higher urinary level of perchlorate, nitrate, and thiocyanate in OP. We found that urinary thiocyanate was positively associated with OP (odds ratio [OR] = 1.06; [1, 1.13]; p = 0.049). Restricted cubic spines revealed that urinary thiocyanate was in a U-shape association with OP. Conclusions: Urinary thiocyanate was in a U-shape association with OP, suggesting that we should keep the exposure of thiocyanate under a reasonable range.