The University of Suwon

Wastewater-based epidemiology (WBE) has proven to be a powerful tool for tracking the spread of viral pathogens, such as SARS-CoV-2; however, as WBE has expanded to include new pathogens, like mpox virus (MPXV), more data is needed to guide practitioners on how to design WBE campaigns. Here, we investigated the decay rates of heat-inactivated MPXV (HI-MPXV) and attenuated vaccinia virus (VV) viral signal in primary influent and settled solids collected from a local POTW at 4 °C, 22 °C, or 35 °C using digital PCR. Subsequently, we studied the solid-liquid partitioning of the viruses in primary influent. Over 30 days, decay rates did not significantly differ between viruses (p = 0.5258). However, decay was significantly higher in primary influent (0.109-0.144/day) than in settled solids (0.019-0.040/day) at both 22 °C (p = 0.0030) and 35 °C (p = 0.0166). Furthermore, as part of the partitioning experiment, we found that HI-MPXV and VV adsorb to the solids fraction of primary influent at higher intensities than previously studied enveloped viruses (K_F = 1000-31,800 mL/g, n = 1.01-1.41). Likewise, it was determined in the partitioning experiment that a concentration of greater than 10³ gc/mL in raw influent was needed for the viable quantification of MPXV and VV DNA in the clarified liquid fraction of raw primarily influent. Our study provides essential insights into informative sample collection and storage conditions for the analysis of wastewater and for transport modeling studies. Due to the slow decay observed in settled solids at all tested temperatures in the persistence experiment, this matrix may be most suitable for retrospective analyses of MPXV infections within a community.

Chlorine is the principal microbial disinfectant used for water treatment. However, chlorine-resistant bacteria such as Mycobacterium spp., can survive chlorine treatment and even grow in the presence of chlorine, posing potential public health risks. In this study, we isolated a Mycobacterium sp. strain from treated effluent and investigated its chlorine resistance and recovery using transcriptomic analyses. Specifically, isolate M1, showing 94.58 % average nucleotide identity (ANI) with Mycobacterium massilipolynesiensis type genome, was exposed to 1 ppm HOCl for 30 min and subjected to RNA sequencing. Genes identified as upregulated compared to control conditions (no HOCl) were involved in detoxification (toxic compound degradation; nemA; log₂ fold-change [FC]: 7.41), redox homeostasis (COQ5; quinone synthesis; log₂ FC 5.70, rosB; riboflavin synthesis; log₂ FC 5.61), protein homeostasis (cysHKO, moeZ cysteine biosynthesis, and arg complex; arginine metabolism), and lipid metabolism (cpnA; 6.95 FC) suggesting a multifaceted adaptation to oxidative stress. Levels of a few membrane transport proteins (czcD, and bcr) were also upregulated, highlighting their role in chlorine exposure. Overall, this study broadens the understanding of chlorine resistance strategies employed by Mycobacterium sp. to combat oxidative stress and the resulting toxic intracellular compounds, and has implications for adjusting water treatment technologies toward eliminating mycobacteria.

Nonthermal plasma (NTP) has garnered attention for its potential in various biological activities. Due to the interactions with proteins and other cellular components, NTPs might stimulate osteoblast activity, but the exact mechanisms are still unknown. This study used two kinds of NTP: dielectric barrier discharge (DBD) and jet NTP (JNTP) to treat serum-free cell culture media at different time intervals. The indirect (plasma-media interfaces of ion exchange) exposure to osteoblasts was analyzed to determine whether it could influence osteoblast behavior and stimulate their osteogenic activity. Among various NTP-ionized media, 25% to 100% of the JNTP medium (JNTPM, treated for 10 min) showed noticeable induction of ALP activity in osteoblasts. 25% JNTPM induced maximum ALP activity and also demonstrated enhanced mRNA expression of osteogenic markers like Runx-2, Osterix, collagen 1α, bone sialoprotein, and osteocalcin. In addition, JNTPM treatment to osteoblasts demonstrated enhanced collagen production and mineralization. The administration of JNTPM to SaOS-2 cells increased Axin-2 reporter activity, along with enhanced stabilization of β-catenin and phosphorylation of GSK3-β, indicating the stimulation of the WNT signaling pathway. Moreover, JNTPM treatment increased osteoblasts' intracellular reactive oxygen species (ROS) levels. Prior exposure of N-acetyl-l-cysteine (NAC) to JNTPM-treated osteoblasts diminished the phosphorylation of GSK3-β, Axin-2 reporter activity, and the stability of β-catenin, highlighting the importance of intracellular ROS in enhancing Wnt signaling and osteogenic activity in osteoblasts in response to JNTPM. Thus, JNTPM treatment induces osteogenic stimulatory properties in osteoblasts through cross-talk between ROS and the Wnt signaling axis, suggesting a potential therapeutic approach for bone formation.