Oxidative stress and inflammation are crucial factors in the development of cardiovascular diseases. In previous research, the oxidative stress and inflammation models have frequently been explored independently. In the current study, we investigated the antioxidant and anti-inflammatory effects of tomato extract and its two main carotenoids (lutein and lycopene) with various concentrations using a rat cardiomyocyte model of co-existing oxidative stress and persistent chronic inflammation. It was discovered that the antioxidant effects of 0.5–5 μM lutein, 0.5–5 μM lycopene, and 50–200 μg/mL tomato extract increased in a dose-dependent manner. However, the pro-oxidation effects emerged by measuring the antioxidant-related indices, including the levels of ROS, SOD, and GPX in H9c2 cells as concentrations exceeded those mentioned above. The anti-inflammatory effects of lutein, lycopene, and tomato extract were simultaneously strengthened with higher concentrations, potentially due to the suppression of the NF-κB signaling pathway. Furthermore, high concentrations of lutein, lycopene, and tomato extract potentially regulated Nrf2/HO-1 and NF-κB signaling pathways dependent on TGF-1β and IL-10 to demonstrate high concentrations of pro-oxidation and anti-inflammation effects. Our findings indicate that the dose–effect regulatory mechanisms of antioxidant and anti-inflammatory properties among lutein, lycopene, and tomato extract will be advantageous in developing more effective therapeutic strategies to prevent cardiovascular diseases. Numerous epidemiological studies have demonstrated that cardiovascular disease (CVD) is among the significant causes of mortality in humans . The occSpecific correlations exist between oxidative stress and inflammation during disease development. For instance, the occurrence of oxidative stress and an inflammatory response would both impede the insulin response and increase the risk of cardiovascular disease and other related illnesses . Likewis−, thus preventing the occurrence of CVD . Af. Af31]. p < 0.05). In this experiment, the concentration dose was determined with a cell survival rate higher than 95%. Therefore, lutein below 20 μM, lycopene below 20 μM, and tomato extract below 400 μg/mL were used for the subsequent experiments (Treatment with different concentrations of lutein (1–100 μM), lycopene (0.1–100 μM), and tomato extract (1–1000 μg/mL) reduced the cell viability in a dose-dependent manner . The celeriments .2O2) compared to the control group (p < 0.05). Pretreatment with different doses of lutein, lycopene, and tomato extract improved the oxidative stress induced by 80 ng/mL TNF-α + 200 µM H2O2 to different degrees (p < 0.05). ROS levels decreased continuously and in a dose-dependent manner when the concentrations of lutein, lycopene, and tomato extract were 0.5–5 μM, 0.5–5 μM, and 50–150 μg/mL, respectively. Instead, as the concentrations of lutein (10–20 μM), lycopene (10–20 μM), and tomato extract (200–400 μg/mL) increased, the ROS levels also increased gradually and in a dose-dependent manner . In a dose-dependent manner, 50–150 μg/mL of tomato extract, 0.5–5 μM lutein, and 0.5–10 μM lycopene increased activity of SOD  showed significantly lower GPX activity compared to the control group (233.84 U/mg protein) (p < 0.05); the groups treated with 0.5–20 μM lutein, 0.5–20 μM lycopene, and 50–400 μg/mL tomato extract all exhibited higher GPX activity compared to the T + H group. Moreover, GPX levels decreased after pretreatment with 15–20 μM lutein, 10–20 μM lycopene, and 200–400 μg/mL tomato extract, suggesting a pro-oxidation effect (The T + H group (80 ng/mL TNF-α+ 200 μM Hn effect .2O2) significantly upregulated the inflammatory VCAM (2294), ICAM (222.89), IL-1β (151.3), and IL-6 (165.70). The expressions of inflammatory proteins were successfully induced. Compared with the T+H group, the expressions of VCAM, ICAM, IL-1β, and IL-6 protein decreased gradually with the increase in lycopene concentration (p < 0.05). Tomato extract decreased the expression of VCAM and IL-6 proteins in a dose-dependent manner  significantly downregulated the expression of TGF-β1 (52.61–78.63) and IL-10 (56.37) anti-inflammatory factor proteins compared to the control group (p < 0.05). Moreover, 0.5–20 µM lutein, 0.5–20 µM lycopene, and 50–400 µg/mL tomato extract alleviated the 80 ng/mL TNF-α and 200 µM H2O2 co-induced downregulation of anti-inflammatory factor proteins (TGF-β1 and IL-10) to some extent (p < 0.05). Secondly, it showed a significant dose-dependent effect on the upregulation of TGF-β1 and IL-10. For example, at a lutein concentration of 0.5 µM, the grayscale values of TGF-β1 and IL-10 proteins expression were 129.31 ± 8.73 and 123.68 ± 1.78, respectively. When the concentration of lutein increased to 20 µM, the TGF-β1 and IL-10 proteins expression increased to 173.54 ± 34.2 and 169.48 ± 18.29, respectively (The T + H group (80 ng/mL TNF-α + 200 µM Hectively .2O2), intracellular iNOS expression gradually decreased with increasing concentrations of lutein, lycopene, and tomato extract (p < 0.05), indicating that 0.5–20 μM lutein, 0.5–20 μM lycopene, and 50–400 μg/mL tomato extract inhibited the expression of iNOS and thus reduced NO production  and iNOS , act as oduction .2O2) group significantly inhibited Nrf2 (43.24 ± 3.07), NQO-1 (58.76 ± 6.96), and HO-1 (53.63 ± 1.06) protein expressions compared with the control group (p < 0.05). In addition, 0.5–20 µM lutein, 0.5–20 µM lycopene, and 50–400 µg/mL tomato extract pretreatment increased the expression of the Nrf2 signaling pathway downstream proteins  compared to the T + H group (p < 0.05), suggesting that 0.5–20 µM lutein, 0.5–20 µM lycopene, and 50–400 µg/mL tomato extract activated the Nrf2 signaling pathway.The T + H (80 ng/mL TNF-α + 200 µM Hp < 0.05), whereas 10–20 µM lutein and 10–20 µM lycopene dose dependently decreased the expression of Nrf2, HO-1, and NQO-1 (p < 0.05). The expression of antioxidant proteins downstream of the Nrf2 signaling pathway showed an increasing trend followed by a decreasing trend. For example, when the concentration of lutein was 0.5 µM, the expression of HO-1 was 69.2 ± 4.9. When the concentration of lutein increased to 10 µM, the expression of HO-1 increased to 211.2 ± 24.5. However, when the concentration of lutein continued to increase to 20 µM, the expression of HO-1 decreased to 41.52 ± 2.7  was activated by 0.5–20 µM lutein. Moreover, the activation trend of 0.5–20 µM lycopene and 50–400 µg/mL tomato extract showed a trend of increasing and then decreasing. This is generally consistent with the changes in intracellular ROS induced by 0.5–20 µM lutein, 0.5–20 µM lycopene, and 50–400 µg/mL tomato extract. The modulation of the Nrf2 signaling pathway by different concentrations of lutein, lycopene, and tomato extract was likely to be the mechanism by which they produced low concentrations of antioxidant effects and high concentrations of pro-oxidant effects.p < 0.05). When the concentration continued to increase, the expression of Nrf2 protein in the nucleus of 10–20 μM lutein, 10–20 μM lycopene, and 200–400 μg/mL tomato extract did not continue to increase  was significantly higher compared with that in the control group (164.3 ± 14.85), indicating that 80 ng/mL TNF-α + 200 µM H2O2 can effectively activate the NF-κB pathway. Lutein, lycopene, and tomato extract pretreatment can inhibit the expression of NF-κB p-p65 to different degrees compared to the T + H group. When the concentration of lutein increased from 10 µM to 20 µM, the grayscale value of NF-κB p-p65 protein decreased significantly from 164.5 ± 18.27 to 37.97 ± 17.35 (p < 0.05). At the same time, lycopene and tomato extract also decreased the expression of NF-κB p-p65 in a dose-dependent manner, indicating that the pathway was inhibited  was significantly higher (p < 0.05). Furthermore, 0.5–20 µM lutein (127.7 ± 25.96–49.58 ± 24.63), 0.5–20 µM lycopene (125.3 ± 2.14–96.16 ± 18.79), and 50–400 µg/mL tomato extract (111.3 ± 4.63–66.15 ± 1.48) significantly decreased nuclear NF-κB expression (p < 0.05)  . QuantitInhibitory modifications to the NF-κB signaling pathway increased with concentration, although the capacity of 10–20 µM lutein, 10–20 µM lycopene, and 200–400 µg/mL tomato extract to activate Nrf2 signaling pathway and downstream protein expression reduced with the increase in concentrations. As a result, it was speculated that increased production of anti-inflammatory molecules (TGF-1 and IL-10) suppressed the expression of Nrf2 signaling pathway-related proteins. In this work, siRNA gene-silencing technology was utilized to silence anti-inflammatory factors TGF-1 and IL-10, and then Q-PCR technology was employed to determine the expression of Nrf2, HO-1, and NF-κB genes.p < 0.05) after treatment with different concentrations of lutein, lycopene, and tomato extract under the premise of TGF-β1 gene silencing. Meanwhile, the expression of Nrf2 was no longer decreased by high concentrations of lutein (10–20 µM) lycopene (10–15 µM), or tomato extract (200–400 µg/mL) pretreatment. This indicates that Nrf2 may depend on TGF-β1 to function. The Nrf2 expression level was downregulated by TGF-β1 at high concentrations of lutein, lycopene, and tomato extract (The expression of the Nrf2 gene in the TGF-β1 gene-silenced model group (75.97 ± 0.57%) was significantly higher than that in the non-silenced model group (49.28 ± 1.52%). The expression of Nrf2 was significantly higher , and HO-1 mRNA expression in high concentrations of lutein (10–20 µM) and tomato extract (200–400 µg/mL) no longer decreased. These results suggested that HO-1 may be dependent on TGF-β1 to exert antioxidant effects.Lutein, lycopene, and tomato extract may be controlled by TGF-β1 to affect HO-1 expression and exert antioxidant effects. After the treatment with different concentrations of lutein and lycopene, HO-1 mRNA expression showed a trend of increasing low concentration and decreasing high concentration compared to the T + group (T + was a model group transfected with TGF-β1 at 80 ng/mL TNF-α + 200 µM Hp < 0.05). The results indicate that the expression of NF-κB was increased after inhibition of TGF-β1, and the NF-κB signaling pathway was dependent on TGF-β1 to play an anti-inflammatory role. However, compared with the T + group, NF-κB mRNA expression after different concentrations of lycopene and tomato extract was not increased  than that in the T + group (157.1 ± 14.85%) and significantly increased . In addition, the expression of Nrf2 no longer decreased at high concentrations of lutein (10–20 µM), lycopene (10–15 µM), and tomato extract (200–400 µg/mL)  A.p < 0.05). In addition, the expression of HO-1 did not decrease after high concentration of lycopene (10–15 µM), indicating that HO-1 may depend on IL-10 to act.Lutein, lycopene, and tomato extract may be controlled by IL-10 to affect the expression of HO-1. High concentrations of lutein, lycopene, and tomato extract may downregulate the HO-1 expression depending on IL-10, thus playing a role in promoting oxidation. Under the premise of silencing the IL-10 gene, HO-1 expression was significantly increased after the treatment with different concentrations of lutein, lycopene, and tomato extract (p < 0.05). In addition, the expression of NF-κB in high concentrations of lutein (10–20 µM), lycopene (10–15 µM), and tomato extract (200–400 µg/mL) no longer decreased, indicating that Nrf2 may depend on IL-10. High concentrations of lutein, lycopene, and tomato extract may depend on IL-10 to downregulate the expression of NF-κB, thus playing an anti-inflammatory role.Lutein, lycopene, and tomato extract may be controlled by IL-10, which may affect the expression of NF-κB and play an anti-inflammatory role. Under the premise of silencing the IL-10 gene, the expression of NF-κB was significantly increased after different concentrations of lutein, lycopene, and tomato extract  caused in H9c2 cells was altered by 0.5–20 µM lutein, 0.5–20 µM lycopene, and 50–400 µg/mL tomato extract. It was discovered that the antioxidant effects of 0.5–5 μM lutein, 0.5–5 μM lycopene, and 50–200 μg/mL tomato extract were continually strengthened. The antioxidant activity of carotenoids was dramatically impacted by the chemical structure, membrane binding site, concentration, and oxygen partial pressure [The DCFH-DA assay has been widely applied in cell studies and used as a marker of cellular oxidative stress. The oxidation of DCFH produces DCF, a fluorescent compound that was originally thought to be useful as a specific indicator of Hther ROS . In thispressure . All C=Cpressure . Accordipressure . The conpressure .The total glutathione peroxidase assay is a simple UV kit used to detect glutathione peroxidase activity in cells. The organic peroxide reagent provided in this kit is cumene hydroperoxide (Cum-OOH). The organic peroxide reagent (Cum-OOH) provided by the GPX Assay Kit does not react with glutathione in the presence of glutathione peroxidase and is not catalyzed by intracellular catalase. Therefore, the activity of glutathione peroxidase can be more specifically detected. The capacity of the main carotenoids of tomatoes to scavenge free radicals reduced when the concentration of lutein was more than 5 µM, that of lycopene was more than 5 µM, and that of tomato extract was more than 200 ng/mL. Moreover, the activity of antioxidant enzymes such as SOD and GPX ceased to rise and even fell. These carotenoids showed pro-oxidative action at high dosages despite having antioxidant activity at low amounts. Previous research indicated that the pro-oxidation effect of carotenoids may be due to the imbalance in the cellular redox state, an excessive concentration of carotenoids, or an excessively high oxygen partial pressure . CarotenIn this research work, lutein, lycopene, and tomato extract enhanced the anti-inflammatory effects in a dose-dependent manner. The anti-inflammatory effects of lutein (0.5–20 μM), lycopene (0.5–20 μM), and tomato extract (50–400 μg/mL) were simultaneously strengthened with higher concentrations. Previous studies have also verified that the anti-inflammatory effect of lycopene is dose dependent. Lycopene at doses of 20 mg/kg and 60 mg/kg could dramatically lower the content of inflammatory factors in serum and boost non-alcoholic fatty liver caused by high intakes of fat and sugar . The reg2O2. This proved that lutein, lycopene, and tomato extract at low concentrations activated the Nrf2 signaling pathway by enhancing Nrf2 nuclear transport. Nevertheless, as lutein, lycopene, and tomato extract concentrations increased, the expression of Nrf2 in the nucleus either stopped growing or showed a trend of decreasing after saturation, which was consistent with the trend of the intracellular ROS level and SOD and GPX antioxidant enzyme activity. These findings confirm that the pro-oxidation of high concentrations of lutein lycopene and tomato extracts are directly related to the Nrf2 signaling pathway. This may be the primary mechanism that exerts antioxidation at low concentrations and pro-oxidation at high concentrations.Nrf2 is a key transcription factor when cells are under oxidative stress and plays an important role in maintaining cell homeostasis . To presLutein, lycopene, and tomato extract have potent anti-inflammatory properties, decreasing the expression of pro-inflammatory factors in H9c2 cells , while increasing the expression of anti-inflammatory factors (TGF-1 and IL-10), and decreasing the activity of the iNOS enzyme may be crucial to the anti-inflammatory activities. In this research work, lutein, lycopene, and tomato extract enhanced the anti-inflammatory effects in a dose-dependent manner. The anti-inflammatory effects of lutein (0.5–20 μM), lycopene (0.5–20 μM), and tomato extract (50–400 μg/mL) were simultaneously strengthened with higher concentrations. This was mainly because IκBα was induced to undergo phosphorylation after stimulation by TNF-α. Phosphorylated IκBα degrades rapidly, releasing NF-κB into the nucleus and controlling transcriptional genes . Lutein,Oxidative stress and inflammation are controlled through functional interactions and cross-talk between Nrf2 and NF-κB pathways to maintain a sustainable balance of cellular redox states . AdditioThe NF-κB signaling pathway was consistently blocked by treatments with lutein (10–20 µM), lycopene (10–20 µM), and tomato extract (200–400 µg/mL), and the results showed a decreasing trend in the expression of the downstream protein of the Nrf2 signaling pathway in H9c2 cells, as well as the accumulation of Nrf2 in the nucleus. As a result, it was hypothesized that increased production of anti-inflammatory factors would suppress the expression of Nrf2 signaling pathway-related proteins. TGF-β1 and IL-10, representative inhibitory cytokines, play crucial roles in the regulation of immune homeostasis and have been shown to have outstanding therapeutic potential in inflammatory diseases. Hence, it was conjectured that the increase in anti-inflammatory factor (TGF-β1 and IL-10) may be responsible for the decline in the downstream protein and nuclear transport of the Nrf2 pathway. The expressions of TGF-β1 and IL-10 mRNA were silenced via the siRNA gene-silencing technique to investigate the regulation of anti-inflammatory factors TGF-β1 and IL-10 on the Nrf2 signaling pathway and NF-κB signaling pathway. p < 0.05). Meanwhile, lutein lycopene and tomato extract may be controlled by TGF-β1 to affect the expression of HO-1 and play an antioxidant role. After inhibiting the expression of TGF-β1 gene, the expression of NF-κB increased, and the NF-κB signaling pathway may depend on TGF-β1 for its anti-inflammatory effects.Under the premise of silencing the TGF-β1 gene, the expression of Nrf2 was significantly increased after treatment with different concentrations of lutein lycopene and tomato extract (p < 0.05). High levels of lutein, lycopene, and tomato extract may rely on IL-10 to suppress the production of HO-1, thus increasing oxidation. The expression of HO-1 was considerably raised following treatment with lutein, lycopene, and tomato extract at different dosages, whereas the IL-10 gene was silenced (p < 0.05). The expression of NF-κB was significantly increased after treatment with lutein, lycopene, and tomato extract under the condition of silencing the IL-10 gene (p < 0.05), indicating that lutein, lycopene, and tomato extract have a significant role in anti-inflammatory activity through the expression of NF-κB by controlling IL-10.Similarly, after silencing the IL-10 gene, the expression of Nrf2 increased considerably following treatment with lutein lycopene and tomato extract at different dosages  can upregulate the expression of Nrf2/HO-1 and NF-κB mRNA to a certain extent. High concentrations of lutein, lycopene, and tomato extract downregulate Nrf2/HO-1 and NF-κB signaling pathways depending on TGF-β1 and IL-10, thus exerting low-concentration antioxidant and anti-inflammatory effects. Antioxidant and anti-inflammatory effects are effective therapeutic targets for CVD, and targeting TGF-β1 and IL-10 to regulate Nrf2/HO-1 and NF-κB signaling pathways may be a new therapeutic strategy for CVD. This finding will assist in properly elucidating the underlying molecular mechanisms of antioxidants at low concentrations and anti-inflammatory effects at high concentrations, and more effectively guide the dose–response relationship between lutein, lycopene, and tomato extract in preventing the occurrence of CVD.It was found that lutein, lycopene, and tomato extract primarily exhibited antioxidant effects at low concentrations and pro-oxidation effects at high concentrations. In addition, the higher the concentration of lutein, lycopene, and tomato extract, the greater the anti-inflammatory activity. Lutein, lycopene, and tomato extract can exert antioxidant effects via activating the Nrf2 signaling pathway and exert anti-inflammatory effects by inhibiting the NF-κB signaling pathway.Furthermore, the expression of Nrf2 downstream proteins  and nuclear translocation of Nrf2 were suppressed after treatment with high concentrations of lutein, lycopene, and tomato extract. This also unequivocally proved that the Nrf2 signaling pathway can function as both a low-concentration antioxidant and a high-concentration pro-oxidant, while the anti-inflammatory effect of high concentrations may occur via the inhibition of the nuclear shift of NF-κB. TGF-β1 and IL-10 can target the regulation of Nrf2/HO-1 and NF-κB to achieve a low concentration of antioxidants and a high concentration of anti-inflammatory effects. Nrf2 and NF-κB signaling pathways, and anti-inflammatory factors such as TGF-β1 and IL-10, may be potential therapeutic targets for CVD.


DndABCDE-catalyzed DNA phosphorothioation (PT), in which the nonbridging oxygen is swapped with a sulfur atom, was first identified in the bacterial genome. Usually, this modification gene cluster is paired with a restriction module consisting of DndF, DndG, and DndH. Although the mechanisms for the antiphage activity conferred by this Dnd-related restriction and modification (R-M) system have been well characterized, several features remain unclear, including the antiphage spectrum and potential interference with DNA methylation. Recently, a novel PT-related R-M system, composed of the modification module SspABCD paired with a single restriction enzyme, SspE, was revealed to be widespread in the bacterial kingdom, which aroused our interest in the interaction between Dnd- and Ssp-based R-M systems. In this study, we discussed the action of Dnd-related R-M systems against phages and demonstrated that the host could benefit from the protection provided by Dnd-related R-M systems against infection by various lytic phages as well as temperate phages. However, this defense barrier would fail against lysogenic phages. Interestingly, DNA methylation, even in the consensus sequence recognized by the Dnd system, could not weaken the restriction efficiency. Finally, we explored the interaction between Dnd- and Ssp-based R-M systems and found that these two systems were compatible. This study not only expands our knowledge of Dnd-associated R-M systems but also reveals a complex interaction between different defense barriers that coexist in the cell.Recently, we decoded the mechanism of Dnd-related R-M systems against genetic parasites. In the presence of exogenous DNA that lacks PT, the macromolecular machine consisting of DndF, DndG, and DndH undergoes conformational changes to perform DNA binding, translocation, and DNA nicking activities and scavenge the foreign DNA. However, several questions remain unanswered, including questions regarding the antiphage spectrum, potential interference by DNA methylation, and interplay with other PT-dependent R-M systems. Here, we revealed that the host could benefit from Dnd-related R-M systems for a broad range of antiphage activities, regardless of the presence of DNA methylation. Furthermore, we demonstrated that the convergence of Dnd- and Ssp-related R-M systems could confer to the host a stronger antiphage ability through the additive suppression of phage replication. This study not only deepens our understanding of PT-related defense barriers but also expands our knowledge of the arms race between bacteria and their predators. Although DndFGH could inhibit phage lysogenization effectively, this defense barrier failed to protect the host on prophage induction. Moreover, by taking advantage of the Dnd R-M systems from Bermanella marisrubri RED65, which recognized 5′-GATC-3′/5′-GATC-3′ motifs, we revealed that the methyl group in 5′-G6mATC-3′/5′-G6mATC-3′ would not inhibit the restriction activity of DndFGH. Finally, we combined Dnd R-M and Ssp R-M systems. We found that these two kinds of PT-associated R-M systems were compatible. These findings not only expand our knowledge of PT-associated R-M systems but also deepen our understanding of the arms race between bacteria and phages.Although the molecular mechanism underlying the DNA damage caused by DndFGH has been well studied, its phage resistance spectrum has not been characterized. Furthermore, the effect of the convergence of Dnd- and Ssp-related restriction–modification  systems is less well known. In this study, we determined the spectrum of phage resistance conferred by DndFGH and demonstrated that the Dnd R-M system could protect the host from various lytic phages  from coli B7A . Based ocoli B7A . HoweverProteus mirabilis 1166 PMIR (Dnd1166 R-M) with undefined modification motifs, the Dnd R-M system from E. coli B7A (DndB7A R-M) recognizing 5′-GPSAAC-3′/5′-GPSTTC-3′, and the Dnd R-M system from B. marisrubri RED65 (DndRED65 R-M) recognizing 5′-GPSATC-3′/5′-GPSATC-3′ , d(GPSA) and d(GPST) were identified in the genome of P. mirabilis 1166 PMIR as well as DH10B(pWHU4386), implying that the motif in Dnd1166 was 5′-GPSAAC-3′/5′-GPSTTC-3′ , 27ProtePSTTC-3′ ; Fig. S1PSTTC-3′ . To quanactivity . Interesle to T7 .1166 R-M and DndB7A R-M prompted us to challenge DH10B(pWHU4386) and DH10B(pWHU4387) with phage cocktails at various multiplicities of infection (MOIs). According to the antiphage performance of Dnd R-M systems during coincubation in the growth curve assay, different phage cocktails were prepared. As shown in 1166 R-M systems exhibited activity against the phage cocktails consisting of T1, T5, and EEP at an MOI of 0.01, which has been determined to be the limit of T5 tolerance for Dnd1166 R-M systems. However, with the addition of T4 and T7, even at an MOI of 0.001, which is the lowest concentration that we tested, the growth curve collapsed similarly to that of the control group. Again, DndB7A R-M systems exhibited strong antiphage activity against the phage cocktails consisting of T1, T5, T7, and EEP even at an MOI of 5, which was the highest concentration that we tested. However, consistent with the phenomenon that we observed in Dnd1166 R-M systems, the growth curve collapsed when strains carrying DndB7A R-M systems were challenged with the all-in-one phage cocktail , DH10B(Dnd1166 R-M), and DH10B(DndRED65 R-M) with EOPs of (1.64 ± 0.58) × 10−2, (3.05 ± 0.22) × 10−1, and (3.13 ± 1.67) × 10−2, respectively, indicating that the Dnd R-M system could protect the host from invasion by temperate phages such as λ (1166 R-M), the lysogenization efficiency was reduced to 2.78% ± 2.15%, and no lysogens were detected in strains harboring DndB7A or DndRED65 R-M , (1.01 ± 1.56) × 10−8, (0 ± 0), and (0 ± 0) for strains carrying pACYC184, pWHU4386, pWHU4387, and pWHU4388, respectively   .1166 or DndB7A R-M systems, and the EOP was (3.05 ± 0.22) × 10−1 or (3.26 ± 0.63) × 10−1 and (1.64 ± 0.58) × 10−2 or (1.93 ± 0.71) × 10−2 for the Dnd1166 and DndB7A R-M systems at 28°C or 42°C, respectively. However, the EOP increased by approximately one order of magnitude from (3.13 ± 1.67) × 10−2 to (3.64 ± 0.47) × 10−1 for the DndRED65 R-M systems as the temperature increased. Considering that this value was close to that from Dnd1166 R-M, we hypothesized that this weakened defense system could still provide sufficient protection  × 10−4 or (4.68 ± 0.29) × 10−4, respectively, which was similar to the situation in DH10B(pACYC184) ([4.30 ± 0.30] × 10−4 versus [6.09 ± 1.52] × 10−4)  .RED65 R-M systems to methylated DNA. First, we determined the modification motif of the DNA methylase (protein_ID = YP_003925.1) encoded by the T1 phage. The genomic DNA of T1 prepared from JW3350 remained sensitive to DpnI, indicating that this Dam methyltransferase could convert 5′-GATC-3′/5′-GATC-3′ to 5′-G6mATC-3′/5′-G6mATC-3′, which is consistent with previous research (dam from the T1 genome using the CRISPR-Cas system to generate CC20 (RED65 R-M) with T1 or CC20, the EOP as well as the efficiency of the center of infection (ECOI) were determined by calculating the ratio of the phage titer measured in DH10B(DndRED65 R-M) to that measured in DH10B(pACYC184) (RED65 R-M) showed similar resistance to both CC20 and T1, with an EOP of (1.11 ± 0.07) × 10−1 versus (1.04 ± 0.13) × 10−1 and an ECOI of (1.23 ± 0.28) × 10−1 versus (1.07 ± 0.19) × 10−1, respectively, implying that DndFGH could scavenge invaders regardless of DNA methylation. Furthermore, to explore the potential influence of the DndRED65 R-M system on phage proliferation in a single life cycle in more detail, one-step growth curves were generated for DH10B(pACYC184) and DH10B(DndRED65 R-M) infected with CC20 and T1, respectively (RED65 R-M). However, without the protection of the DndRED65 R-M system, the burst sizes of T1 and CC20 were 52.26 ± 3.10 and 31.93 ± 2.73, respectively, indicating that the decrease was caused by dam deletion rather than the effect of the DndRED65 R-M system. These data demonstrated that DNA methylation would not interfere with the action of Dnd R-M systems even if both systems recognized the consensus sequence. Surprisingly, the presence of DndRED65 R-M systems showed no significant influence on the burst size of T1 phage , which seemed contradictory to the previous results , which exhibited the strongest protection, was treated as the host. Consistent with the previous results, DndB7A R-M conferred the host with powerful protection with an ECOI of (3.12 ± 0.27) × 10−3, indicating that there was only a narrow chance for T1 to produce at least one active progeny in the presence of DndB7A R-M. However, the burst size of T1 decreased slightly from 52.26 ± 3.10 to 40.65 ± 5.95   . Surprisectively . Compare 0.0185) . These dPSCA-3′ generated by SspABCD and exert nicking activity toward unmodified DNA, thus protecting the host from various phages , , , and . First, we determined the growth rate of these strains to check for potential conflict between these two kinds of PT-based systems. Similar growth curves were observed using either LB broth or M9 broth, suggesting that the combination of these two kinds of PT-related R-M systems did not inhibit cell growth  × 10−2 and (1.07 ± 0.18) × 10−1, respectively, the combination of these two systems significantly decreased the ECOI to (7.94 ± 1.48) × 10−3  × 10−3 . Similar) × 10−3 .The increase in phage resistance caused by the combination of the Dnd and Ssp R-M systems prompted us to perform an qRT-PCR assay, which is an effective and simple method , 35, to In this study, we proved that similar to Ssp R-M systems, DndABCDE-FGH can provide a solid defense against various phages, even phage cocktails. Interestingly, the antiphage and antiplasmid preferences were quite different among different Dnd R-M systems, even when they shared the same DNA motifs. However, although DndFGH exhibited strong resistance to invasion by temperate phages, such as λ, this fortress was not infallible against attack by “internal enemies”. This was reasonable considering the action mode of R-M systems, which distinguish self- and nonself-DNA based on the specific modification tags. A prophage that is successfully integrated into the genome is recognized as self-DNA and modified as well as replicated together with the lysogen. With the protection provided by the PT modification, prophages can escape restriction by Dnd R-M systems. Notably, researchers recently proved that the PT modification could influence the transcription of the genes encoded by the prophage and alter the binding affinity of the repressor associated with prophage activation, indicating a more complex scenario of the interaction between Dnd systems and prophages .3H]-labeled AdoMet . Although these two Dam methylases could both modify 5′-GATC-3′/5′-GATC-3′ to generate 5′-G6mATC-3′/5′-G6mATC-3′, no significant sequence similarity was found between these two kinds of Dam methylases. This prompted us to check the modification status of the CC20 progeny released from BW25113  broth or on LB agar plates unless otherwise indicated. Proteus mirabilis strains were grown in LB broth at 37°C. B. marisrubri strains were grown in DSMZ Medium 514c at 28°C. When necessary, an appropriate concentration of antibiotic (100 µg/mL ampicillin and/or 25 µg/mL chloramphenicol) was added to the medium.All of the strains, bacteriophages, and plasmids used in this study are listed in dndBCDEFGH gene cluster was amplified from the genomic DNA of P. mirabilis 1166 PMIR using the primer pair 1166F/1166R  was diluted 1:100 in LB medium containing 100 µg/mL ampicillin and 25 µg/mL chloramphenicol at 37°C until the optical density at 600 nm (OD600) reached 0.6–0.8. A 300-µL bacterial culture was mixed with molten soft agar (0.75%) and immediately overlaid on prepoured LB agar (1.5%) plates. Phage T1 stocks were subjected to 10-fold serial dilution with SM buffer  and spotted on the upper layer of the plate. After incubation overnight at 37°C, plaques were verified by PCR using the primer pair Dam-LL/Dam-RR  and immediately overlaid on prepoured LB agar (1.5%) plates. Phage stocks were subjected to 10-fold serial dilution with SM buffer. Once the top agar solidified, 5 µL of each 10-fold diluted phage stock was spotted onto the bacterial lawns and cultured overnight at the indicated temperatures.Overnight-cultured E. coli strains were diluted 1:100 in LB medium containing the appropriate antibiotic and incubated until they reached the log phase (~OD600 = 0.6–0.8). The molten soft agar (0.75%) containing 100 µL of serially diluted phage solution and 300 µL of mid-exponential bacterial culture was poured onto a preprepared LB agar (1.5%) plate and incubated overnight at a fixed temperature. EOP was calculated by dividing PFU/mL (on the tested strain) by PFU/mL (on the control strain).Overnight-cultured E. coli or S. enterica strains were diluted 1:100 in LB broth containing 1 mM CaCl2, 1 mM MgCl2, and the appropriate antibiotic at 37°C or 28°C until the OD600 reached 0.5. One milliliter of the bacterial culture was mixed with phage solution to meet the multiplicity of infection (MOI) of 0.1. The mixture was incubated without shaking for 5 minutes for phage absorption. After centrifugation at 10,000× g for 1 minute, the pellet was washed twice with LB broth and resuspended in 1 mL of LB. Samples were subjected to 10-fold serial dilution, mixed with molten soft agar (0.75%) containing the phage-sensitive host, poured onto preprepared LB agar (1.5%) plates, and incubated overnight. The ECOI was calculated by dividing the number of COIs on the sensitive strain by the number of COIs on the resistant strain.Overnight-cultured E. coli strains were diluted 1:100 in LB broth containing the appropriate antibiotic, 1 mM CaCl2, and 1 mM MgCl2 and incubated at 37°C until the OD600 reached 0.6. Phage was added to the log-phase cells to achieve a final MOI of 0.01. After a 5-minute incubation, 1 mL of cell cultures was centrifuged at 10,000× g for 1 minute to remove the unabsorbed phages. The pellet was washed twice, resuspended in 1 mL of LB, subjected to 10-fold serial dilution, and incubated at 37°C. Then, 100 µL aliquots were taken periodically over 60 minutes from the appropriate dilution gradient, mixed with molten soft agar (0.75%) containing DH10B and overlaid onto preprepared LB agar (1.5%) plates. The burst size of the phage was determined by dividing the PFU/mL in the latent period by the PFU/mL in the plateau period.Overnight-cultured E. coli or S. enterica strains were diluted 1:100 in LB or M9 medium containing the appropriate antibiotic and incubated until the OD600 reached 0.5. The log-phase cultures were diluted 1:100 in LB or M9 medium and transferred into a 96-well plate to monitor the growth of samples using a microplate spectrophotometer . The OD600 of each sample was measured at 30-minute intervals during the incubation. For the phage infection growth curve, phages were added at various MOIs to log-phase (OD600 = 0.5) cells and cocultured in a 96-well plate to monitor the growth of the cells by a microplate spectrophotometer .Overnight-cultured E. coli BW25113 or JW3350 was transformed in parallel into chemically competent cells with or without dndBCDE-FGH modules. After overnight cultivation on LB agar plates with or without ampicillin, the transformation efficiency was measured by dividing CFU/mL (from plates with ampicillin) by CFU/mL (from plates without ampicillin).Fifty nanograms of pBluescript II SK+ isolated from 600 = 0.5) E. coli or S. enterica strains were infected with phage T7 at an MOI of 0.5 or phage PT1 at an MOI of 5. Samples were harvested at each time point, and DNA was extracted using the chloroform–phenol method. For each quantitative RT-PCR (qRT-PCR), 1 ng of total DNA was added as a template. The primer pairs RT-T7-F/RT-T7-R and RT-E-gapA-F/RT-E-gapA-R were used to monitor the abundance of DNA from T7 and E. coli, respectively. For the quantification of DNA abundance from S. enterica and PT1, the primer pairs RT-PT1-F/RT-PT1-R and RT-S-gapA-F/RT-S-gapA-R were used instead (−ΔΔCt) method was applied to determine the relative DNA level.Log-phase  analysis, as previously described (escribed . Brieflyg for 1 minute. The culture was diluted to an appropriate gradient and overlaid on LB agar (1.5%) plates. After incubation for 24 hours at 28°C, the colonies were checked by PCR using the primer pair Lambda-F/Lambda-R  plates. The plates were incubated at 28°C or 42°C. The survival rate was calculated by dividing CFU/mL (from plates incubated at 42°C) by CFU/mL (from plates incubated at 28°C).To assess the survival rate of bacteria under the induction effect of λ600  after the temperature was shifted to 42°C. Meanwhile, bacteria were removed by centrifugation at 10,000× g for 1 minute. The supernatant was subjected to 10-fold serial dilution, mixed with DH10B in molten soft agar (0.75%), and immediately overlaid on prepoured LB agar (1.5%) plates. The PFU/mL was calculated after overnight incubation.To evaluate the growth rate of the bacteria and the progeny production efficiency, 1 mL of culture was taken at each time point to monitor the OD


Vibrio vulnificus. We additionally present a survey of reductive activity with eight alternative nitroaromatic substrates, to provide access to alternative ablation prodrugs, and explore applications such as remediation of dinitrotoluene pollutants. The predicted binding modes of four key substrates were investigated using molecular modelling.Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase (“NTR 2.0”) for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases. To provide insight into the enhanced enzymatic mechanism of NTR 2.0, we have solved the X-ray crystal structure at 1.85 Angstroms resolution and compared it to the parental enzyme, NfsB from  Nitroaromatic compounds are relatively rare in nature, but many have been synthesised for industrial purposes, e.g., explosives, dyes and pesticides . NitroreThe ability of nitroreductase enzymes to convert nitroaromatic prodrugs into potent cytotoxins has been exploited to develop cancer therapies that target cancer cells while leaving healthy cells unharmed . NitroreEscherichia coli has been used in partnership with the 5-nitroimidazole prodrug metronidazole [E. coli NfsB or a mutant variant thereof [For most targeted ablation studies in zebrafish, the nitroreductase NfsB from nidazole ) nature nidazole . However thereof  frequentE. coli NfsB [Vibrio vulnificus by introduction of rationally identified F70A and F108Y residue substitutions. NTR 2.0 exhibits faster ablation kinetics and requires lower metronidazole doses, permitting enhanced interrogations of cell function, ablation of previously recalcitrant cell types and novel sustained ablation paradigms that will be useful for modelling of chronic disease states [To resolve these issues, we recently engineered an improved nitroreductase, NTR 2.0, that achieves metronidazole-mediated cell-specific ablation at 100-fold lower metronidazole concentrations than required by oli NfsB . NTR 2.0e states . To bettC 2 2 21 with unit cell parameters a = 68.43, b = 137.36, c = 107.04, α = 90°, β = 90°, and γ = 90°. The asymmetric unit contained two NTR 2.0 molecules, with a Matthews coefficient of 2.32 Å3/Da and a solvent content of 47% [NTR 2.0 was successfully expressed, purified, and crystallised using a sodium acetate and polyethylene glycol precipitant, resulting in crystals that diffracted to 1.71 Å . The NTRV. vulnificus NfsB enzyme [As previously reported, NTR 2.0 is a double mutant that exhibits substantially improved metronidazole reductase activity compared to the parental B enzyme . To inveB enzyme ,10,11, nB enzyme . InsteadB enzyme ,13,14. AV. vulnificus NfsB are compared in In both 6CZP and our NTR 2.0 structure, two monomers interact head-to-tail, resulting in a dimer with a large interface. Each dimer has two active sites, formed at this interface and comprised of residues from both monomers. In both our wild-type and mutant structures, one FMN and one acetate molecule occupy each active site. Inhibition studies have demonstrated that acetate binds only to the oxidised form of a NfsB family nitroreductase and competitively inhibits both substrate and NADPH binding . The terNTR 2.0 and the parental enzyme share high structural similarity (root-mean-square deviation (RMSD) of 0.32 Å); however, significant differences were noted in the active site .In the NTR 2.0 structure, mutation of active site residue 108 from phenylalanine to tyrosine introduces hydrogen bonding from the Y108 hydroxyl group to nearby residue E102. This new bond may stabilise the active site, lowering the energy of the transition state during metronidazole catalysis. Additionally, nearby residue F199 is displaced in NTR 2.0 by 1.5 Å, the significance of which is not readily discernible .Notably, the NTR 2.0 structure also exhibits a widened substrate entrance channel compared to the wild-type, resulting from the displacement of residues F123, F124 and F68 and the removal of the aromatic side chain at residue F70 . It is wConsurf is a computational tool that aligns an input protein sequence against a set of orthologous sequences from a reference database and assigns a conservation score to each residue . FollowiE. coli, which enabled their activity to be monitored by half maximal inhibitory concentration (IC50) growth inhibition assays using nitroreductase-expressing strains.If a widened substrate access channel/reduced steric hindrance is the main driver behind improved metronidazole activity, we might expect NTR 2.0 to be a generally more efficient enzyme that is improved over the parental enzyme in its activity with a broad range of other nitroaromatic substrates. To investigate this, and to assess the potential of NTR 2.0 for applications beyond metronidazole-mediated ablation, we surveyed the activity of NTR 2.0 with a panel including eight alternative nitroaromatic substrates  was most potent, but nitrofurantoin (NTR 2.0 IC50 = 29.7 ± 0.1 μM) was comparable, and might provide a suitable alternative for ablation in any scenarios where metronidazole is ineffective. Overall, our data were consistent with the supposition that the widened substrate access channel is generally beneficial to activity with diverse substrates. Other possible impacts of the F70A and F108Y substitutions on active site configuration and substrate binding were explored by molecular modelling.Relative to unmodified azomycin B. The prE. coli NfsA and NfsB [2O molecules, respectively  for docking into the active site of both NTR 2.0 and the parental enzyme . For thiand NfsB ,20. It sectively A,B, whicMetronidazole (MTZ). The plane-to-plane distance between the imidazole ring of MTZ and isoalloxazine in both enzyme forms is approximately 3.5–3.8 Å  and 4.3 Å (NTR 2.0). The calculated Ein are −80.9 kJ·mol−1 and −84.1 kJ·mol−1, respectively..5–3.8 Å C,D. Howenidazole C,D. The Tinidazole (TNZ). As with MTZ, differences in H-bond formation are observed: in unmodified V. vulnificus NfsB, the H-bonds are formed between the tinidazole sulfonyl group and Lys14 and Lys74, whereas in NTR 2.0, Lys14 interacts with N3 of the imidazole ring, and the sulfonyl interacts with N3-H of isoalloxazine (V. vulnificus NfsB) and 4.7 Å (NTR 2.0), respectively. The calculated Ein are −32.7 kJ·mol−1 and −80.9 kJ·mol−1, respectively.loxazine E,F. The 2,6-Dinitrotoluene . When bound to both enzymes forms, the compound is nearly parallel with respect to the isoalloxazine ring of FMNH2, but is not involved in the π–π interaction with the isoalloxazine or Phe70 of V. vulnificus NfsB  and 5.1 Å (NTR 2.0), whereas Ein are equal to −75.1 kJ·mol−1 and −56.4 kJ·mol−1, respectively. The 4-nitro group forms H-bonds with Lys14 and Lys74.ach case I,J. The V. vulnificus NfsB parental enzyme. Reduction at the 4-nitro position yields a more potent cytotoxin capable of inter-strand DNA cross-linking via an N-acetoxy intermediate [The ability of activated CB1954 metabolites to cause a local zone of cell killing via diffusion from nitroreductase-expressing to neighbouring cells  offers rmediate , whilst rmediate  and tumormediate . It has rmediate , but potrmediate . At thisV. vulnificus NfsB enzyme, NfsA from E. coli and YfkO from Bacillus subtilis were examined .To investigate the reductive preference of NTR 2.0, the relative levels of 2- vs. 4-hydroxylamine end-products generated were measured by reverse-phase high-performance liquid chromatography (HPLC) . In addiectively ,28,29, aE. coli strains 7NT  and the efflux pump gene tolC [The ene tolC ) and BL26 tag at the C-terminus. The expression construct was used to transform E. coli BL21 cells and grown in LB medium containing 50 µg·mL−1 kanamycin at 37 °C. After induction with 0.5 mM IPTG, the culture was incubated for a further 24 h at 18 °C. The culture was harvested by centrifugation at 2600× g for 20 min. NTR 2.0 was purified using nickel affinity chromatography where bound protein was eluted over a two-step wash and elute method, involving an initial wash at 60 mM imidazole to remove loosely bound non-target proteins and a second wash at 1 M imidazole to elute NTR 2.0. For further purification, size exclusion chromatography was performed using a Superdex S200 10/300 GL column  connected to an ÄKTA BasicTM FPLC system and a 40 mM Tris-HCl pH 7.0, 200 mM NaCl buffer. Fractions containing the target protein were assessed for purity by SDS-PAGE analysis and quantified by their 280 nm absorbance trace.The gene encoding NTR 2.0 was ordered pre-synthesised from Twist Bioscience . The gene fragment was cloned into pET28a(+) (Novagen) which expresses a His−1 in a buffer consisting of 40 mM Tris-HCl pH 7.0 and 200 mM NaCl. Initial protein crystallisation trials employed large-scale crystallisation screens covering 288 conditions  and used the sitting drop vapour diffusion method in 96-well plates at 291 K (18 °C). NTR 2.0 protein solution (100 nL) was mixed with precipitant solution (100 nL), and equilibrated beside a 100 µL reservoir of precipitant solution. Further crystallisation screens employed hanging-drop vapour diffusion in 24-well VDX plates  at 291 K. Crystals of NTR 2.0 formed within two weeks by mixing 1 µL of 10 mg/mL protein solution (40 mM Tris-HCl pH 7.0 and 200 mM NaCl) with 1 µL of precipitant solution (0.1 M sodium acetate trihydrate pH 4.5 and 32% (v/v) polyethylene glycol 300) and by equilibrating over a 500 µL reservoir of precipitant solution. Crystals were orthorhombic and yellow in colour, indicating the presence of the bound FMN cofactor in the oxidised state. Crystals were flash-frozen in liquid nitrogen prior to data collection.For crystallisation, NTR 2.0 was concentrated to 10 mg·mL2 gas at 100 K. Data were collected at the macromolecular crystallography beamline (MX1) at the Australian Synchrotron, Melbourne, Australia using an ADSC Quantum 210r detector at a wavelength of 0.9537 Å using 0.5° oscillation per image with a crystal-to-detector distance of 250 mm. A data set was collected to 1.71 Å resolution from a single crystal. All data processing was conducted using software found within CCP4 program suite 6.4.0  [Rmerge was <0.9 and the final structure was solved to 1.85 Å using molecular replacement with the starting model PDB code 1DS7. The data collection statistics are summarised in Due to the high polyethylene glycol concentration in this crystallisation condition, no additional cryo-protectant was required prior to crystal freezing/testing. For data collection, crystals were mounted in a stream of N4, 1994) . The dat4, 1994) . The int4, 1994) . Data weE. coli  as a model. Molecular replacement was carried out using Phaser [The NTR 2.0 structure was solved to 1.85 Å using molecular replacement using the structure of NfsB from g Phaser  in the Cg Phaser  followedE. coli 7NT strain was used, which bears scarless in-frame deletions of seven candidate NTR genes  and the efflux pump gene tolC [E. coli 7NT pUCX:NTR2.0 cells were used to inoculate wells of a sterile 96-well microplate containing 200 µL LB supplemented with 100 µg·mL−1 ampicillin and 0.2% (w/v) glucose. Cultures were incubated overnight at 30 °C, 200 r.p.m. The following morning, cultures were diluted 20-fold in induction medium . Cultures were incubated at 30 °C, 200 r.p.m. for 2.5 hours. Culture aliquots (30 µL) were added to sterile 384-well microplates containing 30 µL induction medium with or without twice the desired final prodrug concentration. For dose–response tests, each culture was exposed, in duplicate, to increasing titrations of prodrug (15 conditions containing two- or 1.5-fold stepwise increases) and one induction medium-only control. The medium was supplemented with DMSO as appropriate, ensuring that the final DMSO concentration remained <4%. OD600 readings were recorded using an EnSpire 2300 Multilabel Reader (PerkinElmer), and initial OD600 values were within the range of 0.12–0.18. Cultures were incubated at 30 °C, 200 r.p.m. for a further 4 hours, after which OD600 readings were recorded once more. The increase in OD600 of challenged and unchallenged wells for each strain was compared and used to calculate percentage growth at each prodrug concentration. GraphPad Prism 8.0 was used to calculate prodrug IC50 values using a dose–response inhibition four-parameter variable slope equation.The gene encoding NTR 2.0 was PCR amplified and cloned into the NdeI and SalI sites of the expression plasmid pUCX (Addgene no. 60681). To assess NTR 2.0 properties in a minimal nitroreductase background, the ene tolC . E. coliv/v) methanol (pH 6.5). A 100 µL volume of each sample was analysed by reverse phase-HPLC employing an Agilent 1200 series system with an Ascents C8 3 µm 150 × 4.6 mm column . The mobile phase used for HPLC analysis was 45 mM ammonium formate buffer (pH 6.5) as aqueous and 80% acetonitrile as organic. The HPLC run parameters consisted of 4 min at 5% organic, a linear increase to 50% organic from 4 to 19 min and a further gradient increase to 70% organic from 19 to 21 min. The flow rate was maintained at 1.5 mL·min−1 throughout, and the eluate was monitored at 262 nm. Elution profiles from each nitroreductase-CB1954 reaction were compared against control reactions for B. subtilis YfkO or E. coli NfsA, which have consistently been shown to exclusively reduce CB1954 at the 4- or 2-nitro positions, respectively [Reaction mixes of 100 µL comprising 10 mM Tris-HCl pH 7.0, 1 mM NADPH and 200 µM CB1954 were initiated by addition of 1.75 µM purified enzyme. Reactions were incubated for 25 min at room temperature before being stopped by addition of one volume ice-cold 100% methanol. Samples were transferred to −80 °C for at least 1 h to precipitate proteins after which samples were centrifuged for 10 min at 12,000× g, 4 °C. The supernatant was decanted and diluted 1:20 in 45 mM ammonium formate buffer, 2.5%  and NTR 2.0 (PDB code 7UWT). A simplified modelling approach was used involving the enzyme active center fragment containing the amino acid residues at a radius of 11 Å around the N5 atom of isoalloxazine ring of FMN [2). All the atoms heavier than hydrogen were frozen, except side chains of amino acids, which can form H-bonds with the substrates . The H2O molecules present in the starting structures were left to move freely in the active center. Previously, we have shown that water molecules are important for prediction of supramolecular host-guest structure [in) were calculated using Equation (1):For computer modelling, we used the crystal structures of g of FMN . FMN wastructure . The corV. vulnificus NfsB enzyme, NTR 2.0 was not impaired with any of the compounds tested and exhibited improved activity with 2,6-dinitrotoluene, 2,4-dinitrotoluene, RB6145, tinidazole and metronidazole. Reduction of CB1954 by NTR 2.0 yielded a 4-hydroxylamine product that is likely to generate a small local bystander effect, which could potentially be leveraged for modelling broader injury/trauma paradigms in transgenic animal models. The X-ray crystal structure of NTR 2.0 revealed a widened substrate access channel compared to the wild-type structure, which may contribute to the generally increased activity. The data from computer simulations show that key nitroaromatic substrates bind in the active sites of both NTR 2.0 and the parental enzyme in productive orientations, with the calculated distances between the N5-H of isoalloxazine and the oxygen of the nitro group varying between 6.7 and 4.2 Å. However, because of the significant data scattering, these data may be considered as approximate only. The inferred energies of nitroaromatic substrate binding are comparable to that of binding of nitrofurantoin to reduced NfsA, −91.6 kJ·mol-1, calculated in a similar way [In this study, we performed structural and activity analyses of NTR 2.0 to decipher its substrate selectivity with a variety of nitroaromatic prodrugs. Relative to the parental ilar way . Most liTogether, these structural and activity analyses shed light on the likely molecular mechanisms underpinning the improved activation of metronidazole, as well as additional substrates that may enhance the versatility of NTR 2.0 for a range of applications.


Recent studies have shown that derangements in kynurenine pathway metabolite levels are associated with various pathologies such as neurodegenerative diseases, schizophrenia, depression, bipolar disorder, rheumatoid arthritis, and cancer. Therefore, reliable, accurate, fast, and multiplex measurement methods for kynurenines have become increasingly important. This study aimed to validate a new mass spectrometric method for analyzing tryptophan metabolites.A tandem mass spectrometric method, including protein precipitation and evaporation steps, was developed to measure serum levels of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. Samples were separated using a Phenomenex Luna C18 reversed-phase column. The kynurenine pathway metabolites were detected by tandem mass spectrometry. The developed method was validated according to Clinical & Laboratory Standards Institute (CLSI) guidelines and applied to hemodialysis samples.The developed method was linear at the concentrations of 48.8 – 25,000, 0.98 – 500, 1.2–5000, 1.2–5000, and 0.98–250 ng/mL for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine, respectively. The imprecisions were less than 12 %. The median serum concentrations of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid were 10530, 1100, 218, 17.6, and 25.4 ng/mL in pre-dialysis blood samples, respectively. They were 4560, 664, 135, 7.4, and 12.8 ng/mL in post-dialysis blood samples, respectively.A fast, simple, cost-effective, accurate, robust, and validated tandem mass spectrometric method was developed, and the method was successfully used for the quantitation of kynurenine pathway metabolite concentrations in hemodialysis patients. Tryptophan and its biological molecules have essential roles in metabolism. It is metabolized by four major biological pathways . However, most of the tryptophan in the body is converted to kynurenine . Tryptopl-Tryptophan (CAS Number: 73–22-3), l-kynurenine (CAS Number 2922–83-0), kynurenic acid (CAS Number 492–27-3), 3-hydroxyanthranilic acid (CAS Number 548–93-6), 3-hydroxy-dl-kynurenine (CAS Number 484–78-6), HPLC grade water (CAS Number: 7732–18-5), acetonitrile (CAS Number 75–05-8), formic acid (CAS Number: 64–18-6) and donepezil hydrochloride (CAS Number 120011–70-3) were obtained from Sigma Aldrich.The separation of analytes was achieved using the C18 HPLC column , and the gradient elution of mobile phases A and B consisted of 100 % HPLC grade water with 0.1 % formic acid and 100 % acetonitrile with 0.1 % formic acid. The time-volume changes in the gradient program were as follows: 0–0.1 min., 25 % B; 1–2 min., 50 % B; 2–4 min., 100 % B; 4–5 min., 25 % B. Initially, isocratic elution was attempted. However, gradient elution was preferred as the metabolites could not be fully separated due to their physicochemical similarities. Different flow rates and run times from 1 min to 30 min were tried. The best peak shape and separation were achieved at a 0.8 mL / min flow rate and a run time of 5.0 min. The Shimadzu HPLC  system, coupled with a mass spectrometer, had a pump unit (LC-20 AD), an autosampler (SIL-20 AC HT), and an online degasser (DGU-20A3) unit. LC gradient for kynurenine pathway metabolites were shown in Kynurenine pathway metabolites were detected using an API 3200 (Applied Biosystems / MDS Sciex) tandem mass spectrometer set to ESI in positive multiple reaction monitoring (MRM) mode. The metabolites were identified by infusing pure standards. The Q1–Q3 ion transitions for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, 3-hydroxykynurenine and donepezil were 205.2/146.2, 190.2/144.0, 209.1/94.1, 154.0/136.0, 225.1/110.0, and 380.2/91.1, respectively. The main method optimization parameters for the mass spectrometer were set as follows: ionspray voltage of 5000 V; ion source temperature of 350 °C; gas1 and gas2 at 50 psi; curtain gas at 20 psi; and collision gas at 6 psi. Declustering entrance, collision cell exit potential, collision energy, entrance potential and dwell time for each analyte are shown in g for 10 min, and the supernatants were evaporated with nitrogen gas at 37 °C. The residues were dissolved with a 200 µL acetonitrile:water  solution, including 0.1 % formic acid, and 30 µL of the mixture was injected into the system.Serum metabolite concentrations were quantified using a modified method . BrieflyThe tandem mass spectrometric method was primarily validated according to the Clinical and Laboratory Standards Institute (CLSI) C62-A: Liquid Chromatography-Mass Spectrometry Methods protocol . Method The calibration standards and working solutions were prepared with the help of a surrogate matrix . Since tResponse serum spike was the peak area of the analyte-spiked serum pool. Response serum blank was the peak area of the analyte in the serum pool, and the surrogate response spike was the peak area of the analyte-spiked surrogate matrix. All metabolites' equivalency (%) ranged from 90 to 104 %. Consequently, the PBS solution, including 1 % BSA and human serum, had a similar matrix effect. Stock solutions of tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine were prepared at final concentrations of 25, 10, 10, 5, and 4 mg/mL, respectively. The calibration standards were prepared at concentration ranges of 48.8–25,000, 0.98–500, 1.2–5000, 1.2–5000, and 0.98–250 ng/mL for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine via serial dilution of the stock solutions with the surrogate matrix. A 100 ng/mL internal standard (donepezil) solution was prepared in methanol. Prepared solutions were stored at 2 to 8 °C until analysis.SPSS statistical software package version 21.0, EP Evaluator Release 8 version , and Excel (2010) were used for the statistical evaluation, and a p-value less than 0.05 was accepted as statistically significant.The linearity study followed the CLSI EP06-A protocol . CalibraThere are various approaches to determining the LLOQ value. The first method is based on determining the lowest analyte concentration that can be quantified with satisfactory precision and accuracy. According to FDA guidelines, data from at least five replicates of the analyte-spiked sample in at least three independent runs should be used. Calculated imprecision values should be ≤ ±20 %, and a deviation in accuracy should be ≤ ±20 % . Therefoblank and LOD = LOB + 1.65SDlow concentration sample  × 100.The recovery study followed the CLSI EP34 protocol . The recC2 was the measured analyte concentration after spiking, C0 was the initial analyte concentration of the solution, and C1 was the added known concentration of standard.The matrix effect is generally evaluated with one of two standard methods: post-column infusion or post-extraction spiking. In this study, the second method was preferred, as reported by Chambers et al. . In thisThe results of the recovery and matrix effect studies are shown in Results of the matrix effect study. Matrix effects were less than 6.7 % for all metabolites.The carry-over study analyzed low- and high-level samples in the order stated in CLSI EP10-A3 . Low conL1-L2-L3-H1-H2-L4-H3-H4-L5-L6-L7-L8-H5-H6-L9-H7-H8-L10-H9-H10-L11.The calculated results from the tandem mass analysis were recorded onto the EP Evaluator Release 8 program  and carry-over results were determined for each analyte. The EP Evaluator program determined the acceptability criteria based on CLSI protocol EP10-A3 guidelines.The carry-over values are shown in g for 10 min. Patient samples were kept at −80 °C until the day of analysis. This study was approved by Selcuk University's local ethics committee . The chromatograms of kynurenine pathway metabolites obtained from a patient serum sample before and after dialysis are shown in The validated method was applied to clinical samples obtained from patients with chronic kidney disease (CKD) who had been undergoing hemodialysis for at least three months in the dialysis unit of Selcuk University Faculty of Medicine Hospital. Two milliliters of blood were drawn into collection tubes, and then centrifuged at 2000 The kynurenine pathway metabolite levels and routine laboratory parameters measured in post- and pre-dialysis serum samples are presented in + [Tryptophan is an essential amino acid that is important for living organisms. Less than 1 % of the available tryptophan is used for protein synthesis . Tryptop+ . Since k+ . TherefoHowever, quantifying tryptophan and metabolite levels is challenging due to their low stability, low blood concentrations, and presence of interferences in biological matrices. Since each of the kynurenine pathway metabolites has different physiologically characteristic functions, it is vital that most of the metabolites are able to be quantified by a simultaneous measurement method. However, an important aspect of the problem is that the physiological concentrations of metabolites vary over a wide range; for example, tryptophan is found in micromolar concentrations, while other kynurenine pathway metabolites are found in nanomolar concentrations. Another critical problem is the need for selective and specific methods because these metabolites have similar physical properties and structures . While HMöller et al. revealed a tandem mass method for simultaneously measuring six different kynurenine pathway metabolites, including tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxyanthranilic acid, and quinolinic acid. Intra- and inter-assay imprecision values of the method ranged from 1.0 % to 9.9 %, and accuracy values ranged from 75 % to 107 %. The mean recovery values were between 96 % and 101 %, and the LLOQ values of tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxyanthranilic acid, and quinolinic acid were 0.0352 μM, 0.035 μM, 0.035 μM, 0.047 μM, 0.084 μM, and 0.096 μM, respectively. The method had advantages such as sufficient sensitivity, good recovery, and accuracy; however, its disadvantages were the required high amount of sample (800 µL), time-consuming pretreatment steps including solid phase extraction (SPE), and long run time 12 min) . Hu et a min . HuThe second important aspect of our study was the clinical application of a validated method for measuring kynurenine pathway metabolite levels in pre- and post-dialysis serum samples from patients with CKD who had undergone hemodialysis. There are few studies investigating kynurenine pathway metabolite levels in patients with CKD. Debnath et al. reported mean serum tryptophan, kynurenine, 3-hydroxykynurenine, kynurenic acid, and quinolinic acid levels in patients with CKD secondary to type 2 diabetes as 10,030, 790.4, 2,336, 26.9, and 611.2 ng/mL, respectively . Pawlak A validated LC-MS/MS method was developed to quantify concentrations of metabolites in the kynurenine pathway. It was then applied to clinical samples obtained from hemodialysis patients. The primary advantages of the method were its short run time, simple pretreatment steps, cost-effectiveness, good extraction recovery, high precision, sufficient sensitivity, and ability to provide multiplex analysis with enough selectivity. The metabolite levels measured in hemodialysis patients were consistent with those of previous studies, meaning that the developed method can be reliably used in clinical samples.Sedat Abusoglu: Conceptualization, Writing – original draft, Visualization. Duygu Eryavuz Onmaz: Conceptualization, Writing – review & editing. Gulsum Abusoglu: Investigation, Methodology. Fatma Humeyra Yerlikaya: Writing – original draft, Visualization. Ali Unlu: Supervision, Project administration.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Adipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown.Using electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons.2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCβ1). Blocking PKCβ1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCβ1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2.Adiponectin enhanced T-type CaOur study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCβ1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.The online version contains supplementary material available at 10.1186/s10194-023-01658-2. Adipor1 gene is potentially associated with the severity of postoperative pain but not cancer pain , where EC50 is the concentration at which the half-maximum effect occurs, X values are logarithms of concentrations, and n is the coefficient. The Boltzmann equation was used to fit plots showing voltage-dependent activation and steady-state inactivation.Data are presented as the mean ± S.E.M. Microsoft Excel, Prism 8.0, and Clampfit 10.2 were used for data collection and statistical analysis. Paired or unpaired IT). The isolated inward currents were not further affected by nitrendipine , a well-described L-type channel blocker, but were dramatically suppressed by TTA-P2 , a specific T-type channel blocker [IT isolation  TG neurons because they play pivotal roles in nociceptive processing , 27, 29. blocker , demonstne 5 μM, .8 ± 1.1%ut Figs. . We furtIT response. Immunoblot analysis demonstrated that adipoR1 , intra-TG injection of chemically modified adipoR1-siRNA significantly decreased the protein abundance of adipoR1  and adipoR2 subtypes have been identified as functional receptors for adiponectin in mammals . Thus, wIT increase. The two adiponectin receptors belong to a novel group of membrane receptors that contain seven transmembrane domains, similar to those in G protein-coupled receptors, but are both structurally and functionally distinct [IT response ; the protein expression level of total CaMKII (t-CaMKII) also remained unchanged  did not affect the adiponectin-induced IT response  were unaffected when 100 nM adiponectin was applied  remained unaffected  firing by 63.3 ± 5.7%  enhanced IT, whereas an inactive counterpart had no effect [IT induced by insulin-like growth factor-1 (IGF-1) in dorsal root ganglion (DRG) neurons [IT induced by M3 muscarinic receptor activation was abolished by PKC antagonists [Xenopus oocytes [V3.2 channels [PKC is a pivotal regulator of T-type channels . Studiesegulated , 58. Froo effect . Similar neurons . In contagonists . Similaragonists . Intrigu oocytes . One of  oocytes . Known P oocytes . Additio oocytes . Furtherchannels . Intereschannels . Howeverchannels . In addichannels . This tr2+ channels in that they function near the resting membrane potential of neurons [Adipor1 gene has been shown to be potentially associated with the severity of postoperative pain [T-type channels are unique among voltage-gated Ca neurons . This lo neurons , 67. Rec neurons , 69. In ive pain , 13. Furive pain . Interesive pain , and obeive pain . Althougive pain . Of noteive pain , 73. Intive pain , 75. Sexive pain , 77. GivIn summary, we present new insights and dissect the molecular components that underlie the effect of adiponectin on Cav3.2 channels. Our study provides evidence that stimulation of adipoR1 in TG neurons enhances Cav3.2 channel currents through CK2α-dependent PKCβ1 signaling. This mechanism is proposed to enhance neuronal excitability and contribute to pain hypersensitivity. Knowledge of the adipoR1-mediated CK2α-PKCβ1-Cav3.2 cascade in peripheral sensory neurons may pave the way for developing potential therapeutic targets in clinical treatment of pain disorders such as trigeminal neuralgia.Additional file 1: Table S1. Primers used for RT-PCR analysis of PKC isoforms in mouse TGs. Table S2. Primers used for RT-PCR analysis of PKC isoforms in HEK293 cells. Fig. S1. Protein expression of adipoR1 and adipoR2 in mouse TGs. Fig. S2. Knockdown of adipoR1 in TGs. Fig. S3. Co-immunoprecipitation analysis of the association of RACK1 with adipoR1 in mouse TGs. Fig. S4. Knockdown of RACK1 in mouse TGs. Fig. S5. Association of CK2 alpha with the adipoR1 in mouse TGs. Fig. S6. Knockdown of CK2α in mouse TGs. Fig. S7. Protein expression of p-CaMKII and t-CaMKII in mouse TGs. Fig. S8. RT-PCR analysis of mRNAs of classic PKC isoforms  in mouse TGs. Fig. S9. Knockdown of PKCβ1 in mouse TGs. Fig. S10. Immunoblot analysis of PKCβ1 expression in cytoplasmic and membrane fractions. Fig. S11. RT-PCR analysis of mRNAs of classic PKC isoforms  in HEK293 cells. Fig. S12. Protein expression of adipoR1 in HEK293 cells transfected with ADIPOR1 cDNA. Fig. S13. The increased expression level of Cav3.2 induced by CCI-ION was attenuated by intra-TG injection of Cav3.2-siRNA. Fig. S14. Protein expression of adipoR1 in mouse TGs after CCI-ION.


The impact of water quality on the survival of human norovirus (NoV) was determined in irrigation water field run-off (tail water) and well water from a representative Central Coast vegetable production site in the Salinas Valley, California.Tail water, well water, and ultrapure water samples were inoculated separately with two surrogate viruses for human NoV—Tulane virus (TV) and murine norovirus (MNV)—to achieve a titer of 1×105 plaque forming units (PFU)/ml. Samples were stored at 11, 19, and 24°C for 28 days. Additionally, inoculated water was applied to soil collected from a vegetable production site in the Salinas Valley or to the surface of growing romaine lettuce leaves, and virus infectivity was evaluated for 28 days in a growth chamber.Virus survival was similar for water stored at 11, 19, and 24°C and there was no difference in infectivity based on water quality. After 28 days, a maximum 1.5 log reduction was observed for both TV and MNV. TV decreased by 1.97-2.26 log and MNV decreased by 1.28- 1.48 logs after 28 days in soil; infectivity was not influenced by water type. Infectious TV and MNV were recovered from lettuce surfaces for up to 7 and 10 days after inoculation, respectively. Across the experiments there was no significant impact of water quality on the stability of the human NoV surrogates.Overall, the human NoV surrogates were highly stable in water with a less than 1.5 log reduction over 28 days and no difference observed based on the water quality. In soil, the titer of TV declined by approximately 2 logs over 28 days, while MNV declined by 1 log during the same time interval, suggesting surrogate-specific inactivation dynamics in the soil tested in this study. A 5-log reduction in MNV (day 10 post inoculation) and TV (day 14 post inoculation) was observed on lettuce leaves, and the inactivation kinetics were not significantly impacted by the quality of water used. These results suggest that human NoV would be highly stable in water, and the quality of the water  does not significantly impact viral infectivity. Human NA genome . GeneralA genome . Human NA genome . The envA genome .via food handlers during final preparation  drinking water quality standards  Produce Safety Rule (PSR) requires monitoring of agricultural water supplies to meet certain microbial standards . The pretandards .California is the leading U.S. producer of several types of fresh market produce, with the Central Coast termed the “nation’s salad bowl” due to the high production of leafy greens in this region . Califorin vitro, murine norovirus (MNV) and Tulane virus (TV) are commonly used as surrogates. MNV is a member of the genus Norovirus and TV is a member of the genus Recovirus, both in the family Caliciviridae . Tail water collected from a commercial farm was used as a “lower quality” water source with a high turbidity and organic load compared to well water sources. Tail water was compared to well water from the same location and to ultrapure water to determine the influence on human NoV stability.22.12 atmosphere. Confluent monolayers of cells were cultured in T-150 flasks with a cell density of approximately 4.6 × 106 cells per 150 cm2 flask. For growing TV stock, MK2-LLC cells in T-150 flasks were washed with Hank’s balanced salt solution (HBSS) and subsequently infected with TV at a multiplicity of infection (MOI) of 0.1. Flasks were incubated for 1 h at 37°C, and then 20 ml of Opti-MEM with 2% FBS was added. The virus was harvested 2 days post inoculation when >80% of cells were detached, and then subjected to three freeze-thaw cycles, followed by centrifugation at 1,811 ×g for 30 min. RAW 264.7 cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% FBS, at 37°C under a 5% CO2 atmosphere. For growing MNV stock, confluent RAW 264.7 cells in T-150 flasks were infected with MNV at a MOI of 0.1. After 1 h of incubation at 37°C, 20 ml of DMEM with 10% FBS was added. As described above, the virus was harvested 2 days post inoculation when >80% of cells were detached, and then subjected to three freeze-thaw cycles and low-speed centrifugation at 1,811 ×g for 30 min.Tulane virus (TV) and murine norovirus-1 (MNV) were propagated in confluent monolayers of the rhesus macaque kidney cell line MK2-LLC and the murine leukemic monocyte macrophage cell line RAW 264.7 , respectively . MK2-LLC2.26 per well. After 24 h of incubation, MK2-LLC or RAW 264.7 cell monolayers were infected with 400 µl of a 10-fold dilution series of TV or MNV, respectively, and the plates were incubated for 1 h at 37°C, with gentle agitation every 10 min. The cells were overlaid with 2.5 ml of Eagle minimum essential medium (MEM) containing 5% FBS, 0.014% sodium bicarbonate, 0.5% penicillin and streptomycin, 2.5% HEPES (pH 7.7), 1% L-glutamine, 14% ultrapure water , and 25% agarose. After incubation for 48 h at 37°C under 5% CO2 atmosphere, the plates were fixed in 10% (wt/vol) formaldehyde and stained with 0.05% (wt/vol) crystal violet for visualization of viral plaques. Viral titers were expressed as mean log plaque forming unit (PFU)/ml ± standard deviation. The limit of detection for the viral plaque assay was determined to be 0.5 log PFU/ml.TV and MNV were quantified by plaque assays in LLC-MK2 and RAW 264.7 cells, respectively . Briefly2.33), phosphate (P), potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), cation exchange capacity (CEC), organic matter (OM), pH, percentage sand, percentage silt, and percentage clay. Irrigation water and tail water samples were tested for pH, electrical conductivity (EC), sodium adsorption ratio (SAR), Ca, Mg, Na, chlorine (Cl), boron (B), bicarbonate (HCO3), carbonate (CO3), total organic carbon (TOC), total nitrogen (N), ammonium (NH4), nitrates (NO3), phosphorus (P), sulfates (SO4), total suspended solids (TSS), and dissolved organic carbon (DOC). Post chemical analysis, all water samples were stored at −20°C prior to use in experiments. Soil samples were stored in sealed plastic bags and held at room temperature prior to use in experiments (up to 12 months).Samples of soil, well water, and tail water were collected from one farm in the Salinas Valley. Bulk soil samples and samples of well water and tail water were collected in March 2016. Tail water was collected in sterile 1-L Nalgene bottles  attached to a telescoping pole to reach the water, as described in EPA method 1603 . An auto2.46 PFU/ml. Aliquots (50 ml) of tail water, well water, and ultrapure water were inoculated with TV or MNV to achieve a starting titer of 105 PFU/ml. Samples were stored for 28 days in incubators set at three different temperatures to mimic the average (19°C), minimum (11°C), and maximum (24°C) water temperatures measured in the reservoir where tail water samples were collected. There were six replicates for TV and MNV in each type of water held at each of the three temperatures. Inoculated well, tail, and ultrapure water held at 19°C was sampled at days 0 (immediately after inoculation), 1, 2, 3, 7, 14, 21, and 28. Inoculated well, tail, and ultrapure water held at 11 and 24°C was sampled at days 0 (immediately after inoculation), 1, 2, 14, and 28. A 2-ml sample was collected at each time point from each replicate and the level of infectious virus was determined by plaque assay. To monitor the pH of each water type, uninoculated samples of water were stored at each of the three temperatures and pH was measured at days 0, 1, 2, 3, 7, 14, 21, and 28.TV and MNV virus stocks were prepared to have a normalized titer of 1 × 102.55 PFU/ml. Treatment groups were TV + well water, TV + tail water, TV + ultrapure water, MNV + well water, MNV + tail water, and MNV + ultrapure water. Each 600-ml sample of virus-inoculated water  was added to bagged soil (2 kg) and then mixed to achieve 100% saturation or 30% water holding capacity (WHC). Aliquots of the inoculated soil were then distributed into plastic containers (500 g of wet soil per container × 6 containers per treatment group). The target titer for each virus was 1.15 × 107 PFU/container (2.3 × 104 PFU/g of soil). Containers were placed on tray flats and held in an environmental chamber  with a light intensity of 230 μmol/m2·s2 using sun white spectrum 12W T5 LED lamps. The chamber was maintained at a constant relative humidity (60% RH) and daily temperatures of 22°C  and 18°C   was then added to separate 30 × 30 cm polyethylene bags. TV and MNV were inoculated into 600 ml of either tail water, well water, or ultrapure water to achieve a titer of 10t light) . The soi2.6Lactuca sativa, Parris Island Cos) were planted in plug trays and germinated in a controlled environmental chamber located in the Plant Sciences Department at the University of California Davis. Ten days after germination, plugs were transplanted into 4-inch (10.6 cm) containers containing Fafard’s 3B potting mix . At 45 days after germination, lettuce plants were transferred to an environmental growth chamber located in a biosafety level–2 laboratory in the Food Science and Technology Department at UC Davis. The lettuce was maintained under conditions of 12 h of light and 12 h of darkness, and daily temperatures of 22°C  and 18°C  and a constant relative humidity of 60%. The lettuce containers were kept in tray flats, and were bottom watered every 5 days with 1,000 ml of ultrapure water.Seeds of romaine lettuce (7 PFU/ml) were diluted 1:10 (v/v) in either tail water, well water, or ultrapure water. Plants were divided into treatment groups TV + well water, TV + tail water, TV + ultrapure water, MNV + well water, MNV + tail water, and MNV + ultrapure water, with six replicates per treatment. Individual leaves were inoculated with 10 × 2-µl drops (2 × 105 PFU/leaf). Plants inoculated with the same organism and water were grouped in one tray flat to avoid cross contamination. Inoculated leaves were sampled at days 0 (immediately after inoculation), 1, 2, 3, 4, 5, 6, and 7. For sampling, inoculated leaves were separated from the plants by hand and transferred into individual 207-ml (7-oz) Whirl-Pak filter bags with 6 ml of PBS per bag. To recover the viruses, the bags containing leaves were pummeled in a stomacher  for 2 min at fast speed. The virus-containing aqueous homogenates collected at days 0, 1, 2, 3, 4, 5, 6, and 7 were then transferred to separate collection tubes for determination of viral titer at different time points by plaque assays. A second experiment was conducted to assess viral infectivity over 28 days. Leaves were inoculated as described above and were sampled at days 0 (immediately after inoculation), 1, 2, 7, 10, 14, and 28.TV and MNV , followed by a multiple comparison with a Tukey test for normally distributed data. Results with 33.1The pH values of water samples measured at day 0 were 7.36 ± 0.78 for ultrapure water (not shown), 7.63 ± 0.15 for well water, and 7.76 ± 0.16 for tail water Table 1.At 11°C, the titers of TV at day 0 were 4.23 ± 0.05 log PFU/ml in ultrapure water, 4.11 ± 0.06 log PFU/ml in well water, and 4.09 ± 0.04 log PFU/ml in tail water Figure 1At 19°C, the titers of TV at day 0 were 3.59 ± 0.11 log PFU/ml in ultrapure water (control), 3.84 ± 0.07 log PFU/ml in well water, and 3.82 ± 0.12 log PFU/ml in tail water Figure 1At 24°C, the titers of TV at day 0 were 3.99 ± 0.00 log PFU/ml in ultrapure water, 4.00 ± 0.02 log PFU/ml in well water, and 3.99 ± 0.04 log PFU/ml in tail water Figure 1Statistically significant differences in the virus titer were observed for both TV and MNV at day 0 compared with day 28 in all water types at each temperature evaluated 2. Stati3.2Based on soil composition analysis Table 2,The titer of MNV detected at day 0 in soil was 4.62 ± 0.02 log PFU/g with ultrapure water, 4.62 ± 0.03 log PFU/g with well water, and 4.62 ± 0.02 log PFU/g with tail water as the suspension medium Figure 33.3In the first trial lasting 7 days, the titer of TV recovered from lettuce leaves at day 0 was determined to be 4.54 ± 0.13 log PFU/leaf using ultrapure water, 4.38 ± 0.16 log PFU/leaf using well water, and 4.23 ± 0.26 log PFU/leaf using tail water as the suspension medium Figure 4The titer of MNV detected at day 0 on lettuce leaves was 2.83 ± 0.12 log PFU/leaf with ultrapure water, 3.33 ± 0.16 log PFU/leaf with well water, and 3.37 ± 0.14 log PFU/leaf with tail water as the suspension medium Figure 4A second trial with lettuce was set up to assess TV and MNV viral infectivity beyond 7 days. The same inoculation procedure used in trial 1 was used in trial 2, but infectivity was assessed for 28 days. The titer of TV recovered from lettuce leaves at day 0 was determined to be 5.20 ± 0.06 log PFU/leaf using ultrapure water, 5.09 ± 0.09 log PFU/leaf using well water, and 5.25 ± 0.09 log PFU/leaf using tail water as the suspension medium Figure 54As obligate intracellular parasites, viruses do not increase in numbers outside of a permissive host cell. In the environment, including water, soils, and surfaces, virus levels only remain static or decline. As such, environmental factors, including temperature, humidity, and UV exposure, play an important role in dictating the infectivity of viruses outside of the host . MoreoveTemperature is recognized as a key variable influencing virus environmental stability . GeneralThe pH of a solution plays an important role in dictating the biochemical interactions of the viral capsid protein with molecules and ions in the environment. The protonation of the capsid proteins suspended in water leads to the formation of electrically charged surfaces . The surUltrapure water is 18.2 MΩ.cm (no other ions present other than those created by the dissociation of water) and has a TOC value below 5 ppb. Unsurprisingly, well water and tail water had significantly higher values of water quality parameters compared with ultrapure water. Tail water had significantly higher values for DOC, TOC, and TSS compared with well water. Total N was higher for well water compared with tail water. However, none of these differences in water quality parameters played a significant role in recovery or infectivity of TV and MNV in the water samples at the temperature ranges evaluated in this study.Infectious TV and MNV were recovered from soil samples at 28 days post inoculation. The level of TV declined by approximately 2 logs and MNV by approximately1 log over 28 days compared with virus levels recovered on day 0 immediately after inoculation. Several factors can influence virus recovery and infectivity in soil, including soil type, water saturation, pH, and organic matter. Fine-textured soils have been reported to adsorb viruses more readily than coarse-textured soils . Fine-teThe soil was maintained at an average soil water saturation of 62% throughout the study period. A highly water saturated soil also allows for more viral movement, as all the pores in the soil are open and the virus has less interaction with the soil particles . The coa2·s2 , is zoonotic and wildlife is known to be a reservoir  for HEV and often impact the microbial quality of surface water . Using tThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ED, LH, and AM contributed to the study conception and design. Funding was acquired by LH. Material preparation, data collection and analysis were performed by XW and TR. The first draft of the manuscript was written by XW, TR, and ED and all authors commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.