We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2–4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Results: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism.  Cisplatin, a highly efficacious chemotherapeutic agent, is used in the treatment of a wide variety of common malignancies ,2,3. CisFrom there, aminopeptidase (AP) converts cysteinyl-glycine-conjugates to cysteine conjugates, reabsorbed into the proximal tubule and converted into nephrotoxic thiols  Figure . This scOur laboratory has identified a new potential target to ameliorate cisplatin nephrotoxicity, Na/H exchange regulatory factor isoform 1 (NHERF1), a scaffolding protein highly expressed in renal proximal tubule cells and critical for the regulation of solute transport and hormone receptor signaling pathways . PreviouThe goal of this work was to explore a novel mediator of susceptibility to cisplatin-induced AKI. We hypothesized that the effects of NHERF1 loss on kidney metabolic pathways affected the renal transport and/or metabolism of cisplatin. 2-4 month-old male WT C57BL/6J and C57BL/6J NHERF1(−/−) KO littermate mice  were houg for 10 min to remove insoluble material. The supernatant was kept and further diluted to 1:100 using the GGT assay buffer and stored at −80 °C until further use. 4 h: (WT vehicle: n = 6), (NHERF1 KO vehicle: n = 6), (WT cisplatin: n = 6), and (NHERF1 KO cisplatin: n = 6) and 24 h: (WT vehicle: n = 5), (NHERF1 KO vehicle: n = 5), (WT cisplatin: n = 7), and (NHERF1 KO cisplatin: n = 8).The activity assay was performed using the GGT colorimetric assay kit  per the manufacturer’s instructions. Tissue samples were prepared using approximately 10 mg of kidney cortex in 200 μL of ice-cold GGT assay buffer. The samples were then centrifuged at 13,000× Paraffin embedded fixed whole kidney tissues were cut in 5 µm thick sections, either transversely or longitudinally from cisplatin treated and vehicle treated WT and NHERF1 KO animals. These sections were stained with hematoxylin and eosin (H&E) as well as periodic acid-schiff (PAS) and sent to a pathologist to evaluate the number of casts, degree of brush border membrane (BBM) sloughing, and acute tubular necrosis (ATN). Images were taken at 20× and 40× (WT vehicle n = 5), (NHERF1 KO vehicle n = 5), (WT cisplatin n = 7), and (NHERF1 KO cisplatin n = 8).IHC was carried out on paraffin embedded fixed whole kidney tissue cut in 5 µm thick sections either transversely or longitudinally from cisplatin treated and vehicle treated WT and NHERF1 KO animals. A primary antibody against GGT (1:50)  and CCBL1  was utilized for staining to evaluate any changes in proximal tubule localization or loss after cisplatin treatment. CCBL1 stained slides were blinded and further evaluated for uniform vs. patchy staining, granular staining, tubule mosaic staining, overall level of proximal tubule staining, and tubule injury. Images were taken at 40×. GGT:  and CCBL: . NGAL was determined using an enzyme linked immunosorbent assay (ELISA) kit  on mouse urine as directed by the manufacturer (WT vehicle n = 5), (NHERF1 KO vehicle n = 5), (WT cisplatin n = 7), and (NHERF1 KO cisplatin n = 8). g for 2 min) and 50 µL of supernatant was transferred to a new tube with preservation solution . Tubes were then stored at −80 °C. In addition, 10 mg of kidney cortex tissue was removed from the kidney and added to the preservation solution  and homogenized. Kidney cortex homogenates were then stored at −80 °C until further use. Blood samples were collected from mice via retro-orbital bleed and added to a tube with 0.5 M borate buffer stock solution and 165 uM γ-glutamylglutamate as the internal standard as described in  within 1Tissue and plasma Cys, CySS, GSH, and GSSG were assayed by HPLC as S-carboxymethyl, N-dansyl derivatives using γ-glutamylglutamate as an internal standard as previously described ,18. The ® Gene Expression Assay Mm00442530_m1, Mm01344332_m1, Mm00802655_m1, Mm01324400_m1, Mm01343626_m1, Mm00515065_m1, Mm99999915_g1; Applied Biosystems), according to the manufacturer’s protocol . Step One Plus Real Time PCR System  was used for qPCR with the parameters: 50 °C 2 min, 95 °C 10 min, followed by 40 cycles of 95 °C 15 s and 60 °C 1 min. Results were analyzed using Step One software version 2.3 . The amplification curves were analyzed by the mathematical equation of the second derivative, and the amounts of Slc7a11, Abcc1, and Gclc mRNA expression were normalized to the housekeeping gene Gapdh mRNA expression. The 2-ΔΔCT method was used to calculate relative quantification. (WT vehicle n = 4), (NHERF1 KO vehicle n = 4), (WT cisplatin n = 4), and (NHERF1 KO cisplatin n = 4).Total RNA was isolated from the kidney cortex using an Ambion mirVana mRNA isolation kit per the manufacturer’s instructions . cDNA was synthesized from 1000 ng total RNA using SuperScript Vilo Master Mix (ThermoFisher) following the manufacturer’s instructions. Real-time quantitative PCR (qPCR) was conducted to measure Slc7a11, Abcc1, Gclc, Gclm, Abcc5, Gss, and GAPDH mRNA expression with TaqMan probes  levels at 24 h: (WT cisplatin: n = 6) and (NHERF1 KO cisplatin: n = 6) and 72 h: (WT cisplatin: n = 8) and (NHERF1 KO cisplatin: n = 8).p-values of <0.05 were considered statistically significant and data were shown as mean ± SEM.A two-way analysis of variance (ANOVA) was used to evaluate changes in GGT activity, changes in body weights of mice, GGT and CCBL in treated kidney cortex by Western blot, reduction/oxidation state of GSH and Cys in plasma and treated kidneys, mRNA expression via qPCR, and urine NGAL from treated mice. A Student’s T-test was used to evaluate Gclc mRNA expression post-hoc and Pt levels between cisplatin treated WT and NHERF1 KO kidneys. p = 0.007) (p = 0.03) (We have previously demonstrated significant differences in kidney injury between NHERF1 KO and WT mice 72 h after cisplatin administration . Kidney = 0.007) . The inc = 0.03) .H&E and PAS staining of the kidneys of cisplatin treated mice after 24 h revealed that NHERF1 KO mice exhibited a greater degree of early histologic injury when compared to WT mice at the same time point . SimilarGSH plays a major role in the maintenance of a normal redox state in kidney cells through its antioxidant capacity ,21. Cispp = 0.0042) (p = 0.013), (CySSG p = 0.003), (GSH p = 0.02), and (GSSG p = 0.002)  A. In res= 0.002) A,B. Howep = 0.027) and Cys levels (p = 0.028) and a higher GSSG level (p = 0.022) relative to vehicle treated WT mice (p = 0.002) and Cys (p = 0.0005) and a significant decrease in CySSG (p < 0.0001) and GSSG in both genotypes (p < 0.0001) (p = 0.003) and increased in both genotypes with cisplatin treatment (p = 0.0002)  C,D. On t 0.0001) D. The ra 0.0002) E. The GSSlc7a11), glutamate-cysteine ligase catalytic subunit (Gclc), glutamate-cysteine ligase modifier (Gclm), glutathione synthetase (Gss), glutathione-s-conjugate-translocating ATPase (Abcc1), and ATP binding cassette subfamily C member 5 (Abcc5). Cisplatin significantly increased Slc7a11 expression in both genotypes (p < 0.0001) but to a greater extent in the NHERF1 KO kidneys (p = 0.03) (Gclc expression was significantly increased with cisplatin treatment in both genotypes (p = 0.0147); however, the expression of Gclc was increased in the cisplatin treated NHERF1 KO kidney to a greater degree than cisplatin treated WT (p = 0.035)  . To inve = 0.03) A. Gclc e= 0.035) B. The exnt group C–F. p = 0.04)  . p = 0.015)  ] A. The 24± 0.02)] B.To determine if the differences in GGT activity were reflected in localization of GGT in WT and NHERF1 KO kidneys, an IHC analysis of kidney slices was performed. GGT staining in the proximal tubules of vehicle treated WT and KO kidneys were similar A,B. Aftep = 0.018)  and  (p = 0.018) , (NHERF1 KO vehicle: 1.1 CCBL/GAPDH ± 0.1)] and  A. Howeve= 0.018) B. As a scaffolding protein, NHERF1 may play a role in the intracellular localization of enzymes involved in cisplatin metabolism such as CCBL. IHC for CCBL in WT and KO kidney slices after vehicle treatment revealed heterogeneity of cell CCBL staining in the distal and cortical collecting tubules of NHERF1 KO vehicle treated kidneys, even within the same tubule A,B compap = 0.95) (p = 0.04)  (KO: 39.6 μg/g ± 2.8) ( = 0.95) . However = 0.04) . The purpose of this study was to address the hypothesis that NHERF1 loss affected the renal handling of cisplatin, as a potential mechanism for the increased susceptibility of NHERF1 KO mice to cisplatin-induced AKI. This hypothesis was based on the principle that the development of cisplatin tubular injury is dependent on cisplatin transport into tubular cells and that NHERF1 affects the expression and function of epithelial transporters ,25. ThisNHERF1 KO mice exhibit greater sensitivity to cisplatin-induced nephrotoxicity at the early time point of 24 h when compared to WT, as manifested in histology and urine NGAL excretion. These data are consistent with the increased sensitivity to cisplatin seen in the previous 72 h study  and suggNHERF1 KO animals demonstrated changes in the levels of glutathione metabolites under baseline conditions, disclosing the existence of underlying oxidative stress. Previous studies have documented that cisplatin produces alterations in glutathione metabolites, confirmed by our findings that plasma from 72 h cisplatin treated mice showed a decrease in GSH, GSSG, CySSG, Cys, and CySS for both WT and NHERF1 KO when compared to vehicle treated ,26,27,28Gclm, Gss, Abcc1, and Abcc5 were not significantly altered with NHERF1 loss or cisplatin treatment. However, Slc7a11 gene expression was significantly increased with cisplatin treatment in both WT and NHERF1 KO kidneys and to a greater extent in cisplatin treated NHERF1 KO kidneys. Slc7a11 plays a key role in antioxidant defense through its mediation of CySS uptake which promotes GSH synthesis and the maintenance of cell survival under oxidative stress [Slc7a11 mediated CySS uptake is important in maintaining redox balance, but it comes at a considerable cost for cancer cells [Slc7a11 must rapidly reduce insoluble CySS to soluble Cys to prevent toxicity. Rapidly reducing insoluble CySS to soluble Cys requires a substantial supply of NADPH from the pentose phosphate pathway [Slc7a11 gene expression supports our previous finding of increased enzyme activity within the pentose phosphate pathway, which we previously proposed could be a compensatory mechanism to increase NADPH production due to oxidative stress. Thus, the increase in Slc7a11 gene expression may be acting in concert with the increase in NADPH production to counteract an increase in oxidative stress and may account for the significantly decreased CySS in both NHERF1 KO plasma and kidneys under baseline conditions. Gclc was significantly increased in the cisplatin treated NHERF1 KO kidneys. It is a key player in regulating cellular Cys concentrations and GSH synthesis. Insufficient GSH results in oxidative stress and upregulation of Gclc gene expression [Slc7a11 and Gclc gene expression with the additional ‘stress’ from cisplatin. Overall, these data are consistent with previous results where NHERF1 KO kidneys appear to function normally under baseline conditions, but when stressed with cisplatin the kidneys are unable to mount an appropriate response to increased oxidative stress demands.Gene expression of e stress . Recentler cells . By acti pathway ,34 and rpression . The higWe also found changes in two of the enzymes, GGT and CCBL, involved in cisplatin metabolism, suggesting an additional mechanism for enhanced cisplatin nephrotoxicity in the NHERF1 KO animals. A robust increase in GGT activity was seen in the 24 h cisplatin treated WT mice, while an increase in GGT activity was not seen in the NHERF1 KO mice, suggesting that NHERF1 is important for GGT’s response to an insult. This finding is somewhat surprising as several studies have indicated that GGT activity and/or expression plays a role in the formation of nephrotoxic cisplatin metabolites ,35,36. TCCBL is the rate limiting enzyme in the metabolism of cisplatin to the ‘reactive thiol’ that has been implicated in nephrotoxicity ,35,36,43Lastly, Pt levels in cisplatin treated WT and NHERF1 KO kidneys were investigated using ICP-MS. We hypothesized that altered transport of cisplatin in NHERF1 KO kidneys, particularly decreased activity of MATE1, the extrusion transport protein in the apical membrane of proximal tubule cells, would result in higher Pt levels and increased toxicity from cisplatin ,13,41. IUltimately, there are still unanswered questions regarding the mechanism of cisplatin nephrotoxicity; however, it is evident that NHERF1 expression plays a vital role in its development. These data have provided more clarity regarding the underlying mechanism of susceptibility to cisplatin in NHERF1 KO mice. Furthermore, future experiments  are integral in providing further insight into the mechanism of susceptibility. These data in combination with our previous discovery of an altered pentose phosphate pathway in NHERF1 KO kidneys  have ledSlc7a11 and Gclc gene expression are increased to enhance cellular uptake of Cys and increase GSH production, expanding the demand for NADPH. Lastly, GGT, an essential enzyme involved in the recycling of Cys for the de novo synthesis of GSH appears to be unable to respond to the increased demand for GSH in response to cisplatin (NHERF1 KO mouse kidneys have increased levels of GSSG, a known pro-oxidant ,31 Figu. Elevateisplatin . Studiesisplatin . NHERF1 In summary, we have demonstrated unique mechanisms for susceptibility to cisplatin-induced AKI related to the scaffolding protein, NHERF1. NHERF1 deficient kidneys exhibit evidence of underlying oxidative stress manifested by a higher level of oxidized glutathione and altered metabolic response to cisplatin manifested by the lack of increase in GGT activity and altered CCBL localization. The former condition likely predisposes to greater injury inflicted by the profound oxidative insult of cisplatin, while the latter defects may contribute by preventing appropriate metabolic handling of the drug. Which of these plays the more important role in enhancing the AKI injury cannot be determined from this study, but the findings highlight further avenues for investigation. Coupled with our prior studies showing increased activity of the pentose phosphate pathway in NHERF1 deficient kidneys, these findings add to a growing body of knowledge implicating NHERF1 in the regulation of cell metabolism. Interestingly, and despite the fact that NHERF1 is best recognized as a scaffolding protein for epithelial transporters, the NHERF1 deficient kidneys show equivalent uptake and even enhanced expulsion of cisplatin, suggesting that altered cisplatin transport does not play a role in this phenomenon. Translation of these findings into the clinical realm may lead to better ways to prevent cisplatin-induced AKI, allowing clinicians to better apply this powerful chemotherapeutic agent.


Effects of nitrogen addition over a period of nine growing seasons on the soil microbial abundance and community diversity in permafrost peatland were analyzed. The results showed that the abundances of soil bacteria, fungi, archaea, nitrogen-cycling genes (nifH and b-amoA), and mcrA increased in N1, N2, and N3 treatments compared to CK. This indicated that nitrogen addition promoted microbial decomposition of soil organic matter, nitrogen fixation, ammonia oxidation, nitrification, and methane production. Moreover, nitrogen addition altered the microbial community composition. At the phylum level, the relative abundance of Proteobacteria increased significantly in the N2 treatment. However, the relative abundances of Actinobacteria and Verrucifera in the N2 treatment and Patescibacteria in the N1 treatment decreased significantly. The heatmap showed that the dominant order composition of soil bacteria in N1, N2, and N3 treatments and the CK treatment were different, and the dominant order composition of soil fungi in CK and N3 treatments were different. The N1 treatment showed a significant increase in the Ace and Chao indices of bacteria and Simpson index of fungi. The outcomes of this study suggest that nitrogen addition altered the soil microbial abundance, community structure, and diversity, affecting the soil microbial carbon and nitrogen cycling in permafrost peatland. The results are helpful to understand the microbial mediation on ecological processes in response to N addition.Nitrogen is the limiting nutrient for plant growth in peatland ecosystems. Nitrogen addition significantly affects the plant biomass, diversity and community structure in peatlands. However, the response of belowground microbe to nitrogen addition in peatland ecosystems remains largely unknown. In this study, we performed long-term nitrogen addition experiments in a permafrost peatland in the northwest slope of the Great Xing’an Mountains. The four nitrogen addition treatments applied in this study were 0 g N·m Since t2·year−1 , and it 2·year−1 . A conti2·year−1 . N depos2·year−1 . The agg2·year−1 . This, i2·year−1 ,10, as w2·year−1 , influen2·year−1 ,13, and 2·year−1 . The abu2·year−1 . Previou2·year−1 ,17. UndeThe response of soil microbe to N addition depends on the microbial species, the N treatment duration and amount, and the ecosystem type. The fungal community is more sensitive than the bacterial community to N deposition . A low a−2·year−1, whereas a 7 °C rise in temperature will increase N mineralization by 9.4 g N·m−2·year−1 in high-latitude regions [nifH, bacteria-amoA (b-amoA), nirK, nirS, mcrA, and pmoA genes. Illumina Miseq sequencing of bacterial 16S rRNA and fungal ITS genes was used to assess the effect of N addition treatments on the soil bacterial and fungal community composition and diversity. In this study, we focused on two aspects: (1) the response of soil microbial abundance to N addition in N-limited peatland; (2) the relationship of soil substrate properties with the composition and diversity of microbial communities under N addition. We hypothesized that N addition would increase soil microbial abundance and community diversity in permafrost peatland by increasing soil nutrient contents, and that the response would be greater than other soil systems due to stronger N limitation.Most permafrost peatlands are located at high latitudes. These peatlands are often N-limited, due to slow decomposition in cold, acidic, and frequently waterlogged soils, along with more N stored in peatland soil tightly bound with organic matter, compared with other ecosystems ,26, whic regions . Thus, g regions . HoweverEriophorum vaginatum L., Vaccinium uliginosum L., Chamaedaphne calyculata L. Moench, Ledum palustre L., and Sphagnum spp.The study was conducted in a permafrost peatland located at the northwest slope of the Great Xing’an Mountains in Northeast China . The active layer is 50 cm to 60 cm above the permafrost layer. The mean annual air temperature is −3.9 °C, and the mean annual precipitation is 450 mm, with 45% falling as rain from July to August . The dom−2·year−1 (CK), 6 g N·m−2·year−1 (N1), 12 g N·m−2·year−1 (N2), and 24 g N·m−2·year−1 (N3); each treatment was applied to three replicated plots. For each N addition treatment, NH4NO3 was first dissolved in 1 L of surface water and then sprayed evenly on the target area. The same amount of surface water without NH4NO3 was sprayed in the CK treatment. The treatments were applied during the growing season (from May to September) of 2012 to 2020. The soil samples were sampled from 0–20 cm below the plant litter layer from each plot on 13 August 2020, and five sampling points were collected with a soil drill from each plot and mixed to form a composite sample. The soil samples were divided into subsamples for further analysis. One of the subsamples was stored at −80 °C to determine the functional gene abundance and community diversity of soil microbes. The second soil subsamples were stored at 4 °C for dissolved organic carbon (DOC), ammonia nitrogen (AN), and nitrate nitrogen (NN) content determination. The remaining soil subsamples were air-dried to determine the soil total carbon (TC), total nitrogen (TN), and total phosphorus (TP) content, as well as soil pH.The experiment was established in autumn 2011. A total of 12 plots were established; each plot was 2 m × 2 m and separated by a 1 m buffer to prevent increased horizontal movement and lateral loss of N. This experiment entailed a total of four N addition treatments: 0 g N·m®Spin Kit for Soil , according to the manufacturer’s instructions. The extracted DNA was stored at −80 °C prior to functional gene and sequencing analysis. The abundances of bacteria, fungi, archaea, nifH, b-amoA, nirK, nirS, mcrA, and pmoA genes were determined by RT-PCR, which was performed on the ABI StepOne instrument  and SYBR green dye. RT-PCR analysis for each soil sample was replicated three times. RT-PCR primers for target gene amplification and the detailed procedure are presented in Escherichia coli competent cells. The plasmids were extracted using the Plasmid Mini Kit . The specificity of plasmids was determined through the Basic Local Alignment Search Tool [DNA from 0.3 g of soil samples was extracted using a FastDNArch Tool , and theFor microbial community analyses, the V3–V4 region of the 16S rDNA gene was amplified with primers 338f (5′–ACTCCTACGGGAGGCAGCAG–3′) and 806r (5′–GGACTACHVGGGTWTCTAAT–3′) , and the−1 KCl and then analyzed with the AA-3 continuous flow analyzer . Soil pH values were analyzed in a 5:1 water–soil solution.The soil total carbon (TC) content was determined via the dry combustion method with the multi N/C 2100 analyzer . Dissolved organic carbon (DOC) in the soil was determined, as per the method described by Ghani et al. . Total NData were analyzed using SPSS software  with an accepted significance level of α = 0.05. One-way analysis of variance (ANOVA) and a post hoc Duncan’s multiple-range test were performed to determine the significant differences between the soil properties from different N addition treatments. A nonmetric multidimensional scaling (NMDS) ordination to illustrate the clustering of bacterial and fungal community composition variation was conducted on the Bray–Curtis distance of the order. Pearson’s correlation coefficients between diversity indices and soil microbial functional group abundance, as well as soil microbial community diversity indices, were calculated. Redundancy analyses (RDA) were conducted on soil chemical properties with microbial diversity; specific bacteria and fungi were analyzed separately. RDA analyses were conducted with CANOCO 5.0 software .−1 to 406.53 mg·g−1. TC content was lower in the N3 treatment than in N1, N2, and CK treatments. DOC content in all treatments ranged from 331.00 mg·kg−1 to 566.47 mg·kg−1. DOC content in the N1 treatment was higher than in CK, N2, and N3 treatments , mcrA, and pmoA were determined using RT-PCR analysis. In all four N addition treatments, bacterial abundance (3.71 × 1012 copies·g−1 to 5.36 × 1012 copies·g−1 dry soil) was more higher than fungal abundance (7.44 × 109 copies·g−1 to 4.92 × 1010 copies·g−1 dry soil) and archaeal abundance (3.10 × 108 copies·g−1 to 9.92 × 109 copies·g−1 dry soil)  . Comparean in CK . The abunificant .In the CK plot, the dominant bacterial phyla were Actinobacteria (22.81%) and Acidobacteria (21.45%), followed by Proteobacteria (18.05%), Chloroflexi (9.6%), Bacteroidota (8.26%), Verrucomicrobiota (4.62%), Desulfobacterota (2.88%), Patescibacteria (2.52%), Myxococcota (1.44%), Firmicutes (1.04%), Gemmatimonadota (0.91%), Nitrospirota (0.88%), and Planctomycetota (1.14%) a. Actinop < 0.05) (p < 0.01)  a. Furthe < 0.01) b.The Shannon, Simpson, Ace, and Chao indices were used to estimate and compare the alpha diversity of bacterial and fungal communities in N1, N2, and N3. We observed that Ace and Chao indices of bacteria in N1 treatment were significantly higher than in CK and differed significantly between N1 and CK treatments C,D. The amoA were significantly and positively correlated to TN. The abundance of b-amoA was significantly and positively correlated to AN , Actinobacteria , Bacteroidota , Myxococcota , and Gemmatimonadota  were positively correlated to TN . The relative abundances of Basidiomycota , Mortierellomycota , and Rozellomycota  were positively correlated to TN. The relative abundance of Mortierellomycota was negatively correlated to pH . The relative abundance of unclassified-k-Fungi was negatively correlated to TC . The relative abundance of Monoblepharomycota was negatively correlated to TC and pH   b.The RDA of soil chemical properties and alpha diversity indices of microbial community in all N treatments is shown in nifH, b-amoA, and mcrA were higher in N addition treatments. In line with our hypothesis, soil nifH (increased 110% to 213%) and b-amoA (increased 567% to 1262%) abundances in permafrost peatland were more sensitive to N addition than agricultural soils  according to a meta-analysis result of 47 field studies [nirK (no effect) and nirS (no effect) genes were less sensitive to N addition in permafrost peatland than in agricultural soils, increasing 53% and 40%, respectively [amoA, and mcrA were found to increase under N addition, indicating that N addition may promote microbial decomposition of soil organic matter, ammonia oxidation, N fixation, and methane production. Previous studies have also demonstrated that the abundances of bacteria, fungi, archaea [amoA [nifH in Antarctic soils [nifH to different levels of N addition was different, related to the cultivar and the amount of added N [nifH gene pool in plant roots was positively correlated with N supply. Coelho et al. [nifH gene abundance in the rhizosphere of sorghum bicolor. We previously found that N addition could increase the biomass of plant roots in the same N addition plots [nifH abundance by promoting plant root growth and secreting more exudates. In contrast, Tian et al. [nirK gene abundance in permafrost peatland soil. Previous researchers have also obtained consistent results suggesting that nitrite reductase (NO-forming) is insensitive to environmental changes [Soil microbes contribute significantly to all the biogeochemical processes of soil ,13, such studies . Howeverectively . The abu archaea , and b-aea [amoA  increaseea [amoA  showed tic soils . The res added N . As repoo et al.  also fouon plots . Therefon et al.  demonstr changes . Wang et changes  reported changes . HoweverThe community structure and diversity of soil microorganisms respond distinctly to different N addition levels . At the Consistent with our initial hypothesis, N addition increased the bacterial and fungal diversity, but this depended on the N addition level. We observed that N1 treatment increased the Ace and Chao indices of bacteria and the Simpson index of fungi in permafrost peatlands. Li et al.  also repN addition could alter soil properties directly or indirectly by affecting soil microbial communities . We obseamoA abundances were closely related to TN, and that soil N is a major factor regulating the abundances of soil bacteria, archaea, and b-amoA [We analyzed soil microbial functional gene abundance and diversity along with soil chemical properties in different N addition treatments. The outcomes suggested that bacterial, archaeal, and b-d b-amoA ,75. We anifH, b-amoA, and mcrA in permafrost peatland, indicating that N addition could stimulate the decomposition of soil organic matter, oxidation of ammonia, fixation of N, nitrification, and production of methane, thereby affecting soil C and N cycling. Soil nifH and b-amoA abundances in permafrost were more sensitive to N addition than agricultural soils, but nirK and nirS abundances were less sensitive to N addition in peatland soil than in agricultural soils. N3 treatment changed the dominant order composition of soil fungi. In addition, N1 treatment increased the Ace and Chao indices of bacteria and the Simpson index of fungi. The positive effects of N addition on diversity indices of soil bacteria and fungi were different from grassland and forests soils, indicating that soil microbial diversity was closely correlated with soil nutrients but not soil pH under N addition in permafrost peatlands. The results highlight the important role of microbes in regulating soil ecological processes in permafrost peatlands under N addition. This study focused only on the effects of N addition on belowground microbial processes; thus, future studies should explore the interaction and response mechanism of aboveground plants and soil microbes to N addition.This study demonstrated that continued N addition for nine growing seasons increased the abundances of bacteria, fungi, archaea,


Introduction: The cardiac electrical conduction system is very sensitive to hypoglycemia and hypoxia, and the consequence may be brady-arrythmias. Weddell seals endure brady-arrythmias during their dives when desaturating to 3.2 kPa and elite breath-hold-divers (BHD), who share metabolic and cardiovascular adaptions including bradycardia with diving mammals, endure similar desaturation during maximum apnea. We hypothesized that hypoxia causes brady-arrythmias during maximum apnea in elite BHD. Hence, this study aimed to define the arterial blood glucose (Glu), peripheral saturation (SAT), heart rhythm (HR), and mean arterial blood pressure (MAP) of elite BHD during maximum apneas.Methods: HR was monitored with Direct-Current-Pads/ECG-lead-II and MAP and Glu from a radial arterial-catheter in nine BHD performing an immersed and head-down maximal static pool apnea after three warm-up apneas. SAT was monitored with a sensor on the neck of the subjects. On a separate day, a 12-lead-ECG-monitored maximum static apnea was repeated dry (n = 6).Results: During pool apnea of maximum duration (385 ± 70 s), SAT decreased from 99.6 ± 0.5 to 58.5 ± 5.5% , while Glu increased from 5.8 ± 0.2 to 6.2 ± 0.2 mmol/l (P = 0.009). MAP increased from 103 ± 4 to 155 ± 6 mm Hg (P < 0.005). HR decreased to 46 ± 10 from 86 ± 14 beats/minute (P < 0.001). HR and MAP were unchanged after 3–4 min of apnea. During dry apnea (378 ± 31 s), HR decreased from 55 ± 4 to 40 ± 3 beats/minute (P = 0.031). Atrioventricular dissociation and junctional rhythm were observed both during pool and dry apneas.Conclusion: Our findings contrast with previous studies concluding that Glu decreases during apnea diving. We conclude during maximum apnea in elite BHD that (1) the diving reflex is maximized after 3–4 min, (2) increasing Glu may indicate lactate metabolism in accordance with our previous results, and (3) extreme hypoxia rather than hypoglycemia causes brady-arrythmias in elite BHD similar to diving mammals. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki. Informed consent, written and oral, have been obtained from the participants. Nine subjects were divers (age 43 ± 3 years), and eight judo athletes matched for morphometric variables  and whole-body aerobic capacity VO2max,  were choAll free divers ranked among the national top 10, three of the participating free divers ranked among the World top 10, and one was a 2016 outdoor free-diving World champion, while one reached third place at the same Championship (no limit depth competition), and one was a World record holder.2) during the test was defined as VO2max. For recognition of true VO2max, three of five criteria had to be met: individual perception of exhaustion, respiratory exchange ratio >1.15, plateau of VO2 curve, heart rate approaching age-predicted maximum, and inability to maintain a pedaling frequency above 70 rpm  .2 and stored at −80°C. Western blotting was performed as previously described in detail  from the lateral vastus of the femoral muscle a.m. Bergstroem . The bion detail . BrieflyThe nine elite breath-hold divers were instructed to refrain from caffeine intake and to fast for at least 6 h before the pool apneas. Any strenuous physical activity was discouraged for at least 1 day before the experiment.A 1.1 mm, 20-gauge catheter was inserted in the radial artery of the non-dominant arm connected to a transducer for continuous flow of saline  for blood pressure monitoring and for collection of blood glucose. Blood glucose analyses were performed immediately after sampling, using an automated self-calibrating blood gas machine  and evaluated for blood gases as well as described previously .°C, 0.8-m-deep indoor pool after a warm-up of three consecutive apneas to maximize the diving response   in a 28°response . They we® 20 monitor. To diminish artifacts from water, heart rhythm (HR) was monitored with electrodes for direct current therapy, and hence only one lead was recording (∼ lead II): electrodes were placed at the sternum and under the left arm in an antero-lateral position. All subjects had normal ECG at rest. Peripheral saturation was monitored with a Covidien ® saturation sensor placed at the neck of the subjects similarly to avoid artifacts from water, but also to ensure valid peripheral saturation, during max apnea and concomitant peripheral vasoconstriction.Arterial blood pressures and heart rates were recorded concomitantly using a Lifepack On a separate day, six of the BHD with the longest self-reported apneas completed a dry apnea after GPI during 12-lead ECG monitoring and after a warm-up of three consecutive apneas to maximize the diving response .® using one-way repeated-measures ANOVA. The Holm-Sidaks method post hoc was used to evaluate differences between the collected data during rest, apnea, and recovery. A P-value < 0.05 was considered statistically significant.Variables are presented as mean ± standard error of the mean (SEM). Data were analyzed by Sigma-Plot P < 0.001), while blood glucose increased from 5.8 ± 0.2 at rest to 6.2 ± 0.2 mmol/l just before the end of apnea (P = 0.009).At the end of maximum pool apnea (314 ± 64 s), peripheral saturation decreased from 99.6 ± 0.5 to 58.5 ± 5.5% (P < 0.001). Just before the end of apnea, junctional rhythm (n = 4), 2nd degree atrioventricular blockade (n = 1) and sinus bradycardia (n = 3) was observed (Heart rate decreased from 86 ± 14 beats per minute (bpm) to 46 ± 10 bpm and remained constant after 4 min (observed .P < 0.001), whereas diastolic blood pressure increased from 76 ± 3 mmHg to a maximum of 113 ± 5 mm Hg after 3 min of apnea (P < 0.001).Systolic blood pressure increased from 157 ± 7 to a maximum of 239 ± 15 mm Hg after 4 min of apnea (P < 0.001).Mean arterial blood pressure increased from 103 ± 4 to a maximum of 155 ± 6 after 3 min of apnea (P = 0.031). Sinus bradycardia (n = 3), 2nd degree atrioventricular dissociation (n = 1), and junctional rhythm (n = 2) were observed (During dry apnea of maximum duration (378 ± 31 s), heart rate decreased from 55 ± 4 to 40 ± 3 bpm (observed . No ST-sAll BHD remained conscious during both maximum pool and dry static apneas.n = 9) and controls : P = 0.121 , (2) had stable or increasing blood glucose, (3) increased in MAP to a maximum after 3 min of apnea without subsequent changing, (4) decreased in heart rate to a minimum of 46 ± 10 bpm and stabilized after 4 min until termination of apnea, (5) junctional rhythm, 2nd degree atrioventricular blockade, and sinus bradycardia were observed concomitantly by 1-lead ECG during maximum pool apnea, and (6) during maximum dry apnea, both junctional rhythm and 2nd atrioventricular dissociations were confirmed with 12-lead ECG. To our knowledge, these combined and detailed hemodynamic observations, and detailed arrythmias in particular have never been demonstrated earlier in elite BHD during static apneas: Although nd degree atrioventricular dissociation has not been reported earlier.After a warm-up of three consecutive apneas and at end of maximum pool apnea (314 ± 64 s) after GPI, the elite BHD (1) tolerated peripheral saturation as low as 58.5 ± 5.5%  mammals  increases during apnea  and decreases in cardiac output, and the consequence is a constant MAP. Of note, HR decreased to a constant level after 4 min of apnea. Hence, we conclude that in elite BHD during maximum apnea and after GPI, the diving response is maximized after 3–4 min and remains constant during apnea.Tissue oxygen delivery from Hb depends on 2,3 diphosphoglycerate, pH, and CO2 . Partialng apnea , and in ng apnea . In the ng apnea : DBP, SYng apnea . As PaCOa in BHD , 2021, t2 of 4.8 kPa, but stable or increasing blood sugar. Our results contrast with previous studies, and the stable blood glucose in this study (1) underlines the possibility of lactate metabolization during maximum apnea in elite BHD as we have sought to determine previously and (2) indicates that hypoxia rather than hypoglycemia is a factor causing brady-arrythmias during maximum apnea in elite BHD. Furthermore, we conclude that the diving reflex is maximized after 3–4 min of apnea, and extreme hypoxia is the key factor causing brady-arrythmias in elite BHD similar to diving mammals.In summary, we found that elite BHD similar to diving mammals have brady-arrythmias during maximum apnea enduring hypoxia of PaOOchotona curzoniae)  . The Pikrzoniae) , 2005, arzoniae) , it may rzoniae) : HIF-1 hrzoniae) . Similarrzoniae) . We suggFuture studies may reveal the content of cardiac HIF-1-alpha in BHD. Furthermore, it is speculated that the understanding of these mechanisms may help in developing new treatments of severe clinical conditions caused by extreme hypoxia, e.g., cardiac surgery, organ transplantation, and in the post-resuscitation care setting. Elite sprint athletes, fighter pilots, and mountain climbers may benefit from apnea training.Data collected in this study are all saved encrypted at hospital servers.The studies involving human participants were reviewed and approved by Ethical Committee of Copenhagen, Denmark. The participants provided their oral and written informed consent to participate in this study.TKj conceived the study and was in charge of the overall direction and planning of the presented ideas and developed the hypothesis. EH, BZ, LG, TKr, JV, and HA carried out the experiments. TKj, EH, AI, KL, TKr, BZ, LG, and HA contributed to sample preparation. All authors provided feedback, assisted in the analysis, critically revised the manuscript, approved the final revised version, and contributed to the interpretation and analysis of the results according to their specific specialized areas.TKj was voluntary (unpaid) board member of the Danish National Diving Federation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Also, GA treatment significantly decreased the Bax/Bcl2 protein ratio and the expression of Cyclin D1, CDK2, CDK4, MMP-9, N-cadherin, and vimentin in SW620 and HT29 cells. Meanwhile, the SIRT3 overexpression could significantly reverse these changes. Moreover, the GA treatment could significantly decrease the weight of xenograft tumor tissues and its SIRT3 protein levels in vivo, while SIRT3 overexpression reversed these effects. Overall, GA inhibited the proliferation, invasion, and migration of colorectal cancer cells, and induced their apoptosis by SIRT3 inhibition.Sirtuin-3 (SIRT3) has been described as a colorectal cancer oncogene and to be regulated by glycyrrhizic acid (GA). However, few studies have explored the interaction between GA and SIRT3. Therefore, in the present study, we showed that GA could significantly decrease SIRT3 protein levels in SW620 and HT29 cells in a dose-dependent manner. Then, we overexpressed SIRT3 by lentivirus infection on SW620 and HT29 cells. We found that,  Colorectal cancer is one of the most common malignant tumors, corresponding to about 1.8 million new cases and more than 800,000 deaths worldwide each year ,3. Besidin vitro [Novel antitumor agents derived and identified from natural sources have attracted increasing attention. These compounds represent a key alternative for the improvement of existing standard cancer therapies. Glycyrrhizic acid (GA) is the main effective ingredient of licorice, a traditional Chinese medicine, which has been proven to have antioxidative stress , anti-inin vitro . HoweverSirtuin-3 (SIRT3) is a NAD-dependent class III histone deacetylase. The main members of this family, such as SIRT1 , SIRT4 , and SIRSW620 (CCL-227) and HT29 (HTB-38) cell lines were purchased from the American Type Culture Collection (ATCC). They were cultured in McCoy’s 5A medium  supplemented with 10% fetal bovine serum  at 37°C and 5% CO2. Different concentrations of GA  were directly added to the medium to perform treatments GA, .Figure First, we harvested and lysed cells to obtain the total protein using the RIPA lysis buffer . Then, we used a BCA kit to detect the total protein concentration and protein levels as previously described . Fifty μ4) were seeded into a Lab-Tek cell culture well . After 24 h of 20 μmol/L GA treatment, we removed the cell culture medium and washed cells 3 times with PBS. After being fixed with 4% paraformaldehyde for 10 min at room temperature, cells were blocked with 5% BSA for 1 h at room temperature. Then, cells were incubated with SIRT3-antibody  overnight at 4°C. On the next day, we removed the SIRT3-antibody, and cells were incubated with Alexa Fluor488 goat-anti-rabbit IgG (H + L)  after being washed 3 times with PBS. Nuclei were counterstained with 5 μg/mL DAPI for 5 min at room temperature. Finally, all samples were analyzed by confocal microscopy.Cells  lentivirus infection. The empty vector control lentivirus (AAV-NC) was used as control. After 48 h of infection with lentivirus, we harvested cells to detect SIRT3 protein levels by Western blot.4 cells/well). After removing the medium, we washed cells 3 times with PBS. Then, 100 μL of cell culture medium supplemented with 10 uL CCK8 solution  were added to incubate cells for 1 h at 37°C and 5% CO2. Finally, we detected the OD600 to calculate the relative cell viability.After 24 h of stimulation with 20 μmol/L GA, we seeded cells into a 96-well cell culture plate . For apoptosis detection, after being washed twice with cold-PBS, we harvested cells to perform the Annexin V-FITC/PI staining following the Annexin V-FITC/PI apoptosis detection kit instructions .5 cells/100 μL medium into the transwell upper chamber. The lower chamber received 600 uL of McCoy’s 5A medium containing 2.5% FBS. After incubation for 24 h at 37°C and 5% CO2, we removed the cell culture medium in the transwell lower chamber, washed it twice with sterile PBS, and fixed it with formaldehyde at room temperature for 30 min. Then, we added 0.1% crystal violet for staining at room temperature for 20 min and washed it 3 times with PBS. Finally, we counted cells under a microscope.We used a transwell chamber test to determine the invasion and migration of colorectal cancer cells . We seedAfter stimulation with 20 μmol/L GA for 24 h, we harvested cells, then detected the caspase 3 activity using a Caspase 3 Activity Assay Kit  and following the manufacturer’s instructions.5 wild type (WT) or SIRT3 colorectal cancer cells cell subcutaneously under the nude mouse armpit. Nude mice in GA and GA+AAV-SIRT3 groups received 50 mg/kg of GA by gavage every day. Mice in the WT group received the same amount of solvent every day. All mice eat freely for 3 weeks, then were euthanized to separate and weigh colorectal cancer xenograft tumor tissues.Forty-eight nude mice  were used to establish colorectal cancer xenograft tumor models. First, we injected 2 × 10First, colorectal cancer xenograft tumor tissues were cut into 3.5 μm slices using Slicer . Then, we detected SIRT3 protein levels using an immunochemistry VECTASTAIN® Elite® ABC Kit  following the manufacturer’s instructions.p < 0.05 was considered as a significant difference.We used GraphPad Prism 5 to analyze the data and construct images. We used the Student’s t-test to compare differences between two groups. Differences between multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. A in vitro. Moreover, overexpression of SIRT3 could attenuate GA’s effects. Finally, we found that GA reduced the weight of colorectal cancer xenograft tumor tissues.In the current study, we hypothesized that GA exerted its anti-colorectal cancer activity by SIRT3 inhibition. First, we tested the effect of GA on SIRT3 protein expression in SW620 and HT29 cells. We found that GA inhibited SIRT3 expression in colorectal cells in a dose-dependent manner. Next, we found that GA inhibited the proliferation, cell cycle progression, migration, and invasion of colorectal cancer cells It has been found that SIRT3 is highly expressed in colorectal cancer tissues and that SIRT3 gene silencing can inhibit the proliferation, invasion, and migration of colorectal cancer cells, besides increasing their apoptosis . Here, wTo study whether GA can affect the biological characteristics of colorectal cancer cells by inhibiting SIRT3 expression, we first overexpressed SIRT3 in colorectal cancer cells through lentivirus infection (AAV-SIRT3). The Western blot results showed that the AAV-SIRT3 lentivirus infection successfully established SIRT3-overexpressed SW620 and HT29 cell lines ). Then, Since cell proliferation is closely related to the cell cycle, we hypothesized that GA affected colorectal cancer cell proliferation by blocking the cell cycle progression. To test this hypothesis, we used flow cytometry to detect the cell cycle of WT and SIRT3-overexpressed (AAV-SIRT3) colorectal cancer cells after stimulation with 0 or 20 μmol/L GA for 24 h. We found that, compared to the WT group, 20 μmol/L GA significantly increased the G1/G0 stage ratio and decreased the S/G2 stage ratio of SW620 ) and HT2in vitro. We found that, compared to the WT group, the stimulation with 20 μmol/L GA for 24 h significantly decreased the number of SW620  and HT2of SW620 ) colorecof SW620 .Figure The evaluation of the induction of cancer cells apoptosis is pivotal during studies with drugs used to treat cancer. Therefore, in the present study, we used flow cytometry to detect the apoptosis of colorectal cancer cells after GA treatment. We found that, compared to the WT group, the stimulation with 20 μmol/L GA for 24 h significantly increased the apoptosis of colorectal cancer cells, and overexpression of SIRT3 attenuated this effect . Additioin vivo environment complexity, many in vitro results can not be repeated in vivo. Thus, to verify whether GA’s effects on colorectal cancer cells are also effective in vivo, we established a nude mouse colorectal cancer xenograft animal model. Colorectal cancer cells were subcutaneously injected and the mice were euthanized after 3 weeks to obtain colorectal cancer xenograft tissues. Compared to the WT group, the GA treatment group presented significant decreases regarding SIRT3 protein levels in colorectal cancer xenograft tissues. However, SIRT3 protein levels in the GA+AAV-SIRT3 group were significantly higher compared to the GA group (Due to the GA group ). MoreovGA group ).FigureIn the present study, we found that GA significantly decreased SIRT3 protein levels in colorectal cancer cells. The silencing information regulator 3 (SIRT3) is one of the seven sirtuins family members of mammals and is homologous to the yeast silence information regulator sir2. Sirtuins (SIRT1-7) are NAD-dependent deacetylated proteins and ADP ribosyltransferases and the main mediators of non-histone acetylation, Therefore, their enzymatic activity is regulated by the NAD/NADH ratio in cells. Moreover, the SIRT3 function can vary between tumor types and may even oppose ,25. For in vitro and in vivo, while the overexpression of SIRT3 reversed this effect. We also found that GA blocks colorectal cancer cells in the G1/G0 phase, inhibiting their proliferation via SIRT3 inhibition. Since SIRT3 is a mitochondrial protein, it also affects mitochondrial energy metabolism. During proliferation, the cell needs to synthesize a lot of proteins and consume a lot of energy [To study the relationship between GA’s anti-cancer properties and SIRT3, we established SIRT3-overexpressed colorectal cancer cell lines. Then, we found that GA inhibited the proliferation of colorectal cancer cells f energy . Therefof energy . Thus, Gf energy , cyclin-f energy , and cycf energy  are impof energy ,35. TherMoreover, the migration and invasion of tumor cells to surrounding tissues characterizes the beginning and the key link of cancer metastasis, the main cause of cancer-related deaths . Thus, wSIRT3 is also related to apoptosis . Previouin vitro results were confirmed in vivo, since GA significantly decreased the SIRT3 protein levels in colorectal cancer xenograft tissues along with their weights. Also, overexpression of SIRT3 reversed GA inhibitory effects on the proliferation of colorectal cancer cells in vivo.Finally, our Overall, GA inhibited the proliferation, invasion, and migration of colorectal cancer cells, and induced their apoptosis by inhibiting SIRT3. Therefore, GA can be a potential drug for colorectal cancer treatment.


Background: Cardiac surgery is associated with a substantial risk of major adverse events. Although carbon dioxide (CO2)-derived variables such as venous-to-arterial CO2 difference (ΔPCO2), and PCO2 gap to arterial–venous O2 content difference ratio (ΔPCO2/C(a−cv)O2) have been successfully used to predict the prognosis of non-cardiac surgery, their prognostic value after cardiopulmonary bypass (CPB) remains controversial. This hospital-based study explored the relationship between ΔPCO2, ΔPCO2/C(a−cv)O2 and organ dysfunction after CPB.Methods: We prospectively enrolled 114 intensive care unit patients after elective cardiac surgery with CPB. Patients were divided into the organ dysfunction group (OI) and non-organ dysfunction group (n-OI) depending on whether organ dysfunction occurred or not at 48 h after CPB. ΔPCO2 was defined as the difference between central venous and arterial CO2 partial pressure.Results: The OI group has 37 (32.5%) patients, 27 of which (23.7%) had one organ dysfunction and 10 (8.8%) had two or more organ dysfunctions. No statistical significance was found (P = 0.84) for ΔPCO2 in the n-OI group at intensive care unit (ICU) admission , and at 4 , 8 , and 12 h post admission . In the OI group, ΔPCO2 also showed the same trend . No statistical difference was found for ΔPCO2/C(a−cv)O2 in the n-OI group (P = 0.46) and OI group (P = 0.39). No difference was detected in ΔPCO2, ΔPCO2/C(a−cv)O2 between groups during the first 12 h after admission (P > 0.05). Subgroup analysis of the patients with two or more failing organs compared to the n-OI group showed that the predictive performance of lactate and Base excess (BE) improved, but not of ΔPCO2 and ΔPCO2/C(a−cv)O2. Regression analysis showed that the BE at 8 h after admission  was a risk factor for organ dysfunction 48 h after CBP.Conclusion : ΔPCO2 and ΔPCO2/C(a−cv)O2 cannot be used as reliable indicators to predict the occurrence of organ dysfunction at 48 h after CBP due to the pathophysiological process that occurs after CBP. Althougon (VO2) , 5 and pon (VO2) , 7, the oversial , 9. CO2-patients . Hyperlapatients . Organ dly small , 13.2-derived variables and organ dysfunction occurring during the early phases after elective cardiac surgery with CPB. The secondary objective was to compare these variables with BE and lactate, which are usually associated with tissue hypoperfusion.The main objective of this study was to investigate relationship between COWe conducted a prospective observational study in a 39-bed mixed intensive care unit (ICU) of a university-affiliated Hospital. Our clinical trial was registered in the China Clinical Trial Registry (registration number: ChiCTR-ROC-17010727). Ethics approval was obtained from the Ethics Committee of Taizhou Hospital, Zhejiang Province. Written informed consent was obtained from all patients. The clinical trial started in February 2017 and ended in August 2018.Adults (≥18 years old) admitted to ICU immediately after CBP, were included if an arterial line and a central venous catheter (confirmed by X-ray) had been inserted. After initiation of the trial, we removed one of the eligibility criteria: “Factors affecting the accuracy of cardiac output monitoring: aortic regurgitation, atrial fibrillation, aortic balloon counter pulsation, high dose norepinephrine,” because it was not necessary and would have reduced our sample size. The exclusion criteria were: emergency surgery, pregnancy, chronic renal insufficiency with dialysis, preoperative acute or chronic liver failure, hematologic diseases, misplacement of the central venous catheter, and death within 48 h.2O, respirations 14–16 times/min, fraction of inspired oxygen 40%. Analgesia was achieved with continuous infusion of fentanyl (0.5–10 mg/kg/h) targeting Critical Care Pain Observation Tool scores of 0–2. Anesthesia was maintained with a continuous infusion of propofol (1–4 mg/kg/h) targeting Richmond Agitation-Sedation Scale scores of−2–0. Vasoactive drugs and fluid infusions were adjusted according to mean arterial pressure of 60–70 mmHg. Infusion of red blood cells was prescribed as needed to maintain hemoglobin (Hb) concentration ≥9.0 g/dL. Crystalloid solution  or 20% albumin was added for volume expansion if necessary.Intraoperative and ICU management was conducted according to local protocols and international guidelines. Ventilator settings were as follows: control mode, tidal volume 6–8 mL/kg, positive end-expiratory pressure ≤ 5 cmHWe prospectively collected preoperative, intraoperative, and postoperative variables, including clinical characteristics, duration of surgery, CBP duration, vital signs, fluid balance, vasoactive-inotropic score, routine blood tests, blood gas results, and clinical biochemistry. The specific data collection time points were at ICU admission and 4 h (H4), 8 h (H8), 12 h (H12), 24 h (H24), and 48 h (H48) after ICU admission. White blood cell count, hemoglobin concentration, platelet count, clinical biochemistry, chest X-rays, or CT scans were reviewed to evaluate organ function at ICU admission, H24, and H48. The length of stay in ICU, length of ventilator use, European System for Cardiac Operative Risk Evaluation II, Acute Physiology and Chronic Health Evaluation Score II (APACHE-II), and sequential organ failure score were recorded at ICU admission, H24, and H48. Paired arterial and central venous blood gases, arterial blood lactate, base excess (BE), and Hb levels were measured at ICU admission, H4, H8, and H12 using an automated analyzer .2-derived and O2-derived variables were calculated as follows:Vasoactive-inotropic score  and CO2-Vasoactive-inotropic score = dopamine (μg/kg/min) + dobutamine (μg/kg/min) + 10 × milrinone (μg/kg/min)+ 100 × epinephrine (μg/kg/min) + 100 × norepinephrine (μg/kg/min) + 10,000 × vasopressin (μg/kg/min)2/fraction of inspired oxygen <300 mmHg or PaO2 <60 mmHg requiring non-invasive ventilation or invasive mechanical ventilation support), acute kidney injury [Kidney Disease Improving Global Outcomes (KDIGO) level ≥1], acute tissue hypoperfusion , cardiac arrest  was used to compare categorical variables. GraphPad prism version 8.0.2 statistical software was used for boxplot charts.According to the occurrence of organ dysfunction at H48, the patients were divided into n-OI and OI groups, with the OI group being further subdivided into OI-1 and OI-2 subgroups according to the number of organs with evidence of dysfunction at H48. Comparisons took place within the OI group, within the n-OI group, between the OI and n-OI groups, and between the n-OI and OI-2 subgroups. The ROC curve analysis was carried out to assess the predictive performance of the variables independently associated with H48 organ dysfunction. The area under the curve (AUC) was calculated based on their 95% confidence intervals (CIs).P-value was < 0.05. A Hosmer–Lemeshow test was used to assess the goodness of fit of the model.In a subsequent analysis, variables were introduced into the logistic regression model if significantly associated with H48 organ dysfunction at the univariate analysis, when P = 0.01], shorter length of ICU stay , and lower H24 and H48 sequential organ failure scores  and . No statistical difference in the duration of mechanical ventilation was detected between the two groups of patients   h. In order to reduce the influence of spontaneous breathing on CO2 of the n-OI group at the four time points were 9.0 (7.0–11.0), 9.0 (7.0–11.0), 9.0 (7.0–11.0), and 9.0 (7.0–11.0) mmHg (P = 0.84), and those of the OI group were 9.0 (8.0–12.8), 10.0 (7.0–11.0), 10.0 (8.5–12.5), and 9.0 (7.3–11.0) mmHg (P = 0.37). The ΔPCO2/C(a−cv)O2 of the n-OI group at the four time points were 2.0 (1.6–2.7), 2.1 (1.7–2.8), 2.0 (1.5–2.3), and 2.0 (1.7–2.4) mmHg/mL (P = 0.46), and those of the OI group were 2.1 (1.7–2.9), 2.2 (1.8–2.5), 2.1 (1.8–2.6), and 1.8 (1.6–2.5) mmHg/mL (P = 0.39) (2-derived variables at any of the four time points (P > 0.05) between the n-OI and OI groups  , respectI groups . Statiste points .2, ΔPCO2/C(a−cv)O2, lactate and BE had no predictive value (P = 0.009) was a risk factor for H48 organ dysfunction after CPB.Regarding specific time point variables, ΔPCOve value . Comparive value . After a2/C(a−cv)O2 and ΔPCO2 did not differ significantly between the two groups at different time points during the first 12 h after ICU admission. Elevated ΔPCO2 and ΔPCO2/C(a−cv)O2 were common phenomena after CPB. The ΔPCO2/C(a−cv)O2 and ΔPCO2 cannot be used to predict H48 organ dysfunction. The predictive performance of lactate and BE was significantly enhanced as the number of dysfunctioning organs increased, although it was limited.We found that the ΔPCO2 and ΔPCO2/Ca−cvO2 on 48 h-organ dysfunction after adult cardiac surgery. In our study, the ΔPCO2 value, CPB time, aortic clamping time, and sequential organ failure score at ICU admission were similar to Guinot et al. study , at a 6.8 mmHg cut off with a sensitivity of 72.0% and specificity of 70.6%. Complications are defined as MMOM  (2 could be predictive of outcome (2/C(a−cv)O2 is not associated with respiratory quotient (2/C(a−cv)O2 was not associated with the existence of tissue hypoxia effectively because of its poor relationship with lactate (R2=0.067). ΔPCO2/C(a−cv)O2 was grouped according to whether it was ≥1.4 mmHg/mL, and no statistical significance were detected in the length of the ventilator, LOS in ICU, or the 24 h and 48 h sequential organ failure score (data not provided). That is probably largely because of the influence of CPB on the activation of inflammation in vivo, ischemia-reperfusion injury, myocardial suppression, and other factors, thus, a microcirculation disorder exists that can last for 72 h  , as foll outcome . A previquotient . In our for 72 h . From ouP itself , transloP itself , 29, andP itself  may dire2 and ΔPCO2/C(a-cv)O2, lactic acid has a moderate ability to predict two or more organ dysfunction. In addition, BE is the amount of base in mmol required to titrate 1 L of whole arterial blood to a pH of 7.40, with the sample fully saturated with oxygen at 37°C and a PaCO2 of 40 mmHg. We also found that BE was a more robust predictor of H48 organ dysfunction than lactic acid, and H8 BE was a risk factor for dysfunction of two or more organs (odds ratio = 1.37), whereas lactic acid was not. A high negative value of BE may be associated with renal dysfunction, metabolic acidosis, and shock, but the factors associated with the reduced systemic BE in patients after cardiac surgery are yet to be clarified , which could also explain the low susceptibility of BE to interference from other factors.Hyperlactatemia is common after cardiopulmonary bypass, and it can be influenced by many factors. Patients undergoing CPB are exposed to the “elution effect,” i.e., the release of lactic acid from the lungs  and the larified . We speccv−aCO2/C(a−cv)O2 cannot replace Pmv−aCO2/C(a−cv)O2 (mv: mixed venous blood) and might have underestimated the CO2 exchange from splanchnic circulation. Finally, it was not easy to detect when the outcome of interest, i.e., organ dysfunction, first occurred relative to the measurement. Continuous measurements or change over time may have more prognostic value than a single time point. However, the dynamic changes of ΔPCO2 at t0 and T6 time points were not found to be associated with postoperative complications or mortality in children after CPB O2 cannot be used as reliable indicators to predict the occurrence of organ dysfunction at 48 h after CBP, which is due to the complex pathophysiological processes after CBP.In conclusion, ΔPCOThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Ethics Committee of Taizhou Hospital, Zhejiang Province. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SZ: data curation, investigation, resources, writing—original draft, validation, and funding acquisition. DZ: data curation, investigation, and methodology. X-QC: data curation, investigation, methodology, and software. Y-PJ, L-ZQ, W-YY, and W-YZ: data curation, investigation, and resources. C-GW: data curation and formal analysis. Q-MZ: data curation and resources. T-HT: design of the study and performed data synthesis of the revised stage. R-HL: conceptualization, data curation, formal analysis, funding acquisition, software, validation, writing—review, and editing. All authors have read and approved the manuscript.This study was supported by the Medical and Health Science and Technology Project, Zhejiang Province (2017KY163). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We evaluated gene expression profiles related to apoptosis, autophagy, and oxidative stress; DNA fragmentation  and caspase activity (CAS3) were also examined. Exposure to 0.42 and 4.2 µg L−1 upregulated the genes coding for tnfa and baxa, sod1, prkaa1, respectively. CAS3 immunohistochemistry revealed a higher number of positive vitellogenic oocytes in ovaries exposed to 0.42 µg L−1. Subsequently, we examined the relationship between CAS3 signaling and DNA fragmentation. Accordingly, DNA fragmentation was observed in vitellogenic follicles of fish exposed to 0.42 and 4.2 μg L−1. Our results demonstrate that follicular atresia can occur after exposure to environmental levels of DiNP for 21 days, which may adversely affect the reproductive performance of female zebrafish in a non-monotonic manner.Di-isononyl phthalate (DiNP) is a plasticizer reported to elicit hormone-like activity and disrupt metabolism and reproduction in fish and other vertebrates. In general, phthalates have been used at high concentrations beyond reported environmental levels to assess their adverse effects on fish gonadal physiology. The present study exposed adult female zebrafish to a wide range of DiNP concentrations [0.42 µg L In adult female zebrafish, DiNP exposure elicited discrete gene expression modifications in the ovaries.2. 21-day DiNP exposure enhanced caspase-3 activity in vitellogenic follicles.3. Environmental concentrations of DiNP induced DNA fragmentation in follicular cells.Approximately 6 million tons of phthalate esters are produced worldwide  and used−1 .Among them, DEHP is one of the most studied phthalates in the aquatic environment , 7, and Recent studies conducted in our laboratory revealed that in zebrafish, DiNP altered the gonadal endocannabinoid system, oocyte growth, and oocyte maturation , 18. TheOne of the reproductive mechanisms in female gonads is the follicular atresia, a process based on follicular reabsorption throughout an interplay between apoptosis and autophagy, ensuring reproductive viability of the follicles in the ovaries , 22, andvia activation of CAS9 and CAS3, involving the mitochondrial outer membrane permeabilization (MOMP) and p53 tumor suppressor  were maintained in 100-L glass aquaria with oxygenated water under controlled conditions . Fish were fed twice a day with a 1:1 mixture of adult zebrafish complete diet  and live brine shrimp nauplii. Females  were exposed for 21 days to three nominal concentrations of DiNP : 0.42 µg L−1 (10−9 M); 4.2 µg L−1 (10−8 M); 42 µg L−1 (10−7 M) and a control (CTL) free of DiNP. The concentration range used was based on our previous studies . Because of its poor water solubility, DiNP was diluted in 1 ml of absolute EtOH and then, added to each aquarium. EtOH concentration (0.001% v/v) was below the activity threshold reported previously , 32.−1) (Sigma-Aldrich). Ovarian samples for molecular analysis were immediately frozen at −80°C with dry ice. Samples for TUNEL and CAS3 techniques were fixed in Bouin’s solution (Bio-Optica) overnight, then rinsed in 70% EtOH and stored at 4°C until needed for further analysis. Experiments were conducted in accordance with the guidelines on the care and use of fish in research, teaching, and testing from the Canadian Council on Animal Care (2005), as well as being in compliance with the University of Calgary animal care protocol.After 21 days, fish were euthanized with an overdose of buffered methane sulfonate MS-222 (300 mg L® reagent (Invitrogen) followed by chloroform disaggregation. RNA was precipitated with isopropanol and washed twice with absolute ethanol. Samples were treated with DNAse, and RNA quantification was determined by spectrophotometry. The quality of mRNA was assessed by electrophoresis in 1% agarose gel.RNA extraction, assessment of the RNA quality/quantity, and cDNA synthesis were performed using the whole ovary following . Brieflyrplp0  and rplp13 . A list of the used primers is provided in The relative quantification of gene transcripts was conducted using SYBR Green dye-based detection in a CFX iCycler thermal cycler (Bio-Rad). All samples were analyzed in duplicates. Each reaction contained: 1 μl of diluted (1/10) cDNA, 5 μl of 2× SYBR Green PCR Master Mix (Bio Rad), 0.1 μl of both forward and reverse primers, and 3.8 μl of milliQ water with a final volume of 10 μl per well. The reference genes used were 2, 0.5 μl digoxigenin-11-UTP, 1.5 μl terminal transferase enzyme, and 33 μl PBS per sample). Next, the slides were washed with EDTA (1 mM in PBS), blocked with sheep serum, and incubated with anti-digoxigenin AP (1:5.000) for 90 min at room temperature (RT). Finally, the staining solution was freshly prepared by dissolving one Fast Red pill in 2 ml of buffer  to mark the reaction. The sections were observed under a Zeiss Axio Imager.M2 coupled with a high-resolution Zeiss Axiocam 105 color camera.Ovaries were processed according to Randazzo et al. . Briefly2O2 at RT for 5 min. Then, to block non-specific bindings, slide sections were treated with 40% calf serum (Sigma-Aldrich). After the blocking, sections were incubated overnight in a humid chamber at 4°C with the primary cleaved CAS3 antibody from rabbit (1:200)  recognizing a cleaved form of Caspase 3 (~17 kDa). The next day, sections were rinsed with PBS buffer (pH 7.5) for 15 min and incubated with the secondary antibody IgG (Alexa Fluor 488) goat Anti-Rabbit diluted at 1:500  at RT for 2 h. Subsequently, the slides were mounted with Fluoroshield mounting medium with DAPI  for nuclei staining. Sections were observed under a Zeiss Axio Imager.M2 microscope, and images were acquired with a high-resolution Zeiss Axiocam 506 monochromatic camera. Images were processed with ZEN 2.3 lite software (Zeiss Microscopy GmbH).Deparaffinized slide sections (5 μm) were rehydrated and treated with citrate buffer  for the antigen retrieval. Next, endogenous peroxidases were inactivated using 3% HThe relative frequency of atretic follicles and vitellogenic follicles (focusing on stages III and IV) with CAS3-positive reaction were evaluated as described previously , 36.post hoc analysis. When data did not accomplish those conditions, Kruskal-Wallis non-parametric test was used. All statistical procedures were run using GraphPad Prism 6. The results are reported as mean ± standard error of the mean (SEM). Statistical significance was set at p < 0.05. Heatmap design was conducted using R software. Briefly, gene expression was normalized and standardized using z-score, and then, the mean was clustered with Euclidean distance matrix method. The results were expressed as a hierarchical clustering dendrogram followed by the heatmap plot. The relative frequency of CAS3 signal was assessed using a generalized linear model (glm) in R software  were not significantly altered, except for prkaa1 which displayed an upregulation in the 4.2 μg L−1 group. Additionally, 0.42 and 4.2 μg L−1 ovaries evidenced increased mRNA levels for baxa and tnfa, genes related to the apoptotic pathway. The gene coding for sod1 was the only oxidative stress marker showing an upregulated expression in the 4.2 μg L−1 group. Gene expressions were visually described as a heatmap , vitellogenic follicles stage IV (VF IV), as well as atretic follicles (AF), displayed a higher frequency of ir-CAS3 in VF IV from 0.42 μg LThe present study extends the knowledge about the effects of DiNP exposure on zebrafish ovarian follicular apoptosis by providing novel information on discrete gene expression modifications, DNA fragmentation, and increased ir-CAS3 in follicular cells.From an experimental standpoint, previous studies demonstrated the ability of phthalates or other xenobiotics, such as DEHP or bisphenol A (BPA), to exacerbate atresia in cultured mouse antral or zebrafish oocytes, respectively , 38. In baxa, tnfa, prkaa1 and sod1. It is likely that the apoptotic activation (extrinsic and intrinsic) involved changes in baxa and tnfa expression. On one hand, the extrinsic apoptotic pathway depends on TNF family for the activation of CAS cascade   . Rats exes (ROS) , 41. Simand cas3 .We here reported that exposure to DiNP altered CAS3 signal and DNA fragmentation in follicular cells and that DiNP could induce apoptotic processes in the zebrafish ovary, accordingly with previous observations in rats treated with DiNP and other phthalate congeners , 40, 41.Studies with phthalates are mainly conducted in mammals with non-realistic concentrations, presumably due to the phylogenetic proximity to humans. Since these compounds are abundantly present in the aquatic ecosystems , there ibecn1, map1lc3c, mtor, uvrag, bid), any change in the proapoptotic factors could alter the homeostatic balance of follicular development and demise. After DiNP exposure, the autophagic gene coding for prkaa1 was the only upregulated marker, indicating possible metabolic stress  as well as in compliance with the University of Calgary protocol.Conceptualization: OC, HRH, FLN, and RM. Data curation: FG and IFP. Formal analysis: FG, IFP, and BR. Funding acquisition: OC. Investigation: FG and IFP. Methodology: FG, IFP, and BR. Project administration, resources, supervision, and validation: OC. Visualization: OC. Roles/writing—original draft: FG, OC, and HRH. Writing—review and editing: FG, IFP, OC, FLN, HRH, RM, BR. All authors contributed to the article and approved the submitted version.Progetti di Rilevante Interesse Nazionale (PRIN) 2010–2011 (prot 2010W87LBJ) to OC, and by Fundação de Amparo à Pesquisa do Estado de São Paulo  to FG and RM.This work was supported by The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The tumor-associated macrophage (TAM) serves as an immunosuppressive agent in the malignant tumor microenvironment, facilitating the development and metastasis of lung cancer. The photodynamic effect destabilizes cellular homeostasis owing to the generation of reactive oxygen species (ROS), resulting in the enhanced pro-inflammatory function of immunocytes. In our previous study, the Ce6-mediated photodynamic effect was found to have kept the viability of macrophages and to remodel them into the M1 phenotype. However, the mechanism remains unrevealed. The present study now explores the mechanism of photodynamic therapy (PDT)-mediated reprogramming of macrophages. As expected, Ce6-mediated PDT was capable of generating reactive oxygen species, which was continuously degraded, causing “low intensity” damage to DNA and thereby triggering subsequent DNA damage response in macrophages. The autophagy was thus observed in Ce6-treated macrophages and was shown to protect cells from being photodynamically apoptotic. More importantly, Ce6 PDT could activate the stimulator of interferon genes (STING) molecule, a sensor of DNA damage, which could activate the downstream nuclear factor kappa-B (NF-κB) upon activation, mediating the polarization of macrophages towards the M1 phenotype thereupon. In addition, inhibition of ROS induced by PDT attenuated the DNA damage, STING activation, and M1-phenotype reprogramming. Furthermore, the silence of the STING weakened Ce6 treatment-mediated M1 remodeling of macrophages as well. Altogether, these findings indicate the Ce6-induced photodynamic effect polarizes macrophages into an M1 phenotype through oxidative DNA damage and subsequent activation of the STING. This work reveals the crucial mechanism by which photodynamic therapy regulates the macrophage phenotype and also provides a novel intervenable signaling target for remodeling macrophages into the M1 phenotype. Macrophages, especially tumor-associated macrophage (TAM), are crucial immunocytes in the lung cancer microenvironment, accounting for a notable proportion, up to 50% in a variety of solid tumors . Their avia ROS, which resulted in the activation of the downstream nuclear factor kappa-B (NF-κB) (Photodynamic therapy (PDT) has been reported to generate reactive oxygen species (ROS) that produce oxidative damage to organelles, especially capable of damaging DNA in the nucleus . The sti (NF-κB) . NF-κB,  (NF-κB) . This isBased on the aforementioned evidence, we speculated that the Ce6-induced photodynamic effect polarizes the macrophage into the M1 phenotype through oxidative DNA damage and subsequent activation of the STING. Herein, the macrophages were remodeled into the M1 phenotype characterized by the enhanced expression of CD80, CD86, MHC-II, iNOS, GBP5, IL-1β, and IL-6 under the treatment of Ce6 PDT. Further, Ce6 PDT was found to facilitate ROS generation and DNA damage without inducing apoptosis of macrophages. The enhanced autophagy also could be observed in Ce6-treated macrophages. Inhibition of autophagy led to the loss of viability and increased apoptosis in macrophages treated with Ce6. Additionally, molecules of the STING and NF-κB were activated by Ce6 PDT. Notably, inhibition of ROS induced by Ce6 PDT attenuated the DNA damage, STING activation, and M1-phenotype reprogramming. Finally, the silence of the STING weakened Ce6 treatment-mediated M1 remodeling of macrophages.2 and 95% air ambience, 37°C). The RPMI-1640 medium was applied to culture RAW 264.7 cells, while the DMEM was used to develop Lewis cells. When the two types of cells were co-cultured, the RPMI-1640 medium was adopted.RAW 264.7 cells , a typical mouse macrophage cell line, were utilized as the macrophage model . Lewis lCe6, used as a photodynamic agent in the present study, was purchased from Macklin . Macrophages were incubated with Ce6 (4 μg/ml) for 12 h and then irradiated with laser  for 20, 40, or 60 s . The cells were then left for 8 h.Proteins of macrophages were harvested. The expression of iNOS and GBP5 was assayed by Western blotting (WB). Alternatively, cells were harvested and washed with PBS three times; the expression of surface MHC-II was detected by immunofluorescence staining and analyzed by flow cytometry . In addition, mRNA levels of IL-6 and IL-1β were detected by RT-PCR . Phagocytosis of macrophages was analyzed by fluorescent latex beats and confocal laser scanning microscopy  . Briefly5 cells per well and treated as mentioned earlier and then left for 2, 4, 6, and 8 h, respectively. The cells were then incubated with 10 μM of 2,7-Dichlorodi-hydrofluorescein diacetate  at 37°C for 30 min before being harvested and assayed by flow cytometry. Alternatively, N-Acetyl-l-cysteine  was used to pre-treat macrophages for 2 h to block the generation of ROS.Macrophages were seeded in 24-well plates with a density of 3 × 10DNA double-strand break (DDSB) was detected by the comet assay. Briefly, macrophages were seeded in 24-well plates and treated with Ce6. Cells in PBS were prepared and mixed with low melting point agarose. The mixture was then dripped onto a glass slide pre-coated with agarose gel and pressed, followed by electrophoresis at 25 V, 300 mA for 25 min. The mixture was then neutralized using tris-Hcl (PH = 6.0) . FinallyMacrophages were seeded in a 6-well plate or on a confocal dish with the treatment of Ce6. The proteins wherein cells were harvested and used for WB to verify the expression of LC3-II/LC3-I, Atg5, and Beclin-1. The cells on the confocal dish were fixed with 4% paraformaldehyde and incubated with the LC-3 antibody and fluorescent secondary antibody and then imaged by laser scanning confocal microscopy . For inhibition of autophagy, wortmannin (300 nM) was applied to incubate with macrophages for 1 h and then removed. The cells were treated with Ce6, as described earlier. The autophagy-related proteins were detected. Cell apoptosis and apoptosis-associated proteins were then assayed.3 cells per well and treated, as mentioned earlier. The cell viability was detected using a CCK-8 kit . On the other hand, proliferation and apoptosis of macrophages were measured by the Annexin-V/PI staining assay and WB analysis of Bax and caspase-3.Macrophages were plated in 96-well plates with a density of 6 × 10Macrophages were seeded in 6-well plates or on a confocal dish and treated, as mentioned earlier. First, the expression of NF-κB, p-NF-κB, STING, and cGAS was detected by WB. Alternatively, the nuclear translocation of NF-κB was analyzed by immunofluorescence staining of NF-κB and confocal microscopy .The STING expression in macrophages was silenced using small interference RNA (SiRNA). The SiRNA nucleotides of targeting mouse STING (5′-GGAUCCGAAUGUUCAAUCAtt-3′) were purchased from GenePharma . SiRNAs were transfected into macrophages in the presence of the lipofectamine 3000 reagent, according to the manufacturer’s recommended procedure. Briefly, 5 μl of lipofectamine 3000 and 100 pM of SiRNA were used to transfect macrophages after co-incubation in 250 serum-free media for 15 min. After 8 h, the SiRNA and transfection reagent were withdrawn. An efficient knockdown of the STING was assayed by WB. The non-silencing SiRNAs (Scramble) were used as a control. After transfection, subsequent Ce6 PDT was processed on macrophages.For the assay of apoptosis, macrophages or LLC was harvested and then washed with PBS three times. Cells were incubated with FITC-Annexin-V  for 10 min and then incubated with PI for another 5 min. Cells were monostained with FITC-Annexin-V or PI as a control for compensation. The cellular fluorescence of Annexin-V and PI was detected by flow cytometry .For the detection of NF-κB, LC-3, and MHC-II expressions or location, cells were incubated with primary antibodies of NF-κB , LC-3 , CD80 , CD86 , and MHC-II  overnight at 4°C and then incubated with the goat anti-rat IgG/Alexa Fluor 488 secondary antibody  for another 60 min and washed three times before confocal microscopy  or flow cytometry . CD80 and CD86 were direct-labeled antibodies, and no secondary antibody incubation was required.4 cells per sample were acquired for every collection. Geometric means (GM) were used to quantify the mean fluorescent intensity (MFI).Ce6 fluorescence was acquired in the APC channel. CFSE fluorescence or Alexa Fluor 488 secondary antibody fluorescence was acquired in the FITC channel. PI fluorescence was acquired in the PE channel. The excitation wavelength was 638 nm, and the emission wavelength was 660 nm in the APC channel, 488 and 525 nm in the FITC channel, 561 and 585 nm in the PE channel, respectively. After cells were processed and collected as described earlier, they were filtered into special tubes for flow cytometry. Each channel was adjusted to the appropriate voltage before collecting cells. At least 1 × 10Cells treated as described earlier were washed three times with PBS and lysed in RIPA buffer with a 1% protease inhibitor. Cell lysates were centrifuged, and the protein concentration was measured using a BCA assay kit. Equal protein aliquots (10 μg) were fractionated by SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked with 3% bovine serum albumin in TBST and incubated with primary antibodies of Bax , p53 , γ-H2A.X , PCNA , LC-3 , Atg-5 , Beclin-1 , NF-κB , p-NF-κB , STING , cGAS , iNOS , GBP5 , Caspase-3 , and GAPDH  overnight at 4°C, and then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody. Finally, protein bands were developed using an ECL, and the films were exposed using a bio-imaging system .mRNAs of cells were extracted and reverse transcribed to cDNA. cDNA was amplified using an SYBR Green qPCR Master Mix kit . RT-PCR was performed using a Bio-Rad CFX Connect optics module, and data were analyzed using Bio-Rad CFXmanager. The specific primer sequences are as follows:Mouse IL-1β Forward: GCAACTGTTCCTGAACTCAACT.Mouse IL-1β Reverse: ATCTTTTGGGGTCCGTCAACT.Mouse IL-6 Forward: CGGAGAGGAGACTTCACAGAG.Mouse IL-6 Reverse: ATTTCCACGATTTCCCAGAG.Mouse GAPDH Forward: AGGTCGGTGTGAACGGATTTG.Mouse GAPDH Reverse: TGTAGACCATGTAGTTGAGGTCA.6 cells/150 μl in PBS)-bearing mice received irradiation  after Ce6 administration (1 mg/kg bw). For about 12 h, the mice were sacrificed, and tumor grafts were harvested. Paraffin sections of tumor tissues were dewaxed, rehydrated, and antigen repaired with sodium citrate for 20 min. The paraffin sections were then incubated in 3% hydrogen peroxide for 12 min at room temperature. The paraffin sections were blocked with 5% BSA for 40 min, stained with primary antibodies overnight at 4°C, and then stained with the secondary antibody  for 1 h at 37°C. Diaminobenzidine  was applied for coloration for 1–3 min at room temperature. Hematoxylin was used to stain the nucleus. Primary antibodies included CD11b , iNOS , GBP5 , MHC-II , γ-H2A.X , p53 , cGAS , STING , NF-κB , LC-3 , Atg-5 , and Beclin-1 . Finally, the paraffin sections were observed by orthomorphic Olympus microscopy .Female C57 mice at 4–6 weeks (20–22 g) were purchased from the Laboratory Animal Center at the Hubei University of Medicine . Animal handling and experimental procedures were in line with protocols approved by the Animal Care Committee at the Hubei University of Medicine. Mice were housed in a temperature-controlled environment with fresh water and rodent diet available at all times. All inoculations and administrations were performed under nembutal anesthesia. To determine the role of Ce6 PDT in the reprogramming of macrophages, DNA damage, levels of autophagy, and activation of the STING, NF-κB, subcutaneous lung cancer cell . Statistical differences between groups were analyzed using one-way analysis of variance (ANOVA). in vivo experiments showed significant upregulation of iNOS, GBP5, and MHC-II in tumor grafts of LLC-bearing mice that received Ce6 PDT, suggesting that more M1-phenotype macrophages may be present in tumor tissues , the levels of ROS generated by Ce6 PDT and its degradation over time were thus investigated initially. As shown in with Ce6 , indicatwith Ce6 . A minorIn addition to inducing apoptosis, the DNA damage response (DDR) on the other hand, has been reported to activate autophagy , which mper se from death.Next, the inhibitor of autophagy, wortmannin, was applied to explore whether autophagy triggered by Ce6 PDT could protect macrophages from death. The results showed Ce6 PDT induced upregulation of Atg-5, and LC-3 II could be impaired by wortmannin , an indiin vitro results, the evidence of IHC staining also showed strong activation of the STING and NF-κB in tumor grafts of LLC-bearing mice that received Ce6 PDT, implying that STING and NF-κB molecules were activated of cells (both macrophages and tumor cells) in the tumor grafts (In addition to activating the autophagy signaling pathway, broken fragments from DNA damage also have the ability to activate the intracytoplasmic STING and subsequent NF-κB molecules . The actr grafts . In geneThe findings in the aforementioned section have strongly spoken that Ce6 PDT promoted ROS production in macrophages, damaged DNA, activated the STING, and reprogrammed macrophages into the M1 phenotype. To validate that Ce6 treatment-mediated oxidative damage is an essential driver of STING activation and reprogramming of macrophages to a pro-inflammatory phenotype, we next inhibited ROS in macrophages and then observed whether Ce6 treatment could still cause DNA damage, STING activation, and remodeling of the M1 phenotype. The results in Apart from the driving factor of oxidative damage, the STING and its downstream NF-κB are key molecules that mediate the pro-inflammatory response of macrophages . The infIn the present study, oxidative DNA damage was found to be triggered by the action of PDT on macrophages, which on the one hand activated autophagy and protected macrophages from death. On the other hand, DNA damage also activated the STING molecule, which serves as the crucial inflammatory response by activating its downstream NF-κB, thus polarizing macrophages into the M1 phenotype. Activation of STING and NF-κB molecules was critical for macrophage phenotypic reprogramming .per se. High intracellular concentrations of ROS typically result in “high intensity” DNA damage, which is so severe that DDR is unable to repair the cell in time and apoptosis occurs thereupon (The photodynamic action initiated by the catalytic intracellular photosensitizer under certain conditions produces low concentrations of ROS and has little cytotoxicity. In the present study, we continued to use Ce6-mediated PDT to intervene in macrophages using the conditions from the previous study . Intereshereupon . In contThe downstream signaling of DDR is complicated, which includes the PI3K signaling pathway associated with autophagy . Our stuMacrophages undergo autophagy in response to PDT treatment, maintaining their viability, which is a prerequisite for their phenotype to be remodeled. Another downstream molecule of DDR is the STING, a crucial molecule mediating the inflammatory response and the immune response to infection . The STIIn summary, Ce6-mediated PDT stimulates macrophages undergoing oxidative DNA damage, which activates not only autophagy but also the STING molecule. Enhanced autophagy maintains viability of macrophages. Activation of the STING molecule promotes the inflammatory response of macrophages, leading to their reprogramming toward the M1 phenotype thereupon . The pre


DNA-protein cross-links (DPCs) are extremely bulky adducts that interfere with replication. In human cells, they are processed by SPRTN, a protease activated by DNA polymerases stuck at DPCs. We have recently proposed the mechanism of the interaction of DNA polymerases with DPCs, involving a clash of protein surfaces followed by the distortion of the cross-linked protein. Here, we used a model DPC, located in the single-stranded template, the template strand of double-stranded DNA, or the displaced strand, to study the eukaryotic translesion DNA polymerases ζ (POLζ), ι (POLι) and η (POLη). POLι demonstrated poor synthesis on the DPC-containing substrates. POLζ and POLη paused at sites dictated by the footprints of the polymerase and the cross-linked protein. Beyond that, POLζ was able to elongate the primer to the cross-link site when a DPC was in the template. Surprisingly, POLη was not only able to reach the cross-link site but also incorporated 1–2 nucleotides past it, which makes POLη the most efficient DNA polymerase on DPC-containing substrates. However, a DPC in the displaced strand was an insurmountable obstacle for all polymerases, which stalled several nucleotides before the cross-link site. Overall, the behavior of translesion polymerases agrees with the model of protein clash and distortion described above. The sequences of the oligonucleotides used in this study are shown in Yeast DNA polymerase η (yPOLη), human DNA polymerase η (hPOLη), human DNA polymerase ι (hPOLι), four-subunit yeast DNA polymerase ζ (yPOLζ), and escribed ,43,44. O4 cross-linking and purified, as previously described [32P-labeled primer for 30 min at room temperature.Model DPCs between the oligonucleotides and Fpg were prepared by NaBHescribed ,45. The 2 or 0.5 mM MnCl2. The reaction was allowed to proceed at 37 °C for 2, 5, or 30 min. At these times, aliquots were withdrawn and an equal volume of a loading solution  was added, followed by 2 min of heating at 95 °C. The reaction products were resolved by 21% denaturing polyacrylamide gel electrophoresis and visualized by phosphorimaging . The lengths of the extension products were determined from comparisons with the mobility markers (Primer extension reactions (20 μL) contained 10 nM DPC substrate (see above), DNA polymerase , the dNTP mixture of 50 μM of each, 30 mM HEPES (pH 7.4), 5% glycerol, 0.1 mg/mL of bovine serum albumin, and 10 mM MgCl markers  and partThe schemes of the model substrates are shown in 2+ or Mn2+ ions   photoproducts, and 1,2-intrastrand cisplatin cross-links [POLζ is a processive family B DNA polymerase that participates in translesion DNA synthesis, usually as an “extender” polymerase ,51. Howess-links ,52,53, pss-links ,54, sugg2+ ions  does not involve translesion synthesis. However, in our previous work  it was scis-syn dimers [POLη is an archetypal eukaryotic translesion DNA polymerase of family Y. The best-known POLη role is the protection against UV lesions by accurately copying opposite cyclobutane pyrimidine dimers ,58. Human dimers , with re2+ ions  can use a DNA lesion “skipping” mechanism due to Streisinger slippage or dNTP-stabilized misalignment on a template with repetitive bases ,81,82,83The situation when a DPC is located in the displaced strand rather than in the template is of a particular interest. Despite some DNA polymerases being well known for their strand displacement activity, the consequences of having a DPC in the displaced strand remain largely unexplored. Strand displacement may occur in many biologically relevant situations, such as repair synthesis, viral DNA replication, etc., and the presence of a DPC could interfere with these processes. In contrast to the situation when a DPC is located in the template, bypassing a DPC in the displaced strand does not involve dNMP insertion opposite a covalent protein adduct. Moreover, the presence of a cross-link could partially melt and unwind the DNA, which may ease strand separation. However, almost all DNA polymerases studied here and in , and priOverall, it is clear that POLι, POLη, and POLζ, despite their ability to incorporate dNMPs opposite to a wide variety of lesions, cannot bypass the model DPC, and likely other DPCs as well. The only DNA polymerase that shows this property in vitro, albeit with quite a low efficiency, is PrimPol . These o