Macrobrachium rosenbergii remains poorly understood. In this study, osmoregulation, physiological metabolism, antioxidant capacity, and apoptosis were examined over 96 h of acute salinity stress. Hemolymph osmolality increased with increasing salinity. After 48 h of salinity exposure, the glucose, triglycerides, total protein, and total cholesterol contents in two salinity stress groups  were significantly lower than those in the 0‰ salinity group. The highest levels of these parameters were detected at 6 h; however, superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were the lowest at 96 h in the 13‰ salinity group. The activity of immunity-related enzyme alkaline phosphatase (AKP) showed a decreasing trend with increasing salinity and remained at a low level in the 26‰ salinity group throughout the experiment. No significant differences were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT), or lysozyme (LZM) among the three treatments at 96 h. After 96 h of salinity treatments, the gill filament diameter significantly decreased, and a more pronounced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive signal was detected in the 13‰ and 26‰ groups compared to that in the 0‰ group. Expression levels of apoptosis-related genes, including Cysteine-aspartic acid protease 3 (Caspase 3), Cysteine-aspartic acid protease 8 (Caspase 8), Cytochrome c (Cyt-c), tumor suppressor gene (P53), Nuclear factor kappa-B (NF-κB), and B cell lymphoma 2 ovarian killer (Bok) were significantly higher in the 26‰ salinity group than in the other groups at 24 h, but lower than those in the 0‰ salinity group at 96 h. Cyt-c and P53 levels exhibited a significantly positive relationship with MDA, AST, and LZM activity during salinity stress. In the 13‰ salinity group, Bok expression was significantly correlated with SOD, T-AOC, AKP, acid phosphatase, and LZM activity, whereas in the 26‰ group, the AST content was positively correlated with Caspase 8, Cyt-c, and P53 expression. A significant negative relationship was observed between Caspase 3 expression and catalase (CAT) activity. These findings provide insight into the mechanisms underlying the response to acute salinity stress and will contribute to improving M. rosenbergii aquaculture and management practices.Salinity stress can trigger a series of physiological changes. However, the mechanism underlying the response to acute salinity stress in  Salinity is regarded as a crucial environmental factor that influences various aspects of aquatic animal life, including reproduction, growth, development, and survival ,2,3. Som+-K+-ATPase (NKA) is typically an essential enzyme that plays a crucial role in regulating ions and maintaining osmotic pressure balance within intra- and extracellular fluids [Hemolymph and blood osmolality are commonly measured to assess the osmoregulatory capacity of aquatic animals. At salinities below and above the isosmotic concentration, the hemolymph becomes hyperosmotic and hypo-osmotic, respectively, in the surrounding medium . Na+-K+-r fluids . Numerour fluids ,13.The hepatopancreas is an important digestive gland in crustaceans that plays a critical role in physiological metabolism and antioxidant defense in response to environmental stress . Under sFurthermore, these stress-induced challenges often lead to an increase in reactive oxygen species (ROS), ultimately inducing oxidative stress, which adversely affects crustaceans ,20. ManyMacrobrachium rosenbergii) is an economically important cultured species worldwide. It grows and develops in freshwater and migrates to estuaries for reproduction, where water of a specific salinity level is necessary for larval metamorphosis and hatching. During migration, this species develops high tolerance to a wide salinity range [M. rosenbergii as it transitions from freshwater to brackish water. Therefore, further investigations are needed to explore the mechanisms underlying the response to acute salinity stress.The giant freshwater prawn  at the Pearl River Fisheries Research Institute, CAFS. A total of 360 healthy cultured prawns with an average body weight of 12.49 ± 3.08 g were obtained from Liantang Town, Zhaoqing City, Guangdong Province. Prior to the experiment, the prawns were acclimatized in an indoor aquarium for 5 days during which the water temperature was maintained at 30 ± 0.5 °C, dissolved oxygen levels were maintained at 6 mg/L, and the pH ranged from 7.0 to 8.0. The prawns were fed a commercial artificial compound diet twice daily at 8:00 a.m. and 5:00 p.m., and residues and excreta were removed through regular water changes. Before the start of the experiment, the prawns were fasted for 24 h.Following acclimation, the prawns were randomly assigned to three treatment groups: 0, 13, or 26‰ salinity. The salinity was measured by using a salinometer . There were 3 treatments and a total of 9 tanks, with 40 individuals per tank. The control group was maintained in freshwater , and the higher-salinity groups (13‰ and 26‰) were achieved by diluting the seawater stock solution with fully aerated dechlorinated tap water.When the experiment commenced, six prawns  from each treatment group were randomly selected and anesthetized within an ice bath for tissue collection at five time-points .The hemolymph samples were taken from the base of the prawn’s fifth walking limb using a 1 mL syringe washed with heparin sodium salt, placed on ice, transported to the laboratory, and maintained for 1–2 h.w/v) physiological saline. After centrifugation, the supernatant was diluted and stored in centrifuge tubes for subsequent analysis.The hepatopancreas tissues were frozen in liquid nitrogen and then stored at −80 °C for physiological assays and RNA extraction. Part of the hepatopancreas tissues were thawed and homogenized at 12,000 rpm for 10 min at 4 °C with 1:9 .After centrifugation at 10,000× The levels of GLU, triglycerides (TG), total protein (TP), and total cholesterol (T-CHO) in the hepatopancreas after 6, 12, 24, 48, and 96 h of salinity stress were determined via commercially available kits .The contents of anti-oxidative parameters including total superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content, and the immunity index containing aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) in hepatopancreas were determined to evaluate the antioxidant activity under salinity stress using commercially available kits .After dehydration, the samples were transparented in xylene, embedded in paraffin, and sectioned (5–6 μm) using a Leica RM2016 Microtomes paraffin slicer . Paraffin sections were deparaffinized and rehydrated before staining with hematoxylin–eosin (HE) solution . The stained sections were sealed with neutral resin and then visualized under light microscopy . At least six gill filaments were randomly selected for measurement in each visual field, and each gill filament was measured three times. The gill filament diameter was measured by using CaseViewer 2.4 software .Tissue cell apoptosis was assessed using the terminal-deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay following the protocol of the apoptosis detection kit  . BrieflyTotal RNA was isolated from the hepatopancreases using TRIzol™ reagent . RNA integrity was assessed using 1% agarose gel electrophoresis. Total RNA (1 µg) from different tissues were digested with DNase I . First-strand cDNA was synthesized from 1 μg of total RNA using an M-MLV Reverse Transcriptase Kit  following the manufacturer’s instructions.® Green Supermix, 0.5 μL of each primer (10 pmol/μL), and 3 μL of double-distilled water to a final volume of 10 μL. The reaction protocol was as follows: 95 °C for 3 min; 35 cycles of 95 °C for 40 s, 60 °C for 45 s, and 72 °C for 30 s; and 72 °C for 10 min for data acquisition, and then 95 °C for 5 s, 60 °C for 30 s, and 95 °C for 15 s to obtain the melt curve. β-actin was selected as the internal reference because of its stable expression.The primers for apoptosis-related genes and reference genes used in this study are listed in p-values less than 0.05 referred to significant differences (*), while values less than 0.01 referred to highly significant differences (**) between two variables.Pearson’s correlation analysis was performed to explore the correlations between antioxidant parameters and expression levels of key genes in salinity-stressed groups. The heatmap was created in R 4.2.3 with the pheatmap package, and p-value < 0.05. The mRNA abundances of genes were calculated using the 2−ΔΔCt method [Differential analysis of hemolymph osmolality, anti-oxidative, and physiological metabolism parameters under different salinity challenges was performed using analysis of variance (ANOVA). Data are presented as the means ± standard error (SE) of three replicates, and the statistical significance is represented by a t method . All stap < 0.05). However, there was no difference between the 0‰ and 13‰ salinity groups.The hemolymph osmolality displayed an increasing trend with water salinity . A signip < 0.05). At 6 h, the GLU content was significantly lower in the 0‰ group compared to the other groups. Similarly, at 24 h, the 13‰ group displayed a significantly lower GLU level than the other groups (p < 0.05). After 96 h, both salinity stress groups exhibited significantly lower GLU levels than the 0‰ group (p < 0.05). Furthermore, significant differences in TG contents were detected at 48 h, with significantly higher levels than those in the other two groups (p < 0.05). Significant differences in the TP content were found among the three groups at all time-points except 12 h (p < 0.05). Specifically, the 13‰ group displayed significantly lower TP levels at 6 h, and the 26‰ group exhibited significantly lower TP levels at 24 h compared to that of the other groups (p < 0.05). However, no significant difference was observed between the 0‰ and 26‰ groups at 48–96 h (p > 0.05). Following 12–24 h of salinity stress, the 13‰ group showed significantly lower T-CHO contents than the 0‰ group (p < 0.05), and the T-CHO content in the 0‰ group was significantly higher than those in the other groups (p < 0.05).Several physiological metabolic parameters of the hepatopancreas were driven by the salinity challenge with significant differentiation at some time points . Signifip < 0.05). At 6, 12, and 24 h, no significant difference was observed between the 0‰ group and the other groups (p > 0.05). Significant differences in CAT activities were observed among the three groups at 6, 12, and 24 h (p < 0.05). The highest CAT activities were observed in the 13‰ group at 12 and 24 h (p < 0.05), but no significant differences were found thereafter (p > 0.05). There were no significant differences in GSH-Px activities at 6 or 12 h among the three groups (p > 0.05). GSH-Px activities in the 26‰ group increased over time and were significantly higher at 96 h in both the 26‰ and 0‰ groups compared to those in the 13‰ group. Compared to the 0‰ group, GST activity in the 13‰ group and 26‰ group was significantly lower at 24, 48, and 96 h (p < 0.05). Significant differences in T-AOC activity were observed among the three groups except at 24 and 48 h. T-AOC activity in the 13‰ group was significantly higher than those in the 26‰ group at 6 and 12 h (p < 0.05). However, at 96 h, the T-AOC activity in the 13‰ group was significantly lower than those in the 0‰ and 26‰ groups (p < 0.05). Following 24–96 h of salinity stress, the MDA content in the 0‰ group was significantly higher than that in the 13‰ group (p < 0.05). The MDA content showed a decreasing trend in the 26‰ group, with no significant difference compared to the 0‰ group (p > 0.05).The antioxidant enzyme activities in the hepatopancreas after salinity stress varied at different time points . The SODp < 0.05). However, there was no significant difference between the 26‰ and 13‰ groups (p > 0.05). At 0‰ salinity, ALT activity was significantly higher after 24 h of exposure compared to that in the 13‰ group (p < 0.05). However, after 48 h of exposure, the ALT activity in the 0‰ and 13‰ groups was significantly lower than that in the 26‰ group (p < 0.05). Significant differences in AKP activity were observed among the three groups at all time points, with higher activity in the 0‰ group compared to those in the 13‰ and 26‰ groups after 24–96 h of salinity stress (p < 0.05). The ACP activity showed a decreasing trend in the 13‰ group, and the opposite trend was observed in the 26‰ group. Significant differences in ACP activity were detected among the three groups at other time points except 24 h (p < 0.05). At 12 h, ACP activity was significantly lower in the 26‰ group than in the other groups, and there was no significant difference between the 26‰ and 0‰ groups at 48 and 96 h. The LZM activity in the 13‰ group displayed a decreasing trend, and significant differences were observed among the three groups at 6, 24, and 48 h (p < 0.05). At 48 h, the LZM activity was significantly lower in the 13‰ group than in the other groups. However, there were no significant differences between the 0‰ and 26‰ groups at 24–48 h (p > 0.05).The immunocompetent response of the hepatopancreas to salinity stress exhibited different trends at different time points . After 2p < 0.05).Significant histological differences were observed in the gills among the different salinity treatments . After 9To further investigate the effects of acute salinity stress on gill tissues, we examined the apoptosis signals using fluorescence microscopy . After 9Caspase 3 and Caspase 3), Cytochrome c (Cyt-c), tumor suppressor gene (P53), Nuclear factor kappa-B (NF-κB), and B cell lymphoma 2 ovarian killer (Bok) exhibited significantly higher expression levels in the 26‰ salinity group compared to the other groups at 24 h, but showed lower levels at 96 h (Caspase 3, Cyt-c, and P53 in 13 and 26‰ groups were significantly higher than those in the control group. However, no significant difference was observed in NF-κB and Bok expression levels at this time point. After 12 h of salinity treatment, Caspase 3 and NF-κB were significantly upregulated in the 26‰ group, and Caspase 8 and Cyt-c displayed the opposite expression trends. There were no significant differences for Bok and P53 between the groups. Caspase 8 in the 26‰ salinity group remained low, except at 24 h. After 48 h of exposure to 13‰ salinity, all six genes showed significantly lower expression levels than those in the 0‰ group.The apoptosis-related genes, Cysteine-aspartic acid protease 3 and 8 ( at 96 h . At 6 h,Caspase 3, Caspase 8, Cyt-c, P53, NF-kB, Bok, and antioxidant and immune enzymatic activities (Cyt-c, Bok, and P53 mRNA and those of MDA, AST, and LZM. Additionally, Caspase 8 expression was positively correlated with AST activity. In the 13‰ group, the Bok expression level was positively correlated with SOD, T-AOC, AKP, ACP, and LZM activities. The level of Caspase 3 showed a significant positive correlation with MDA and AKP. The P53 expression level was only positively correlated with MDA content. In the 26‰ stress group, Caspase 8, Cyt-c, and P53 expression levels were positively correlated with AST. However, a significant negative correlation was observed between Caspase 3 and CAT levels.There were significant correlations between apoptosis-related genes tivities . A signi+/K+-ATPase genes in gills in response to salinity stress [M. rosenbergii demonstrated that hemolymph osmolality can be maintained at around 420 mOsmolkg-1 at salinity < 18‰ by gradually increasing the salinity, but that the osmotic capacity becomes severely challenged as salinity increases [M. Rothbergii to estuarine regions with salinity changes during their early life stages.Migratory crustaceans often exhibit high tolerance to salinity stress because of their enhanced capacity for hemolymph osmoregulation . The hemy stress . Our resy stress ,38,39. Sncreases . Our stuEnhanced physiological metabolism produces more energy and substances to help organisms cope with salinity challenges . The hepAcute salinity stress increases the concentration of ROS, resulting in oxidative stress in aquatic animals ,44,45. AClarias gariepinus [Eriocheir sinensis [M. rosenbergii. In contrast to our results, AKP and ACP activity decreased in response to low salinity stress in the inshore shrimp [M. rosenbergii exhibits a stronger tolerance to high salinity than lower salinity.Environmental stress often triggers non-specific immune responses in aquatic animals . AST, ALriepinus . Furthersinensis . The ACPe shrimp , suggeste shrimp ,57. Our e shrimp . These rAs respiratory and osmoregulatory organs, gills exhibit rapid responses to salinity stress, primarily through significant morphological changes, such as gill filament shrinking and deformation, reductions in gill cilia, and cell apoptosis . In our Caspase 3, Caspase 8, Cyt-c, P53, NF-κB, and Bok, showed an initial increase followed by a decrease under salinity exposure. Down-regulation of those apoptosis-related genes indicates that the late stage of the apoptosis process was reached at the end of the high-salinity exposure experiment. Caspase 3 and Caspase 8 are crucial molecules in apoptosis, with Caspase 8 initiating the caspase cascade and activating downstream Caspase 3 to induce apoptosis [Caspase 3 level in the 26‰ group suggested that apoptosis reached its final stage after 96 h. Interestingly, we found a significant negative relationship between Caspase 3 transcription abundance and CAT activity in the 26‰ salinity group. The active Caspase 3 can inhibit CAT activity and induce proteolysis of the CAT protein [Caspase 3 and CAT may play antagonistic roles in response to higher-salinity challenges.The prolonged oxidative stress promotes cell death by activating the apoptotic pathway . In our poptosis . Caspasepoptosis . The low protein . TherefoCyt-c, is a crucial step in cell apoptosis. As a transcription factor, the Cyt-c mRNA expression influences apoptosis activation in the mitochondrial pathway [Cyt-c and P53 expression was significantly related to MDA and AST levels under salinity stress, indicating that high salinity induced apoptosis in gills through the mitochondrial pathway. P53 has been well established to play a vital role in cell death by regulating the apoptosis pathway [P53 in a marine crab at low salinity from 3 h to 24 h is opposite yet analogous to our findings, suggesting a conserved role of P53 in responding to salinity changes among different crustaceans [P53 actively participates in the cell cycle and apoptosis process by promoting the transcriptional regulation of downstream target genes [The activation of caspases, induced by the release of  pathway . In our  pathway . The sigstaceans . Furtheret genes .NF-κB is a highly conserved transcription factor, and activation of the NF-κB signaling pathway induced by environmental stressors usually triggers the expression of downstream genes involved in various cellular events, including apoptosis [NF-κB expression was up-regulated under salinity and temperature stresses in a coral [poptosis . The NF- a coral .Bok is a proapoptotic protein that promotes cell death by interacting with specific anti-apoptotic proteins. Its overexpression has been reported to promote the repair of oxidative damage caused by hydrogen peroxide [Bok expression levels may inhibit the oxidative damage repair. In the 13‰ group, Bok expression level was significantly correlated with SOD, T-AOC, ACP, AKP, and LZM, suggesting that the oxidative damage and inflammation caused by salinity stress may have stimulated damage repair mechanisms by inducing Bok expression; however, this requires further investigation.peroxide . Based oM. rosenbergii gill tissue (M. rosenbergii and enhance our understanding of the potential molecular mechanisms underlying the adaptation to acute salinity stress in crustaceans.Acute salinity stress induces an increase in hemolymph osmotic pressure, triggers antioxidant responses, and elicits immune stress, leading to structural damage and apoptosis of l tissue . These f


In critical care patients, the “”temporary inactivity of the diaphragm caused by mechanical ventilation (MV) triggers a series of events leading to diaphragmatic dysfunction and atrophy, commonly known as ventilator-induced diaphragm dysfunction (VIDD). While mitochondrial dysfunction related to oxidative stress is recognized as a crucial factor in VIDD, the exact molecular mechanism remains poorly understood. In this study, we observe that 6 h of MV triggers aberrant mitochondrial dynamics, resulting in a reduction in mitochondrial size and interaction, associated with increased expression of dynamin-related protein 1 (DRP1). This effect can be prevented by P110, a molecule that inhibits the recruitment of DRP1 to the mitochondrial membrane. Furthermore, isolated mitochondria from the diaphragms of ventilated patients exhibited increased production of reactive oxygen species (ROS). These mitochondrial changes were associated with the rapid oxidation of type 1 ryanodine receptor (RyR1) and a decrease in the stabilizing subunit calstabin 1. Subsequently, we observed that the sarcoplasmic reticulum (SR) in the ventilated diaphragms showed increased calcium leakage and reduced contractile function. Importantly, the mitochondrial fission inhibitor P110 effectively prevented all of these alterations. Taken together, the results of our study illustrate that MV leads, in the diaphragm, to both mitochondrial fragmentation and dysfunction, linked to the up-/down-regulation of 320 proteins, as assessed through global comprehensive quantitative proteomics analysis, primarily associated with mitochondrial function. These outcomes underscore the significance of developing compounds aimed at modulating the balance between mitochondrial fission and fusion as potential interventions to mitigate VIDD in human patients. Mechanical ventilation (MV) is a double-edged sword. Although life-saving for patients in intensive care unit, MV induces diaphragm weakness. This side effect appears to decrease their ability to be successfully weaned from the ventilator with a major impact on clinical practice, patient's morbidity, and the healthcare resource utilization. We have identified the presence of a mitochondrial fragmentation in the diaphragm, induced by MV. We have also observed that this fragmentation, called fission, leads to dysfunctional mitochondrial with production of pathological oxygen species. This results in an impaired ability of diaphragmatic muscle fibers to modulate intracellular calcium fluxes, leading to their weakness. We finally demonstrate that prevention of mitochondrial fission may provide a novel therapeutic approach to prevent MV-induced diaphragm weakness.A substantial portion of critically ill patients admitted to the intensive care unit for acute respiratory failure necessitating mechanical ventilation (MV) experience diaphragm dysfunction.2+ homeostasis disturbance, and calpain activation [The use of MV leads to sudden diaphragm contractile inactivity and weakness, which can appear as early as the first few days . The moltivation . Most oftivation , highligtivation .Indeed, it has been reported that mitochondria are the main source of reactive oxygen species (mROS) generation during VIDD but the mechanism triggering mROS production is poorly known , 7.Mitochondria are vital cellular organelles whose structure and role are intricately intertwined. Well-coordinated fusion and fission processes sculpt mitochondria to meet metabolic requirements and facilitate the removal of impaired organelles . The acuInterestingly, mROS production, which is a main cellular mechanism implicated in the development of VIDD , has beeThe mitochondrial dynamic apparatus comprises a group of shaping proteins often referred to as the dynamin-like guanosine triphosphatases family (GTPases), which facilitate the fusion and fission of mitochondrial membranes . MitofusFission, on the other hand, is executed by dynamin-related protein 1 (DRP1) , a cytosIn a state of mitochondrial fission/fusion equilibrium, DRP1 is evenly distributed in the cytoplasm, with some DRP1 molecules localized to specific punctate foci on mitochondria.When there is a transition to extensive mitochondrial fission, DRP1 is markedly recruited to the outer mitochondrial membrane, forming multi-unit rings responsible for constricting and dividing the mitochondrial tubule into two smaller mitochondria .The recruitment of cytosolic DRP1 to this organelle is facilitated through a variety of DRP1 binding receptor proteins located on the outer mitochondrial membrane, including FIS1 , 17.We hypothesized that inhibiting the interaction between DRP1 and its receptor FIS1 may limit mitochondrial fission, mROS production, and the subsequent diaphragmatic dysfunction induced by MV. To test this hypothesis, we used a cell permeable peptide called P110 that inhibits mitochondrial fragmentation by blocking DRP1 binding to its mitochondrial receptor FIS1 .Mitochondria are situated in close proximity to the redox-sensitive ryanodine receptor type 1 in the sarcoplasmic reticulum (SR), which is responsible for calcium (Ca2+) release.2+ leak from the SR and contractile dysfunction, and has been proposed as an early event involved in VIDD [2+ handling, secondary activation of the calpain pathway, and finally atrophy [2+ leak, and preserves diaphragm strength [Its MV-induced oxidation results in abnormal Ca in VIDD . It has  atrophy . We recestrength .2+ homeostasis impairment, thereby a more effective therapeutic target, we first performed, for the first time in mechanically ventilated mice, diaphragm proteomic profiling to verify if MV may alter mitochondrial pathways related to mitochondrial fission and adenosine triphosphate (ATP) synthesis. Then we evaluated whether preventing mitochondrial fragmentation with P110 may attenuate mROS production and sufficiently limit RyR1 oxidation as well as the subsequent Ca2+ leak that underlies the development of VIDD.Therefore, to evaluate whether mitochondrial dynamics can be considered as an upstream cellular pathway to CaP-value < 0.05, fold-change ≥ 1.5, and unique peptide ≥ 1, 319 differentially expressed unique proteins (control vs. MV) were identified and selected for further analysis . A volcano plot of all of these proteins is displayed in Fig. is Table . Among t2+ dependent protein binding  signaling pathway, previously reported in VIDD [2+ homeostasis pathways, RyR1 and ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting were down-regulated while STAT3, STAT5B, and STAT1 (major components of the JAK-STAT pathway) were up-regulated following MV in comparison to controls, which confirms their implication in VIDD  with the canonical pathway and GO gene sets  mitochondria were only observed in a longitudinal orientation Fig. C. SS mitP110 is a cell permeable peptide that inhibits mitochondrial fragmentation by blocking DRP1 binding to its mitochondrial receptor FIS1. We therefore evaluated the effect of P110 on the aforementioned DRP1-FIS1 interaction in the MV diaphragms. We observed that P110 successfully abolished DRP1 recruitment to the mitochondria compared to the MV untreated group in both the SS and IMF mitochondrial populations Fig. .To investigate the potential role of P110 in the regulation of other major mitochondrial shaping proteins involved in mitochondrial dynamics, we evaluated the total levels of the MFN1 and MFN2 ratio in MV diaphragms. We observed that MV significantly decreased MFN1 expression levels but not MFN2. P110 treatment had no significant effect on either protein's expression levels Fig. . MoreoveIndeed, the dynamin-like GTPase OPA1 serves as a mediator of mitochondrial fusion and plays a crucial role in shaping mitochondrial cristae morphology and enhancing resistance to apoptosis .Within the inner mitochondrial membrane, two peptidases, namely OMA1 and the I-AAA protease YME1L, convert long OPA1 forms (L-OPA1) into shorter forms (S-OPA1) .The fusion of mitochondria relies on L-OPA1, whereas S-OPA1 is linked to mitochondrial fission . We obse2+ content, changes in which could be linked to the mitochondrial morphological alterations. We observed that the mitochondrial Ca2+ level was decreased mainly in the SS subpopulation  oxidative marker , in the diaphragm induced by mitochondrial ROS production  and is agms Fig. A. Furtheker Fig. B–F. When0 is consistent with pathological SR Ca2+ leak. Interestingly, P110 treatment reduced RyR1 oxidation, enhanced calstabin1 binding to the channel, reduced the SR Ca2+ leak, and reduced total proteins oxidation  are also important to take into consideration when reversing aberrant mitochondrial dynamics induced by MV.Mitochondrial function and structure are intimately linked. Previous studies have demonstrated that reducing mitochondrial fragmentation by modulating the fission/fusion balance is sufficient to restore mitochondrial function , 29. In 2+ channels [2+ leak and diaphragm weakness prior to diaphragmatic injury and atrophy. Here, we show, for the first time, that P110 reduced ROS production and prevented RyR1 oxidation, nitrosylation, and the subsequent diaphragm weakness induced by 6 h of MV in mice. Thus, mitochondrial fission and ROS production are likely upstream signaling of Ca2+ dyshomeostasis in VIDD. Indeed, a growing body of evidence indicates that RyR1 is tightly regulated by posttranslational modifications involving remodeling of the RyR1 macromolecular complex [2+ release channels and muscle function [2+ leak remains to be investigated in future studies.We previously reported oxidative damage to other components of the contractile machinery of the diaphragm during VIDD including RyR1 Cachannels . RyR1 ox complex  which cofunction . As prevfunction , 20, we The inhibitory peptide P110 comprises a 7-amino acid sequence that shares homology between DRP1 and FIS1 . In a clIndeed, administering P110 to wild-type mice for a period of five months is well-tolerated and does not impact mitochondrial structure or function .Cells stress markers, bodily metabolism, and behavior were also not affected . MoreoveAlthough the primary cause of mitochondrial oxidative stress remains unclear , we sugg2+ leak to explain the subsequent muscle weakness induced by MV. However, our results on the early effects of MV on diaphragmatic proteomics also show early deregulation of cellular pathways that converge toward a deep mitochondrial alterations, Ca2+ dyshomeostasis, and activation of the JAK-STAT pathway. JAK-STAT and Smad3 pathways might be the signals linking mechanical inactivity to mitochondrial abnormalities. This pathway is stimulated early during MV [In the present study, we only focused on two posttranslational modifications of RyR1 which contributed to the SR Cauring MV , 21, anduring MV . Consisturing MV . InteresFinally, Den Berg et al. have reported diaphragm weakness and atrophy with preserved mitochondrial function and morphology in ventilated critically patients . This isThe inactivity of the diaphragm induced by MV is an important signal that induces proteomic alterations which mainly affect the mitochondria and trigger its fission to meet the sudden decrease of metabolic demand. In this study, we observed that 6 h of MV in mice induces excessive mitochondrial fragmentation leading to mROS production, which is involved in a rapid remodeling of the RyR1 in the diaphragm and subsequent VIDD. These results are summarized in Fig. The study adhered to the animal experiment protocols outlined by the institutional animal care committee in Montpellier, France, in accordance with the recommendations of the World Medical Association, documented under reference #CEEA-LR-12078. Healthy male C57/BL6 mice (aged 10–12 weeks and weighing 25–30 g) were acclimatized in our animal facility for one week before initiating the experiments.n = 8), (2) mice mechanically ventilated for 6 h and treated by P110 , and (3) control (n = 8). P110 molecule was dissolved in Ringer's lactate (RL) solution and delivered in a bolus (loading) dose ((3 mg/kg) intraperitoneal (IP) injection) 1 h before initiation of MV. A constant IP injection (3 mg/kg/h) of P110 was maintained throughout MV.As previously described in our earlier study , mice weRL was used as vehicle for MV group. Airway pressure and hemodynamic parameters were monitored and maintained within the physiological range as previously reported . P110 isThe cell permeable peptide, TAT-P110 (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-Asp-Leu-Leu-Pro-Arg-Gly-Thr), molecular weight = 2426.7 g/mole, was synthesized by Genemed Synthesis Inc.  to >99% purity as determined by high-performance liquid chromatography.2O by placing the expiratory port under a water seal. General care for the MV groups included continuous maintenance of body temperature using a homeothermic blanket , and hourly IP injections of 0.05 ml of ringer lactate solution were administered to maintain hemodynamic stability and compensate for insensible losses. Additionally, bladder expression, ocular lubrication, and passive limb movements were performed as part of the care protocol. Anesthesia was sustained by IP injection of pentobarbital sodium (5 mg/kg body weight/hour) throughout the 6-h duration of MV.Mice in the MV groups underwent anesthesia through an IP injection of pentobarbital sodium (50 mg/kg body weight) and were orally intubated using a 22-gauge angio-catheter. No neuromuscular blocking agents were administered. MV was conducted using a small animal ventilator  with the following settings: a fraction of inspired oxygen of 0.21 (room air), controlled volume mode with a tidal volume of 10 µl/mg body weight, a respiratory rate of 150 breaths/min, and positive end-expiratory pressure maintained at 3–4 cm HFor transmission electron microscopy (TEM) analysis, diaphragm muscle strips (5 × 15 mm) were fixed in a 2% glutaraldehyde solution in 0.1 M cacodylate buffer (pH 7.4). The samples underwent a series of processing steps. Initially, they were fixed with 1% osmium tetroxide and then dehydrated using acetone. Following dehydration, the samples were impregnated with resin. Smaller portions of diaphragm tissues were subsequently embedded in 100% resin either in cross-sectional (transverse) or longitudinal orientations. To ensure the proper orientation and quality of sections, 1-μm-thick sections were cut and stained with a solution containing 1% toluidine blue and 1% borax. Ultrathin sections measuring 70 nm were then sliced using a diamond knife on a Leica ultramicrotome (Leica-Reichert Ultracut E). These sections were stretched with chloroform vapor and mounted on copper grids coated with Pioloform film before being stained with 2% aqueous uranyl acetate and lead citrate.Subsequently, ultrathin sections were examined using a Tecnai F20 transmission electron microscope at 200 kV, located in the CoMET Montpellier Rio Imaging facilities at the Institute for Neurosciences of Montpellier, France. Digital micrographs were captured from each sample at magnifications of 13,500× and 19,000×. Tissues from four mice per group were collected, imaged, and analyzed for both cross-sectional and longitudinal orientations.For each animal, randomly selected mitochondria-rich oxidative muscle fibers were analyzed in both transverse and longitudinal sections as previously described in detail . In shorIn total, an observer who was unaware of group assignments analyzed 2,349 IMF mitochondria from the control group, 1,494 IMF mitochondria from the MV group, and 2,099 IMF mitochondria from the MV + P110 group. The assessment of SS mitochondrial morphology was conducted at a magnification of 19,000× in the longitudinal orientation.Mitochondrial perimeters were determined using Image J software  by manually delineating the borders of intramyofibrillar (IMF) and SS mitochondria, which exhibited well-defined outlines in TEM micrographs.To assess interactions between intramyofibrillar (IMF) and SS mitochondria, we examined micrographs of longitudinally aligned myofilaments obtained from four mice per group. They were captured at magnifications of 13,500× for IMF mitochondria and 19,000× for SS mitochondria.In the IMF compartment, a total of 689 (control), 648 (MV), and 534 (MV + P110) Z-lines possessing mitochondria on both sides, either as pairs of discrete organelles, as a continuous mitochondrion, or as interacting organelles joined by an electron-dense IMJ, were analyzed. The proportion of Z-lines spanned by continuous or interacting mitochondria is reported as a percentage of the total number of Z-lines analyzed. In addition, the proportion of electron-dense physical contact sites between adjacent mitochondria was determined in all the groups in both orientations as described previously .® instrument. Tissues were then lysed in 600 µl extraction buffer ), and agitated for 45 min by rotation. Membrane and cytosolic proteins were collected from the supernatant after 5 min of centrifugation at 10,000×g at 4 °C.Proteins were extracted from frozen costal diaphragms (20 mg) homogenized with a manual polytrong for 10 min at 4 °C. The mitochondria pellet was suspended in 4 ml of Bovine Serum Albumin (BSA) buffer  and nargase (5 mg/g) (Sigma 9014-01-1) was added and centrifuged at 12,000g for 15 min at 4 °C. The new pellet was resuspended in 2 ml of BSA buffer and centrifuged at 1000g for 10 min at 4 °C. The supernatant was then centrifuged at 12,000g for 15 min at 4 °C and the pellet containing IMF mitochondria was resuspended in 100 μl of homogenate buffer and used for mitochondrial ROS production assay. The supernatant obtained at the first step was centrifuged at 12,000g for 15 min at 4 °C and the pellet containing SS mitochondria was resuspended in 100 μl of homogenate buffer and used to measure the SS mitochondrial ROS production.Diaphragms were minced and homogenized in 1 ml of homogenate buffer -N,N,N′,N′-tetraacetic acid (EGTA) 0.2 mM, pH 7.2) with a polytron homogenizer . The homogenate was centrifuged at 1,000Mitochondrial ROS production was measured using the 2′7′-dichlorofluorescein (DCF)-diacetate (DA) method. 50 μg of SS or IMF mitochondria were incubated at 37 °C for 1 h with 20 μM H2DCF-DA (Thermo Fisher Scientific), a compound that becomes highly fluorescent as a result of activation by ROS, producing a fluorescent molecule known as DCF. The fluorescence emitted by DCF was then recorded at 490/528 nm for a duration of 80 min using a multimode plate reader.g for 5 min. The pelleted mitochondria were resuspended in HCl 0.6 N, homogenized, and sonicated. The mitochondria were then incubated in boiling water. The mitochondria lysates were then centrifuged for 5 min at 10,000g, the supernatant was recovered, and its pH was adjusted to between 6 and 8. Ca2+ content in the supernatant was determined spectrophotometrically (Tecan) using an o-cresolphthalein Complexone assay kit . Results were expressed in nanomoles of Ca2+ per milligram of protein.Isolated mitochondria were suspended in homogenate buffer  without EGTA, and an aliquot of this suspension was taken to determine protein concentration by a bicinchoninic acid assay kit. The mitochondria were washed with 1 ml of homogenate buffer (without EGTA) and then centrifuged at 16,0004, 5 mM ethylenediaminetetraacetic acid), and 50 mM Tris-HCl) and the protease subtilisin A (0.1 mg/g of tissue) were added to the mince, and the solution was incubated for 5 min in ice. Reaction was then stopped with 10 volumes of ATP buffer and the solution was homogenized with a polytron homogenizer (15 s at 40% of full power). The homogenate was centrifuged at 8,500g for 10 min at 4 °C, supernatant was removed, and the pellet resuspended with 10 volumes of ATP buffer and again centrifuged at 800g for 10 min at 4 °C. The supernatant obtained was recovered and centrifuged for 10 min at 9,000g at 4 °C. Once again, the supernatant was removed, and the pellet was resuspended with 3.5 ml of ATP buffer and centrifuged at 9,000g for 10 min at 4 °C. Finally, supernatant was eliminated, and mitochondria were resuspended in 30 µl of resuspension buffer propanesulfonic acid, pH 7.4). The mitochondrial protein content was assessed using the Bradford assay, and the results were quantified as milligrams of mitochondrial proteins per gram of muscle wet weight.The mice were euthanized by cervical dislocation. The costal diaphragm was surgically removed. The diaphragm was gently minced with a scissor for 2 min and then weighed. Four volumes of ATP buffer -1-piperazineethanesulfonic acid (HEPES), 110 mM sucrose, and 1 g/l BSA, pH 7.1 [Mitochondria oxygen consumption was measured using the high-resolution Oxygraph-2k . Mitochondria were placed inside two sealed, temperature-controlled chambers (37 °C) containing 2 ml of MIRO5 respiration medium  was assessed when mitochondria were exposed to a solution containing 2.5 mM malate and 5 mM pyruvate. The adenosine diphosphate (ADP)-stimulated rate (state 3) was measured following the addition of 0.25 mM ADP.It represents the recovery rate after completion of ATP synthesis (defined as oxygen consumption of mitochondria after the depletion of exogenous ADP). The acquisition and analysis of data were conducted utilizing Oxygraph-2k-DatLab software version 4.3, provided by ORO-BOROS Instruments. The RCR was defined as the ratio of oxygen consumption during state 3 to oxygen consumption during state 4.For quality control purposes, control diaphragms were always assessed along the ventilated or the ventilated and treated diaphragms at the same day and under the same condition and instrument calibration.Isolated IMF and SS mitochondria were mixed with sample buffer and boiled for 5 min before gel loading. Mitochondrial proteins were transferred onto nitrocellulose membranes for 2 h at 200 mA. Immunoblots were developed using anti-DRP1 antibodies: .3VO4, 1% Triton X-100, and protease inhibitors (Roche)) overnight at 4 °C.As described in our previous study , RyR1 waRyR1-1327 is a rabbit polyclonal antibody that was purified for its binding affinity against a keyhole limpet haemocyanin-conjugated peptide with the amino acid sequence CAEPDTDYENLRRS. The sequence corresponds to amino acid residues 1,327–1,339 of the mouse skeletal RyR1. An additional cysteine residue was added to the amino terminus, and the antibody was subsequently purified using the unconjugated form of the peptide.As outlined in the methodology section of our prior study , the immFor mitochondrial shaping proteins analyses, muscle lysates extracted from diaphragms were combined with sample buffer and subjected to boiling for 5 min prior to loading onto the gel. The concentrations of primary and secondary antibodies were prepared in accordance with the manufacturers' recommendations. The quantification of distinct protein bands was carried out using the Odyssey Infrared Imaging System , with glyceraldehyde-3-phosphate dehydrogenase serving as a loading control to normalize for potential variations in total protein loading.The following proteins were detected: MFN2 , PGC1-α , and OPA1 .The mice were euthanized by cervical dislocation. The costal diaphragm was surgically removed to permit measurements of isometric contractile properties as described in our previous study . The musAfter completion of the measurements of contractile properties, muscle was measured for its length, dried for 5 min to remove the surface buffer, and weighed. The muscle's cross-sectional area was calculated by dividing its weight by the product of its length and tissue density (1.056 g/cm³). Subsequently, diaphragmatic force production was adjusted by normalizing it to the muscle's cross-sectional area, resulting in specific force measurements expressed in Newtons per square centimeter (N/cm²).×g) for 20 min at 4 °C. The supernatant was ultracentrifuged at 32,000 rpm  for 1 h at 4 °C. The final pellet containing microsomal fractions enriched in SR vesicles was resuspended and aliquoted in 300 mM sucrose, 5 mM Pipes (pH 7.0) containing protease inhibitors. Samples were frozen in liquid nitrogen and stored at −80 °C.Using the same methodology we described previously , diaphra2, pH 7.35 as cis solution and 53 mM Ca(OH)2, 50 mM KCl, 250 mM Hepes, pH 7.35 as trans solution [Employing the methodology we previously detailed , planar solution .2+ in the cis chamber was calculated using the WinMaxC program . SR vesicles were added to the cis side, and fusion with the lipid bilayer was induced by making the cis side hyperosmotic through the addition of 400–500 mM KCl. After the appearance of potassium and chloride channels, the cis compartment was perfused with the cis solution. Single-channel currents were recorded at 0 mV by using a Bilayer Clamp BC-535 amplifier (Warner Instruments), filtered at 1 kHz and digitized at 4 kHz. All experiments were performed at room temperature. Data acquisition was performed using Digidata 1440A and Axoscope 10.2 software, and recordings were analyzed using Clampfit 10.2 (Molecular Devices). Open probability was identified by 50% threshold analyses using a minimum of 2 min of continuous record. At the conclusion of each experiment, ryanodine (5 µM) was added to the cis chamber to confirm channels as RyRs.The concentration of free CaFor global quantitative proteomics of fresh frozen diaphragms from controls and MV mice, diaPASEF   and maintained at 37 °C with continuous shaking at 1,400 rpm. Subsequently, peptides were acidified by introducing 1% trifluoroacetic acid (TFA), followed by vortexing and purification via StageTip clean-up using styrenedivinylbenzene RPS. The peptides were loaded onto a StageTip plug with a 14-gauge needle and subjected to two washes with 200 µl of 1% TFA/99% ethyl acetate and 200 µl of 0.2% TFA/5% acetonitrile (ACN), respectively, in a centrifuge at 3,000 rpm. Afterward, elution was performed using 60 µl of a solution containing 1% ammonia and 50% ACN into Eppendorf tubes, followed by drying at 45 °C in a SpeedVac centrifuge. Samples were then resuspended in 10 μl of liquid chromatography buffer (3% ACN/0.1% formic acid), and the peptide concentrations were determined using NanoDrop. For subsequent Parallel Accumulation Serial Fra gmentation (PASEF) and diaPASEF analysis on the timsTOFPro instrument, 200 ng of each sample was utilized.For spectral library generation, 10 μg of each digested tissue sample was pooled and dried in a speedVac. Pooled dried peptides were resuspended in 100 μl of 1% TFA, pH 2 and subjected to fractionation with mixed mode styrenedivinylbenzene SCX StageTip . PeptidePeptides were separated over a period of 120 min at a flow rate of 400 nl/min. This separation was performed on a reversed-phase C18 column equipped with an integrated CaptiveSpray Emitter . The mobile phases consisted of A, which was 0.1% formic acid in water, and B, which was 0.1% formic acid in ACN. The fraction of mobile phase B was increased linearly from 2 to 23% over 90 min, followed by a 10-min gradient to 35%, and finally, an increase to 80% before re-equilibration. The timsTOF Pro instrument operated in PASEF mode with specific settings as follows:m/z, 1/K0 start 0.6 V s/cm2, end 1.6 V s/cm2, ramp time 100 ms, lock duty cycle to 100%, capillary voltage 1,600 V, dry gas 3 l/min, dry temp 200 °C, PASEF settings: 10 Tandem Mass Spectrometry (MSMS) frames (1.16 s duty cycle), charge range 0–5, active exclusion for 0.4 min, target intensity 20,000, intensity threshold 2,500, and Collision-induced dissociation energy 59 eV. We applied a polygonal filter in both the m/z and ion mobility dimensions to isolate features that were more likely to correspond to peptide precursors rather than singly charged background ions.Mass range 100–1,700 m/z 400–1,200. In order to customize the MS1 cycle time for diaPASEF, we configured the repetitions as follows: two repetitions were set for the 16-scan diaPASEF scheme, and four repetitions were established for the 4-scan diaPASEF scheme in these experiments.DiaPASEF  experime−2 to 20 eV at 1/K0 = 0.6 V s cm−2. To construct sample-specific spectral libraries, we conducted searches on the acquired PASEF raw files and diaPASEF raw files against the UniProt mouse database using the Pulsar search engine. This was accomplished by utilizing the hybrid spectral library generation feature of Spectromine with default settings [The collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K0 = 1.6 V s cmsettings .The raw protein intensities were determined by adding together the intensities of the constituent peptides.DiaPASEF data were analyzed with Spectronaut Pulsar X , a mass We estimated the FDR using the mProphet approach, aiming to establish it at 1% at both the peptide precursor and protein levels. The data generated from Spectronaut underwent additional analysis utilizing the Spectronaut statistical package.P-value < 0.05, corrected through permutation-based FDR, a fold-change ≥ 1.5, and unique peptides ≥ 2.The threshold criteria for identifying differentially expressed proteins comprised a The significantly changed proteins between controls and MV diaphragms were used for volcano plot, heatmap, GO, and KEGG analysis. We also conducted GSEA to pinpoint the statistically significant gene sets within a ranked list of genes.P < 0.05, determined through Student's unpaired t-test or analysis of variance (one-way or two-way), with subsequent Bonferroni-selected comparison tests.Data are presented as mean values with the standard error of the mean (SEM). Statistical significance was defined as pgad336_Supplementary_DataClick here for additional data file.


Furthermore, genes associated with immune checkpoints and T cell activation, such as PD−1, PD−L1, CD8A, CD8B, CD28, and GZMM, were shown to be highly expressed in GC subgroups with high LKB1 expression. Compared with fresh gastric cancerous tissues, LKB1 was highly expressed in CD3+CD8+ and CD3+CD8+CD28+ T cells in fresh adjacent non-cancerous tissues. CD3+CD8+ T cells produced an IFN−γ anti−cancer immune response. Furthermore, the proportion of CD3+CD8+ T cells that expressed LKB had a positive correlation with IFN−γ expression. Moreover, GC patients with low LKB1 expression had a poor objective response rate, and worse progression-free survival and overall survival when treated with pembrolizumab. In conclusion, LKB1 may be a potential immune checkpoint in GC patients.Liver kinase B1 (LKB1) is a tumor suppressor gene, the inactivation of which occurs frequently in different tumor types. However, whether LKB1 is associated with the clinical features of gastric cancer (GC) and regulating tumor immunity is unknown. In this study, we showed that LKB1 is highly expressed in the serum of healthy individuals ( Worldwide, gastric cancer (GC) is a public health issue and is the fifth most frequently diagnosed malignancy ,2. AlthoLiver kinase B1 (LKB1), also known as STK11, is a serine/threonine kinase that is widely present in numerous tissues . IncreasIn this study, we demonstrated an association between LKB1 expression and clinical outcomes in GC patients. In addition, we demonstrated that low LKB1 expression promotes an immunosuppressive microenvironment and might result in a poor prognosis among GC patients. Moreover, GC patients with high expression of LKB1 received greater benefit from pembrolizumab treatment. The prognostic value and potential role of LKB1 in tumor immunology are discussed herein and may contribute to understanding a possible mechanism underlying GC.This retrospective study was approved by the local Ethics Committee of Zhejiang Cancer Hospital (IRB−2021−154). We collected blood samples from 176 healthy adults and 416 GC patients between January 2015 and August 2022 and collected 26 pairs of fresh cancerous tissues and adjacent non−cancerous tissues from 26 GC patients. Patients with a history of other malignant tumors were excluded.A commercial ELISA test kit  was used to evaluate the level of LKB1 expression in the plasma samples according to a standardized protocol. Briefly, corresponding plasma samples (50 μL) and standard model proteins (50 μL) were added to 96−well plates. Next, 100 μL of an HRP−labeled antibody was added and the 96−well plates were incubated for 1 h at 37 °C. Then, the plates were washed three times with washing buffer. The substrate mixture (100 μL) was added and the plates were analyzed in a microplate reader at OD 450.t-test was used to analyze the LKB1 expression in different subgroups of GC patients.Chemiluminescence was used to determine CEA , CA19−9 , and AFP expression . SPSS 19.0 software  was used to determine the area under the receiver operating characteristic (ROC) curves of CEA, CA19−9, AFP, and LKB1. Based on TNM pathologic stage, FIGO stage, and HER2 expression, 416 GC patients were divided into different subgroups. A paired http://www.camoip.net/) (accessed on 1 January 2023) is a tool for analyzing expression and mutation data from The Cancer Genome Analysis (TCGA) and the immune checkpoint inhibitor (ICI)−treated projects. A standard processing pipeline was used to analyze immunogenicity and pathway enrichment in GC patients. CAMOIP was also used to analyze different immune cell expression according to LKB1 expression in GC patients.Comprehensive Analysis on Multi−Omics of Immunotherapy in Pan−cancer [CAMOIP] (https://xena.ucsc.edu/public) (accessed on 1 January 2023) is a cancer genomics data analysis platform that supports the visualization and analysis of histologic data from multiple cancer samples. The UCSC Xena database provides LKB1, T−cell immune checkpoint genes, T−cell activation, and antigen presentation of mRNA expression. A complex heatmap was made using R language (4.2.1). SPSS 19.0 software  was used to analyze LKB1 expression based on quartiles. We divided the GC patients into LKB1 high and low expression groups according to the mediate value of LKB1 expression. A t-test was used to analyze T−cell immune checkpoint genes, T−cell activation, and antigen presentation of mRNA expression in GC patients into LKB1 high and low expression GC patient subgroups. p values < 0.001 were considered statistically significant.The UCSC Xena database (n = 26) and fresh adjacent non−cancerous tissues (n = 26) obtained intraoperatively were ground into a tissue milling solution within 30 min. Milling solution and peripheral blood mononuclear cells (PBMCs) (1 × 106 cells/mL) were collected in phosphate-buffered saline (PBS)  supplemented with 0.5% bovine serum albumin (BSA)  and incubated with anti−human monoclonal antibodies for 15 min. The anti−human monoclonal antibodies were used, as shown in Fresh cancerous  and fresh adjacent non−cancerous tissues (n = 26). The difference between LKB1+ and PD1+LKB1+ immune cell proportions in fresh cancerous and fresh adjacent non-cancerous tissues were analyzed using a paired t-test. SPSS 19.0 software  was used to determine the correlation between immune cell proportions and cytokines in fresh cancerous and fresh adjacent non-cancerous tissues.A n = 26) and fresh adjacent non-cancerous tissues (n = 26) was used in this study. An IHC kit  was used to perform IHC in accordance with the manufacturer’s instructions. Briefly, paraffin sections were successively deparaffinized, rehydrated, and boiled for antigen retrieval. The primary LKB1 antibody  and interferon-gamma (IFN−γ) antibody  were incubated for 2 h at room temperature. The sections were washed three times with PBS. A histochemical polymer enhancer (400 μL) was added to paraffin sections for 20 min, followed by 3 washes with PBS. Next, secondary antibodies were added to the paraffin sections, and incubated for 20 min, followed by washing, DAB staining, counterstaining, and mounting.The Biobank of Zhejiang Cancer Hospital provided the fresh cancerous  were obtained from a previous report . SPSS 19p values < 0.05 were considered statistically significant. Furthermore, Kaplan−Meier Plotter (http://www.kmplot.com) (accessed on 1 January 2023) was used to confirm the effect of LKB1 (high vs. low expression) on the survival of GC patients. Python was used to reflect the clinical features and parameters of the GC patients.Statistical analyses were performed using SPSS 19.0 software . Patient survival was confirmed by telephone, and was analyzed using the Kaplan−Meier method with a log−rank test. p < 0.05, p < 0.05, p < 0.001, and p < 0.01, respectively; In this study, 416 GC patients  and 176 healthy individuals  were included. The characteristics of healthy individuals and GC patients are summarized in According to the TCGA databases, the T−cell receptor complex was significantly different between GC patients with high and low LKB1 expression A. We furTo determine LKB1 expression in the immune microenvironment, we measured the expression of six immune infiltrating cells in peripheral blood (B), fresh cancerous (T) and adjacent non−cancerous tissues (N), and 12 types of cytokine expression in GC patients A. We alsWe assumed that LKB1 expressed in immune cells correlates with immune cell function. Based on the results, we analyzed the association between immune infiltration cell (LKB1+) proportion and cytokine expression A. A prevhttp://kmplot.com/) (accessed on 1 January 2023) also indicated that LKB1 expression significantly affected the prognosis of GC patients, with significantly lower overall survival in those patients with low LKB1 expression  cells had a positive correlation with IFN−γ expression in fresh tissues of GC patients. Thus, we inferred that LKB1 might impede T cell  secretion and is positively associated with anti−tumor IFN−γ expression. The underlying mechanism by which LKB1 is associated with cytotoxic T−cell activation warrants further investigation.Recently, immunotherapy has shown great advantages in the clinical treatment of GC ,33. PD−1+T cells . In addi


HNF1B gene. This gene is expressed in the early phase of embryonic development in the pancreas, kidneys, liver, and genital tract; therefore, kidney or urinary tract malformations are associated with diabetes mellitus. The 17q12 deletion syndrome is a cause of maturity-onset diabetes of the young subtype 5 that should be considered.Maturity-onset diabetes of the young comprises a large group of autosomal inherited gene mutations. Maturity-onset diabetes of the young subtype 5 is caused by mutations in the HNF1B. Therefore, a diagnosis of deletion syndrome was made.We present the case of a 35-year-old Hispanic female patient with a history of bicornuate uterus and polycystic renal disease that required kidney transplant. She had insulin-dependent diabetes, with her mother, maternal grandmother, and great-grandmother showing a similar clinical manifestation. Molecular analysis showed a deletion in chromosome 17q12 involving 15 genes, including HNF1B. Principally, it is characterized by the combination of genitourinary tract malformations and diabetes mellitus, similar to our patient.The 17q12 deletion syndrome represents a rare genetic syndrome that involves different genes, including  HNF1B gene.MODY subtype 5 is caused by mutations in the MODY should be suspected in individuals who are lean and lack ethnic T2DM risk factors.Deletion syndrome has different phenotypic expressions based on the genes involved. Diabetes mellitus (DM) has a worldwide distribution and an increasing incidence. The most prevalent types are type 1 (T1DM) and type 2 (T2DM). Each type has a precise etiology  and clinical characteristics [MODY classically presents in individuals with hyperglycemia before the age of 25 years, does not require insulin, and has evidence of autosomal dominant inheritance . MODY isHNF1B gene, located on chromosome 17q12. This gene is expressed in the early phase of embryonic development in the pancreas, kidneys, liver, and genital tract. Its prevalence is low within the already small prevalence of MODY [HNF1B deletion has recently been shown to be associated with 17q12 deletion syndrome in virtually all cases [MODY involves a wide group of genetic mutations with autosomal dominant inheritance . MODY su of MODY . HNF1B dll cases . We discA 35-year-old Hispanic woman with mild mental retardation was referred for poorly controlled DM. She was diagnosed with polycystic kidney disease and DM at ages 9 and 11 years, respectively. She subsequently developed a rapid progression to diabetic nephropathy, requiring a living donor kidney transplant at age 12. In her early 30s, she was diagnosed with a bicornuate uterus. Within her family history, her mother, maternal grandmother, and great-grandmother also had early onset diabetes with death before 60 years of age  of 30.1 kg/m2) and poor glycemic control. Her initial average glycosylated hemoglobin (HbA1c) value was 8.0%, with high variability requiring Degludec insulin plus Aspart insulin up to 1 UI/kg. Despite strict follow-up and diabetes education that included bolus delivery according to carbohydrate count, only partial improvement was achieved. Her C-peptide level was measured, and its value was 0.44 ng/mL (0.5–2.0 ng/mL). Dulaglutide and dapagliflozin/metformin were initiated, achieving an HbA1c of 6.47%.The patient underwent an extra-institutional endocrinology follow-up since her diagnosis. When she turned 30 years old, she was referred to our clinic with obesity  [Due to the phenotype of her diabetes evolution, next-generation sequencing (NGS) was performed. Exome sequencing identified copy number variants in the genes AATF, ACACA, C17orf78, DDX52, DHRS11, DUSP14, GGNBP2, HNF1B, LHX1, MRM1, MYO19, PIGW, SYNRG, TADA2A, and ZNHIT3 [The 1.4 Mb deletion located on the long arm of chromosome 17, involving the genes d ZNHIT3 , explainHNF1B belongs to the hepatocyte nuclear factor family, which is expressed in the pancreas, kidney, liver, bile ducts, and urogenital tract. It is located on chromosome 17q12 and encodes a protein that contains a homeodomain [HNF1B is a key member of the network of transcription factors controlling the differentiation of acinar, ductal, and endocrine cells [HNF1A. Most point mutations are described in this domain, especially in exon 2. Non-sense mutations, deletions, or insertions in this gene have been related to MODY type 5. They are associated with loss of protein function, haploinsufficiency, or negative dominance mechanisms [eodomain . The latne cells . It alsochanisms , 12.HNF1B mutations, age at diabetes diagnosis was found to be greater compared to patients with T1DM  [Within the broad clinical spectrum described by patients with us T2DM) , 14 Apprus T2DM) . TherefoP = 0.00001 each). This suggests that pathogenic mutations of HNF1B cause an earlier or more severe impairment of insulin secretion than mutations in the HNF4A gene [et al., who demonstrated that patients with HNF1B-MODY have reduced insulin sensitivity of endogenous glucose production but normal peripheral insulin sensitivity [et al., 83.3% of patients with HNF1B-MODY underwent insulin treatment from the onset of diabetes and could not be withdrawn from insulin treatment, which suggests that insulin secretion deficiency is a characteristic feature of this mutation [et al. compared C-peptide levels in patients with HNF1B-MODY and T1DM and showed a trend toward a higher mean level at diagnosis in the first group. However, this trend did not reach statistical significance [Regarding treatment, the percentage of patients with HNF1B-MODY treated with insulin was significantly higher than that of patients with HNF4A-MODY when compared with patients with T1DM (median 65.7% versus 36.4%, F4A gene . This issitivity . Likewismutation . In our ificance . Our patificance , 19.et al. described a 26-year-old male with hydronephrosis, non-insulin-dependent diabetes, and detectable C-peptide with a pathogenic 1.39 Mb deletion of 17q12 containing 20 genes, including HNF1B, which is different from our case with a deletion of 1.48 Mb involving 15 genes, with detectable C-peptide being an insulin-dependent diabetes patient report [Almost 276 cases of chromosome 17q12 deletion syndrome were reported in the literature as of 2020 [The 17q12 deletion syndrome is caused by a deletion on the long arm of chromosome 17. Patients may present with diabetes and dysmorphia, such as macrocephaly, prominent forehead, full cheeks, arched brow ridges, bilateral epicanthal folds, and low nasal bridge. It is associated with diabetes in approximately 70% of cases and exhibits variable clinical presentation.


Pelteobagrus fulvidraco. According to histological findings, ammonia nitrogen stress caused structural damage to the intestine and reduced the number of mucous cells. Enzyme activity analysis revealed that the activity of bactericidal substances  had decreased. The ultrastructure revealed sparse and shortened microvilli as well as badly degraded tight junctions. Immunohistochemistry for ZO-1 demonstrated an impaired intestinal mucosal barrier. Furthermore, qRT-PCR revealed that tight junction related genes  were downregulated, while the pore-forming protein Claudin-2 was upregulated. Furthermore, as ammonia nitrogen concentration grew, so did the positive signal of Zap-70 (T/NK cell) and the expression of inflammation-related genes . In light of the above findings, we conclude that ammonia nitrogen stress damages intestinal mucosal barrier of Pelteobagrus fulvidraco and induces intestinal inflammation.Nitrogen from ammonia is one of the most common pollutants toxics to aquatic species in aquatic environment. The intestinal mucosa is one of the key mucosal defenses of aquatic species, and the accumulation of ammonia nitrogen in water environment will cause irreversible damage to intestinal function. In this study, histology, immunohistochemistry, ultrastructural pathology, enzyme activity analysis and qRT-PCR were performed to reveal the toxic effect of ammonia nitrogen stress on the intestine of   Five fish were randomly selected for examination, no bacteria or parasites were found. These fish were kept in a circular tank (600 L water volume) with constant aeration for 1 week at 23°C–24°C. Floating commercial feed was provided satiably twice a day (8 a.m. and 6 p.m.), with one third of the water was changed daily. The approximate composition of commercial feed is as follows: 40% crude protein, 5% crude fat, 8.1% ± 0.3% crude fibr and 16% crude ash. Feeding was ceased 1 day before ammonia nitrogen exposure.50). They were kept in three circular tanks, and were continuously aerated. Then, based on previous research  0 mg/L), 0.5 mg/L group , and 2.5 mg/L group , and posterior intestine tissue samples were immediately collected for downstream analysis.Eighteen intestine samples (6 samples per group) were obtained for histopathological analysis. Briefly, the posterior intestines were removed with sterile forceps, immediately prefixed in 4% paraformaldehyde for 24 h, rinsed in running tap water for 24 h, and then routine dehydration and paraffin embedding were performed. Subsequently, the tissues were sliced with a thickness of 5 μm , and stained with classical hematoxylin and eosin (H & E). Photographs were taken under an optical microscope . Each tissue section was divided into 4 areas , and a sThe above paraffin sections of intestinal samples were dewaxed in xylene, hydrated, and stained for 20 min with Alcian blue solution (pH 2.5). Later, the samples were oxidized in Periodic acid (5 g/L) for 5 min, rinsed with distilled water for 10 min, and immersed in Schiff’s reagent for 20 min under dark conditions before being washed with distilled water. Finally, the samples were stained with Hematoxylin for 2 min before being sealed with neutral gum. Nikon Eclipse E200 (Japan) was used to evaluate tissue slides. Each tissue section was divided into 4 areas , and a sThe above paraffin sections were dewaxed to water and then placed in a repair box containing citric acid antigen repair buffer (pH 6.0). Then, the slices were placed in 3% hydrogen peroxide solution for 25 min before being rinsed three times in PBS (pH 7.4) for 5 min each to block endogenous peroxidase. Afterward, the tissues were evenly covered with 3% BSA  and sealed at room temperature for 30min. The primary antibodies Zap-70 Rabbit monoclonal antibody  and ZO-1 Rabbit polyclonal antibody  were added and incubated overnight in a wet box at 4 °C. After washing, tissues were covered with Goat Anti-Rabbit IgG (H&L) Alexa Flour 488secondary antibody , and incubated at room temperature for 50min. Following PBS washing, sections were immersed in diaminobenzidine hydrochloride (DAB) and re-stained with hematoxylin. The sections were observed under Nikon Eclipse E200 (Japan). Each tissue section was divided into 4 areas , and a sEighteen intestine samples (6 samples per group) were selected for transmission electron microscopy (TEM) analysis. The samples were fixed in 2.5% glutaraldehyde for 24 h at 4°C before being washed with PBS (pH 7.2). They were then fixed with 1% osmic acid, washed with PBS, dehydrated with continuous acetone, embedded, sliced, and stained with uranium acetate and lead citrate. Micrographs were taken with TEM  operating at 80 kV. The length of microvilli and the diameter of mucinous granules were measured and recorded using ImageJ software. The ultrastructural pathological changes of intestinal tissue were evaluated, including necrosis, tight junctions fuzzy, vacuolization, mitochondrial cristae contraction and swelling, endoplasmic reticulum swelling, mitochondrial necrosis, mitochondrial myelinoid lesions, autophagy and apoptosis. The samples were assessed using a scoring system  ranging 2 in a critical point dryer (HCP-02 Hitachi). The samples were scanned at 20 kV with a FEI Inspect S50 SEM . The density of microvilli on the surface of intestinal cells (0.25 μm2 region) was assessed using SEM images .SEM analysis was performed on eighteen intestine samples (6 samples per group). The intestines were cut into 5-mm size pieces, washed in 1% S-carboxymethyl-L-cysteine for 30s to remove mucus before being stored in 2.5% glutaraldehyde sodium bicarbonate buffer (0.1 M pH 7.2). Subsequently, the samples were dehydrated in ascending series of ethanol before being dried with liquid COEighteen intestine samples (6 samples per group) were selected for biochemical analysis. After homogenizing the samples with ice-cold physiological saline , they were centrifuged at 8,000 rpm for 15 min. The Lysozyme (LZM), alkaline phosphatase (AKP) and acid phosphatase (ACP) activities of tissue supernatant were examined with lysozyme assay kit (A050-1-1), alkaline phosphatase assay kit (A059-2-2) and acid phosphatase assay kit (A060-2-1) respectively, following the manufacturer’s instructions .TM Mix and 6 μL of RNase-Free ddH20.Nine intestine samples (3 samples per group) were randomly selected for extraction of RNAs using TRIzol reagent  according to the manufacturer’s instructions. Subsequently, the concentration and purit of RNA as well as the RIN value were determined using a Nanodrop 2000 spectrophotometer , a 1.2% (w/v) agarose gel electrophoresis, and an Agilent 2,100, respectively. To remove gDNA, an equal amount of total RNA (1g) was incubated with RNase-Free ddH20 and gDNase Mix. Using the Superscript first strand synthesis system , reverse transcription was performed in a 20 μL reaction volume containing 10 μL of the RNA template, 4 μL of the 5 x RO-EasyIL-1β, IL-10, IL-8, TNF-α) and tight junctions-related genes . The β-actin gene of yellow catfish was employed as an internal reference to normalize gene expression levels  was performed to analyze the expression of immune-related genes In this study, all data were presented as mean ± SD (standard deviation). SPSS 27.0 software  was used to assess the statistical differences. GraphPad Prism (United States) and Adobe Illustrator (United States) software were used to create the charts. After normality test, the one-way ANOVA analysis was used to evaluate the significant difference.  and scanning electron microscopy (SEM) were performed. According to the TEM results, in the control group , the freThe results of SEM showed that the intestinal microvilli of the control group  were neaTo investigate whether ammonia nitrogen exposure could cause damage to the tight junctions between intestinal mucosal epithelial cells, immunohistochemical analysis and qRT-PCR were conducted. In the control group , the mucOccludin, Claudin-1, ZO-1)   decreaselaudin-2 , a pore-To investigate whether ammonia nitrogen stress could cause intestinal inflammation, immunohistochemical studies and qRT-PCR were conducted. In the three groups , T/NK ceTNF-α, IL-1β, IL-8 and IL-10 increased significantly, especially in the 2.5 mg/L group. In light of the aforementioned findings, it is possible that ammonia nitrogen stress triggered severe inflammatory response.According to the results of gene expression, as ammonia nitrogen concentration increased, the expressions of key inflammatory biomarkers  such as et al.  was upregulated, while the gene expression of pore-forming protein (Claudin-2) was downregulated. These results indicate that ammonia nitrogen stress damages the integrity of intestinal mucosal physical barrier, enhances paracellular permeability, and may induce inflammation.Tight junctions, including claudin family proteins, occludin, and ZO-1, are beneficial to maintain the physical integrity of the intestinal epidermal barrier. Disruption or defects in the intestinal barrier integrity may cause microbial imbalances and other harmful substances to cross the epithelial barrier, leading to activation of immune cells and intestinal inflammation . Khan etet al.  reportedg et al.  found thTNF, IL-1β and IL-8 in Pelteobagrus fulvidraco  rose with the increase of ammonia nitrogen concentration, and the expression level of tight junction related genes  decreased first and then increased. Moreover, the high ammonia concentration group also had the highest expression of anti-inflammatory factor IL-10. Considering the above points, we believe that at the sampling time point, fish in the high concentration group were in the later stages of inflammation resolution and tissue repair period, whereas fish in the low concentration group were still in the major inflammatory stage.An interesting finding in this study was that, the low concentration group (0.5 mg/L) had more severe damage to the intestinal physical barrier than the high concentration group (2.5 mg/L), including lower expression of tight junction related genes, sparser and shorter microvilli, and more blurred mucosal barrier. However, the high concentration group (2.5 mg/L) showed a trend of mucosal barrier repair. In fact, a growing number of studies have shown that certain inflammatory cytokines contribute to barrier protection. Although IL-17A is known to be an inflammatory cytokine, but it also protects the mucosal barrier by affecting pression . Shih et (et al.  revealedy et al.  revealed3 levels in intestinal chyme are very high (Under natural conditions, TAN and pNHery high , exceediery high . HoweverIn this study, we systematically evaluated the pathological changes after ammonia nitrogen challenge in yellow catfish after an ammonia nitrogen challenge at the histological, molecular, and ultrastructural levels, as well as the inflammation-related immune response. In conclusion, this investigation demonstrates that ammonia nitrogen stress can damage the intestinal mucosal barrier of yellow catfish and induce intestinal inflammation.


Low-level BaP exposure resulted in the highest proportion of lysogenic phages (88%), and prophages expressed auxiliary metabolic genes (AMGs) associated with nutrient transformation . In contrast, high-level BaP exposure (200 mg kg−1) disrupted microbial metabolism and suppressed the antiphage systems (n = 29), leading to the increase in phage-bacterium association (37 phage-host pairs) and conversion of lysogenic to lytic phages (lysogenic ratio declined to 43%). Despite fluctuating phage-bacterium interactions, phage-encoded AMGs related to microbial antioxidant and pollutant degradation were enriched, apparently to alleviate pollution stress. Overall, these findings expand our knowledge of complex phage-bacterium interactions in pollution-stressed worm guts, and deepen our understanding of the ecological and evolutionary roles of phages.The earthworm gut virome influences the structure and function of the gut microbiome, which in turn influences worm health and ecological functions. However, despite its ecological and soil quality implications, it remains elusive how earthworm intestinal phages respond to different environmental stress, such as soil pollution. Here we used metagenomics and metatranscriptomics to investigate interactions between the worm intestinal phages and their bacteria under different benzo[a]pyrene (BaP) concentrations. Low-level BaP (0.1 mg kg Earthworms are vital to soil ecosystems due to their widespread distribution and major contributions to the maintenance of the physical structure, drainage, nutrient availability, and microbial activity in soils . MoreoveThe virome contains the most abundant and genetically diverse biological entities in the biosphere . There iWe used a multi-omics approach, including metagenomics, viromics, metatranscriptomics, and bioinformatics, to investigate the earthworm intestinal virome and its interaction with bacteriome under environmental pollution stress. Benzo[a]pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon (PAH) was selected as a test soil pollutant because of its widespread presence and recalcitrance in soils  as well −1 for resident soil [Metaphire guillelmi from Yilong Earthworm Farm in Jiangsu, China) with clear clitellum and uniform body weight (~4 g each) were placed in each microcosm for BaP exposure. The microcosms with punctured lids were incubated at 20 °C in darkness for 28 days with a constant humidity of 15–20% [Surface farmland soil depth 0–20 cm) without BaP contamination was collected from Nanjing, China . The United States Environmental Protection Agency (USEPA) screening levels of BAP is between 0.1 and 1.8 mg kg cm withoAfter 28 days of incubation, earthworms were collected and the intestinal contents were obtained as previously described . Briefly−1 C6H5K3O7, 1.92 g l−1 Na2HPO4·12H2O, 0.24 g l−1 KH2PO4, pH = 7), and then centrifuged at 7000 rpm 4 °C for 10 min to obtain the supernatant. The supernatant was filtered through 0.22 µm filter  and then concentrated by tangential flow filtration  [Earthworm intestinal contents (0.25 g) from each treatment were collected for the extraction of total bacterial DNA using DNeasy PowerSoil Pro Kit . The qualified DNA was used for determining bacterial biomass in earthworm gut by quantitative PCR using universal primers targeting the V4 region of the 16S rRNA gene. Primer sequences were 515F (5’-GTG CCA GCM GCC GCG GTA A-3’) and 806R (5’-GGA CTA CHV GGG TWT CTA AT-3’). For phage DNA extraction, virus-like particles (VLP) in the earthworm intestinal contents were first purified and concentrated by filtration and centrifugation following the previously described protocol , 24. BriBoth bacterial and phage sequencing libraries were generated using NEB Next Ultra DNA Library Prep Kit for Illumina  following the manufacturer’s recommendations. The libraries were then sequenced using the NovaSeq6000 system  to yield 150 bp paired-end reads. This approach yielded 78,071,036–108,731,722 reads  for metagenome and 36,296,118–38,203,828 reads  for virome, respectively. Trimmomatic (v0.36) was used for raw data processing  and BWA-https://github.com/BioInfoTools/BBMap) as default parameter in order to determine the abundance of genes in each sample. MetaPhlAn2  [MetaGeneMark (v3.38)  was usedaPhlAn2)  was usedaPhlAn2) . FunctioaPhlAn2)  and eggNaPhlAn2) .p ≤ 0.05 in DeepVirFinder were considered as phages. The phage contigs detected by one or both methods were classified by querying protein sequence against the HMM models with specific phage taxon from the ViPhOG database using “hmmscan” from HMMER (v3.1b2) [https://swissmodel.expasy.org/, accessed on 11 December 2021) was then used to predict the quaternary structure of each protein with a Global Model Quality Estimation (GMQE) score above 0.5. Then BPROM server  and FindTerm server  were used to predict the promoter and terminator of AMGs, respectively [CD-HIT (v4.7) was used to cluster the contigs longer than 5 kb of all samples to obtain unique phage contigs with parameter -c at 0.95 and -aS at 0.8. Two independent methods, VirSorter2 (v2.1.0)  based on(v3.1b2) . The BLA(v3.1b2)  was used(v3.1b2) , soil [3(v3.1b2) , honey b(v3.1b2) , and Ref(v3.1b2)  database(v3.1b2)  and DRAM(v3.1b2) . The posectively .−3, identity ≥70%, and matching length ≥2500 bp [https://defense-finder.mdmparis-lab.com/, accessed on 13 August 2022) was used to identify antiphage defense systems in bacteria [Three prognostic computational approaches  were adopted to improve the accuracy of phage host prediction . Specifi≥2500 bp . Phage h≥2500 bp . Phages ≥2500 bp . To bettbacteria , which c−5 when the Deephage probability score was not deemed confident [https://pro-hunter.genomics.cn/index.php/Home, accessed on 4 May 2022).To assess the symbiosis of bacteria and phages in different treatments, the proportion of lysogenic phages in the gut virome and lysogenized bacteria in the gut bacteriome were separately identified. Specifically, lysogenic phages were identified by Deephage (v1.0) using 0.6 as the cutoff. The lysogenic marker proteins in phage sequence ; Table onfident . AdditioThe transcriptome of earthworm gut microbiome was obtained to investigate the expression of bacterial genes and phage-encoded AMGs at different BaP levels. In brief, the earthworm gut contents were frozen in liquid nitrogen immediately after dissection. Then total RNA was extracted using RNeasy Mini Kit  from 30 mg sample, which quality and quantity were determined by Agilent 4200 system  and NanoDrop2000 , respectively. Whole mRNAseq libraries were generated using NEB Next Ultra Nondirectional RNA Library Prep Kit for Illumina (New England Biolabs) following manufacturer’s recommendations. The rRNA transcripts were eliminated by ALFA-SEQ rRNA depletion Kits (for Bacteria). The mRNA was converted into cDNA, followed by purification and amplification to obtain the sequencing library. Paired-end sequencing was conducted on a NovaSeq6000 system to generate 150 bp paired-end reads with low quality and short filtered with Trimmomatic (v0.36). Clean reads were mapped to NCBI Rfam (v14.9) databases to remove non-protein-coding rRNA sequences from eukaryotes and prokaryotes by Bowtie2 (v2.33). Afterwards, the remaining clean reads were assembled into a de novo transcriptome using Trinity (v2.4.0) with the parameters: --pre_correction --mink 20 --maxk 60 --step 10. Prodigal (v2.6.3) was used to predict ORFs for assembled Scaftigs longer than 200 bp and the unigenes were clustered using methods consistent with metagenomics . BLASTx https://www.omicshare.com/tools/). The significance of differences in parameters were compared using t-test in SPSS 26.0. Data visualization was performed in the website: https://www.chiplot.online/ and R (v4.0.3).The alpha diversity indexes of the microbes in each sample were determined using the “diversity” function in vegan package in R (v4.0.3). ANOSIM analysis was used to determine the significance of the differences in intestinal microbial communities. Differential expression (DE) of two treatments was estimated using “DEseq” function in DEseq package in R (v4.0.3). KEGG enrichment analysis of significantly augmented genes were implemented using Fisher’s Exact Test  as BaP concentrations increased  BaP exposure  [Although low-level BaP did not noticeably change ROS production, high-level BaP significantly induced ROS production in the earthworm gut. When earthworms were exposed to 2.0, 20.0, and 200 mg kgely Fig. . In resped Figs.  and S2. ely Fig. . Howeverdomonas) , 57 weredomonas) .Fig. 1Top < 0.05), but were significantly depleted when BaP increased further to 200 mg kg−1  increased with BaP concentration and was 1.65-fold higher than that of control at 200 mg kg−1 BaP  , but theKEGG enrichment analysis was conducted for the genes that were significantly enriched in each BaP concentration to explore the major metabolic pathways where these genes are located Figs. . Low-levSiphoviridae, Microviridae, Myoviridae, and Podoviridae   Fig. , and was05) Fig. . Moreove05) Fig. , suggest and ssb  and deplsb [lexA  indicatedcd, dut, and nrdD that are related to pyrimidine metabolism are known to promote bacterial genome replication and reparation [lpxD, vanY, and NEU1 that were related to lipopolysaccharide biosynthesis, peptidoglycan biosynthesis, and sphingolipid metabolism, respectively [dfrB, queC, queD, and queE etc.), which has potent antioxidant properties that alleviate ROS stress [queC, queD, and queE were located in the same genomic neighborhood and shared the same promoter and terminator  [ubiE at Phyre2 showed 99.8% confidence, suggesting the AMG sequence could encode a structurally intact protein and perform its function   were alsion Fig. . This isanthrene .Fig. 4Ph−1, respectively  werWith the expression of approximately half of the annotated AMGs in viromes, the transcriptomic results verified the important roles of these AMGs in mediating bacterial metabolism in worm gut, especially bacterial resistance to BaP stress. Those AMGs that were not detected in the transcriptome could still serve as functional repositories and play an ecological role in the microbial community through horizontal gene transfer (HGT) . To captMimivirus, Myoviridae, Podoviridae, and Siphoviridae to 4 host phyla and 37 genera  , which was consistent with the least phage-bacterium association in all samples. As expected, the bacteria carrying the dominant antiphage defense systems were mainly distributed in the phylum Actinobacteria, which was different from the predicted dominant prokaryotic hosts  Fig. . These rly) Fig. . The ressts Fig. . The relsts Fig. . This wasts Fig.  and S6 [VBR was also determined to explore phage-bacterium interactions. The VBR was 0.01, 0.1, and 0.6 in clean, low, and high BaP contamination, respectively Figs.  and S10,We propose that phage-bacteria interactions and phage-encoded AMGs play important roles in facilitating microbial adaption to BaP stress in earthworm intestines. Low-level BaP stress can stimulate microbial metabolism through exerting a bell-shaped stimulatory effect in earthworm intestinal bacterial community, which enhanced the antiphage defense system and cooperative interaction between phages and bacteria. In contrast, high-level BaP exposure damaged bacterial metabolism and the antiphage systems, resulting in the shift from cooperative to antagonistic relationship between phages and bacteria. Despite the fluctuating interactions between phages and bacteria, the active expression of phage-encoded AMGs related to pollutant resistance and degradation could assist the host bacteria in alleviating pollution stress. Collectively, these results reveal the PtW life history strategy adopted by the earthworm intestinal phages under BaP exposure, and demonstrate the important ecological functions of intestinal phages in assisting symbiotic bacteria to survive pollution stress.Supporting texts 1-2, Supplementary figures 1-10Supplementary table 1-10


The use of multistage barriers as a driver to resist electrochemical interference is a promising approach.Detecting heavy metals in seawater is challenging due to the high salinity and complex composition, which cause strong interference. To address this issue, we propose using a multistage energy barrier as an electrochemical driver to generate electrochemical responses that can resist interference. The Ni-based heterojunction foams with different types of barriers were fabricated to detect Cr(VI), and the effects of the energy barriers on the electrochemical response were studied. The single-stage barrier can effectively drive the electrochemical response, and the multistage barrier is even more powerful in improving sensing performance. A prototype Ni/NiO/CeO The pollution of seawater by heavy metals, including Cr(VI), is a significant and pressing issue ,2,3. Cr, including fluorescence probe, colorimetry, and so on ,9, but t2O3 p-n junction interface has been shown to promote the catalytic process of dimethyldichlorosilane [Semiconductor heterojunctions have been widely applied in fields such as photoelectric materials, sensors, batteries, and more, as they can effectively regulate electron transport through their interfacial effect ,14,15. Frosilane , while Rrosilane . Specialrosilane . In the 2 (an n-type semiconductor) can form a p-n junction [2, respectively [2/Au heterojunction with multistage barriers. Polyaniline (PANI) has abundant amine and imine groups, which allow it to selectively adsorb Cr(VI) [Two typical barriers that can be formed are p-n junctions and Schottky junctions. NiO (a p-type semiconductor) and CeOjunction . Au, witectively ,21,22. Tb Cr(VI) .2/Au/PANI foam was prepared for direct Cr(VI) detection. A multistage barrier was used as an electrochemical driver to generate an electrochemical current response to Cr(VI) by combining it with a selective adsorption film. Different types of barriers were investigated, and the sensing performance was improved significantly by using multistage barriers. The trace detection of Cr(VI) with a high recovery rate (93.63–104.79%) in seawater was achieved by resisting the strong interference of seawater. Using a multistage barrier as an electrochemical driver is an effective approach to resist interference.Herein, the Ni/NiO/CeO3)2·6H2O, CoSO4·7H2O, CuSO4·7H2O, SnCl2·2H2O, Bi(NO3)3·5H2O, Fe(NO3)3·9H2O, and K2Cr2O7 were all purchased from Aladdin Reagent  Co., LTD. Polyvinylpyrrolidone (PVP), hemoglobin (Hb), and dopamine (DA) were purchased from Alfa Aesar. Ce(NO3)3·6H2O, acetone, ethanol, and hydrochloric acid (HCl) were all bought from Sinopharm Chemical Reagents Co., LTD. . Ni foam was purchased from Hengxin Research Metal Co., LTD. . All of the water used was deionized water.All the reagents used in this research were of analytical grade. Uric acid, xylene, aspartic acid, hydroquinone, p-phenylenediamine, KCl, NaCl, AgCl, Zn. Scanning electron microscopy (SEM) images were captured using an S-4800 SEM equipment . X-ray diffraction (XRD) analysis was conducted using an Ultima IV X-ray diffractometer  . Infrared spectroscopy data were collected using the Nicolet iS10 Fourier Transform Infrared (FTIR) spectrometer .−1. Scanning was performed 32 times.During the FTIR test in a dry environment, the attenuated total reflectance (ATR) accessory was placed in the optical path of the spectrometer, and the air background was scanned. The surface of the bulk sample (1 cm wide and 1 cm long) was snared to the crystal surface of the ATR accessory. The infrared spectrum of the sample was collected with a resolution of 4 cmIn the XRD test, the sample was cut into a small piece with a width and length of 1cm to ensure that the test surface was smooth, and the sample was fixed on the sample table with plasticine. Then, the XRD test was started.The SEM test involved directly gluing the sample to the conductive adhesive and using the scanning electron microscope to photograph the sample morphology and energy spectrum mapping.The Ni foam (1 × 1.5 cm) was first cleaned sequentially in propanone, ethanol, and deionized water using ultrasonic cleaning for 15 min each. After drying with nitrogen, it was transferred to a tube furnace and heated to 700 °C within 300 min; then, it was held at that temperature for 240 min. The Ni/NiO foam was obtained after cooling to room temperature.3)3·6H2O and 0.06 g of PVP were dissolved in 50 mL of deionized water and stirred at ambient temperature for 30 min. The resulting mixture was then transferred into a 100 mL TEFLON reactor. Ni/NiO foam was added and subjected to a hydrothermal reaction at 180 °C for 12 h. After cooling, the sample was removed, and the surface impurities were cleaned using ethanol and deionized water. The Ni/NiO/CeO2 foam was obtained after drying at 60 °C.Furthermore, 0.185 g of Ce with a pH of 5.8 was used as the electrolyte.A three-electrode system was used for electrochemical testing, with the Ni/NiO/CeO2/Au foam is presented in 2 nanolayer was deposited on the NiO surface but not entirely covering it. Afterward, the Ni/NiO/CeO2 foam was sputter-coated with Au nanoparticles to form the Ni/NiO/CeO2/Au foam. The SEM images in 2 cubes, with an edge length of approximately 200–300 nm, are uniformly distributed on the NiO nanofolds but not completely covering them  acts as a hindrance to electron transport, and any change in its height (ΔΦ) can affect the electrochemical current response by altering the impedance. The equipotential point for Hb is at pH = 7.4, and when the pH is 5.8, the ion is positively charged . This ca2 foam without an interfacial barrier  produced a larger response than the prepared foam with a p-n junction barrier (Ni/NiO/CeO2 foam), and the foam with a multistage barrier (Ni/NiO/CeO2/Au foam) had the most significant response.The electrochemical current response to DA was used as a reference basis, and the results are shown in  barrier a,b, the on of Hb c–f. Thes2/Au foam with a Schottky barrier , a decrease in impedance, and an increase in current response. In contrast, for the Ni/NiO/CeO2 foam with a p-n junction barrier  and one p-n junction barrier (NiO/CeO2), are present. The electrostatic induction reduces the height of all three barriers simultaneously (△Φm = △Φs1 + △Φs2 + △Φpn), leading to the largest change in electrochemical current response. The above results demonstrate that the barrier can be employed as an electrochemical driver to generate an electrochemical response, and the performance can be enhanced by employing a multistage barrier.The above results can be explained by referring to the energy band diagram presented in  barrier a, the po barrier b, since  Φpn < 0 , leading barrier c, three 4− at 1 < pH < 6.8 [4− [4− to form a negatively charged layer, which obtains holes from the semiconductor  to maintain the charge balance. The conduction bands of the semiconductors reduce as they gain electrons from the negatively charged layer, leading to changes in barrier height, which generate the driving force for the electrochemical current response (The form of Cr(VI) in solution is dependent on the pH value, existing as HCrOpH < 6.8 . Under a 6.8 [4− . The proresponse b,c. Thus2/Au/PANI foams at different pH values, and the optimal pH for the reaction was obtained through experimental evidence  (0–4500 nM). The lowest detection limit (LOD) is 244.43 nM. The Ni/NiO/Au/PANI foam with a Schottky barrier: the sensitivity is 3.44 × 10−3 μA·nm−1·cm−2. The linear regression equation is I = 0.00344C + 51.9129 (R = 0.9829) . LOD is 175.51 nM. The Ni/NiO/CeO2/Au/PANI foam with a multistage barrier: the sensitivity is 16.92 × 10−3 μA·nm−1·cm−2. The linear regression equation is I = 0.01692C + 411.0720 (R = 0.9948) . LOD is 35.68 nM. From the above results, it can be found that the order of Cr(VI) detection performance is as follows: the Ni/NiO/CeO2/PANI foam < the Ni/NiO/Au/PANI foam < the Ni/NiO/CeO2/Au/PANI foam. Compared with much of the previous work presented in 2/Au/PANI foam is able to maintain a lower detection limit while detecting a wide concentration range.The Ni/NiO/CeO4− to form a negatively charged layer. For the Ni/NiO/Au/PANI foam, NiO gains electrons from the layer, and the conduction band reduces. Therefore, the height of the Schottky barrier decreases, and the current response increases (△ΦS < 0). For the Ni/NiO/CeO2/PANI foam, NiO and CeO2 gain electrons from the negatively charged layer, and their conduction bands reduce. In this case, the height of the p-n junction barrier also decreases (△Φpn < 0) [S| > |△Φpn |. Therefore, the sensing performance of the Ni/NiO/Au/PANI foam is better than that of the Ni/NiO/CeO2/PANI foam. For the Ni/NiO/CeO2/Au/PANI foam, NiO and CeO2 gain electrons from the negatively charged layer, and their conduction bands reduce. In this case, the height of two Schottky barriers  and one p-n junction barrier (NiO/CeO2) decreases simultaneously (△Φm = △Φs1 + △Φs2 + △Φpn), so the electrochemical current increases even more. The above theoretical analysis is consistent with the DPV results and the electrochemical impedance spectroscopy test (The sensing mechanism is further discussed in Φpn < 0) , resultiopy test .2/PANI foam) were also tested in Cr(VI) detection. As shown in 4− concentration, indicating that the interfacial barrier is the electrochemical driver to generate electrochemical response.In addition, the controls without interfacial barriers  in seawater can be affected by various metal ions and organic pollutants, making it crucial to ensure interference resistance. As depicted in 2/Au/PANI foam is capable of resisting common interferences in seawater.Real-life samples from tap water and seawater were examined, and the results are shown in 0T) [The resistance to interference in seawater is mainly attributed to the following factors. Firstly, the multistage barrier is used as the electrochemical driver to generate the electrochemical response, which is different from the traditional electrochemical response based on a redox reaction. This sensing mode relies on physical electrostatic induction, which shields the interference of many chemically active substances. In addition, the electrochemical current response produced by changing the barrier takes the form of exponential amplification: J∝-exp(Φ/2k0T) ; thus, o2/Au/PANI foam was studied using the cyclic electrode method. As shown in 4− is measured every 2 days for 20 days. The final electrochemical response remains above 80%  by resisting the strong interference of seawater. The obtained outstanding LOD (35.68 nM) and recovery (93.63–104.79%) make it promising for practical applications. The development of electrochemical sensors using multistage barriers as electrochemical drivers to resist interference represents a new approach.The Ni based foams with different interfacial energy barriers were prepared for Cr(VI) detection. It was found that a barrier can be used as an electrochemical driver to generate an electrochemical response. The electrostatic interaction of Cr(VI) regulates the energy barrier without causing redox and improves the specificity in seawater. Different types of barriers have different levels of performance. Compared with the traditional single-stage barriers, the multistage barriers designed in this experiment can improve the sensing sensitivity by 5–7 times. The fabricated Ni/NiO/CeO