H2S protects rat hippocampal neurons (RHNs) against hypoxia-reoxygenation (H/R) injury by promoting phosphorylation of RhoA at Ser188. However, effect of H2S on the phosphorylation of ROCK2-related sites is unclear. The present study was designed to investigate whether H2S can play a role in the phosphorylation of ROCK2 at Tyr722, and explore whether this role mediates the protective effect of H/R injury in RHNs. Prokaryotic recombinant plasmids ROCK2wild-pGEX-6P-1 and ROCK2Y722F-pGEX-6P-1 were constructed and transfected into E. coli in vitro, and the expressed protein, GST-ROCK2wild and GST-ROCK2Y722F were used for phosphorylation assay in vitro. Eukaryotic recombinant plasmids ROCK2Y722-pEGFP-N1 and ROCK2Y722F-pEGFP-N1 as well as empty plasmid were transfected into the RHNs. Western blot assay and whole-cell patch-clamp technique were used to detect phosphorylation of ROCK2 at Tyr722 and BKCa channel current in the RHNs, respectively. Cell viability, leakages of intracellular enzymes lactate dehydrogenase (LDH), and nerve-specific enolase (NSE) were measured. The H/R injury was indicated by decrease of cell viability and leakages of intracellular LDH and NSE. The results of Western blot have shown that NaHS, a H2S donor, significantly promoted phosphorylation of GST-ROCK2wild at Tyr722, while no phosphorylation of GST-ROCK2Y722F was detected. The phosphorylation of ROCK2wild promoted by NaHS was also observed in RHNs. NaHS induced more potent effects on protection against H/R injury, phosphorylation of ROCK2 at Tyr722, inhibition of ROCK2 activity, as well as increase of the BKCa current in the ROCK2Y722-pEGFP-N1-transfected RHNs. Our results revealed that H2S protects the RHNs from H/R injury through promoting phosphorylation of ROCK2 at Tyr722 to inhibit ROCK2 activity and potentially by opening channel currents.The RhoA-ROCK signaling pathway is associated with the protective effects of hydrogen sulfide (H Cerebral ischemia-reperfusion (I/R) injury is mainly caused by the restoration of blood perfusion following brain tissue ischemia . The pat2S is a gaseous signaling molecule found in mammals that is involved in the regulation of various physiological functions and pathological processes in the nervous system and cardiovascular system [2S is catalyzed by cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercap topyruvate thitransferase (3-MST) [2S protects endothelial cell from hypoxic injury by inhibiting the RhoA-ROCK signaling pathway [2S can inhibit the RhoA-ROCK signaling pathway in cerebrovascular vascular smooth muscle, cells, and cause hyperpolarization and vasodilation [2S can improve cerebral vasoconstriction and diastolic dysfunction in cerebral ischemic mice and play a protective role against cerebral ischemic injury [Hr system . Endogen (3-MST) . Previou pathway . Endogendilation ,9. Moreoc injury .1 and ROCK2, among which ROCK2 is mainly distributed in the brain and blood vessels [2S can inhibit the RhoA-ROCK signaling pathway and produce a protective effect against ischemic brain injury, but the detailed mechanism by which H2S inhibits the RhoA-ROCK signaling pathway is not clear [2S can significantly promote phosphorylation of the RhoA protein at Ser188 and inhibit RhoA activation [The RhoA-ROCK signaling pathway composed of RhoA and ROCK is an important target in cells. ROCK has two subtypes, ROCK vessels . The ROC vessels ,13, suggot clear .Phosphorot clear . Our rectivation .2 protein can reduce the activation of ROCK2 [2S can affect tyrosine phosphorylation at Tyr722 of ROCK2 protein to regulate the ROCK2 activity. Therefore, the present study was designed to investigate the ROCK2 inhibition mechanism of H2S protecting RHNs from H/R injury, especially focusing on the effect of H2S on phosphorylation of ROCK2 at Tyr722.Tyrosine phosphorylation at tyrosine residue 722 (Tyr722 or Y722) of ROCKof ROCK2 . HoweverE. coli and empty plasmid-transfected E. coli. Meanwhile, a significant different band appeared in the lysate precipitate of the ROCK2wild-pGEX-6P-1- or ROCK2Y722F-pGEX-6P-1-transfected E. coli induced by isopropyl-beta- D-thiogalactopyr-anoside (IPTG), and molecular weight of the band is about 178 KDa. The use of RIPA was not associated with an effect on the expression of the target protein, and glutathione S-transferase (GST)-ROCK2wild or GST-ROCK2Y722F was not detected in the lysate supernatant of the transfected E. coli. Our results showed that GST-ROCK2wild and GST-ROCK2Y722F were successfully expressed in E. coli transfected with prokaryotic plasmids. Perhaps, these two expressed proteins exist in the form of insoluble protein in E. coli, so they did not appear in the supernatants and cannot be further purified. Therefore, lysate of E. coli was used for phosphorylation experiment in vitro. To explore effect of H2S on phosphorylation of ROCK2 at Tyr722, lysate of the ROCK2wild-pGEX-6P-1- and the ROCK2Y722F-pGEX- 6P-1-transfected E. coli were used for phosphorylation in vitro. As shown in 2wild (p < 0.01) in lysate of the ROCK2wild-pGEX-6P-1-transfected E. coli, and the NaHS, a H2S donor, markedly promoted the phosphorylation (p < 0.01), but NaHS was not observed to promote the phosphorylation of GST-ROCK2Y722F. The results indicated that H2S can promote the phosphorylation of GST-ROCK2 at Tyr722.As shown in 2 protein expression  and promoted the phosphorylation of ROCK2 at Tyr722  in normal RHNs. Moreover, 2 activity . The results demonstrated that H2S not only inhibited the ROCK2 protein expression, but also promoted the phosphorylation of ROCK2 leading to the inhibition of ROCK2 activity.As shown in 2Y722-pEGFP-N1, and ROCK2Y722F-pEGFP-N1 were transfected into RHNs respectively. The expression of green fluorescent protein (GFP) was used to determine transfection efficiency. As shown in Empty plasmid, ROCK2 and p-ROCK2Y722 were detected, and NaHS  significantly increased the ratio of p-ROCK2Y722/ROCK2 . In the GFP-ROCK2Y722 group, ROCK2, p-ROCK2Y722, GFP-ROCK2Y722, GFP-p-ROCK2Y722 were detected, and NaHS  markedly increased the ratios of both p-ROCK2Y722/ROCK2 and GFP-p-ROCK2Y722/GFP-ROCK2Y722 . In the GFP-ROCK2Y722F group, ROCK2, p-ROCK2Y722, GFP-ROCK2Y722F were detected, NaHS  obviously increased the ratio of p-ROCK2Y722/ROCK2 . The results demonstrated that H2S can promote phosphorylation of ROCK2 or GFP-ROCK2 at Tyr722 in RHNs.In the empty plasmid group, ROCK2 activity ; in the GFP-ROCK2Y722 group, NaHS  obviously inhibited ROCK2 activity ; in the GFP-ROCK2Y722F group, only 200 μmol/L NaHS inhibited ROCK2 activity (p < 0.05). The results suggested that ROCK2 Tyr722 may mediate in the H2S-inhibited ROCK2 activity in RHNs.As shown in p < 0.01), and promoted the release of LDH (p < 0.01) and NSE (p < 0.01) in transfected RHNs. After treatment with NaHS , the protective effect against H/R injury was reflected in increased cell viability and inhibition of LDH and NSE release . Therefore, the lysate of E. coli was used to carry out phosphorylation in vitro.In the present study, the prokaryotic plasmids, ROCK2 at Tyr722 occurs in the presence of kinase Src [2 did not undergo autophosphorylation. In the presence of Src, NaHS, a donor of H2S, significantly promoted phosphorylation of GST-ROCK2wild, but Src did not cause any phosphorylation of GST-ROCK2Y722F in the presence or absence of Src or Src + NaHS. The results demonstrated that NaHS could promote phosphorylation of GST-ROCK2 at Tyr722. In the present study, it was further found that NaHS not only induced phosphorylation of ROCK2 at Tyr722 in RHNs, but also inhibited protein expression and activity of ROCK2. A previous study indicated that phosphorylation of ROCK2 at Tyr722 can reduce ROCK2 activity [2S can promote the phosphorylation of ROCK2 at Tyr722 to inhibit ROCK2 protein expression and activity in RHNs.Phosphorylation of ROCKnase Src . Our resactivity . Thus, o2Y722-pEGFP -N1 and ROCK2Y722F-pEGFP-N1 plasmids as well as empty plasmid were transfected into the RHNs. Western blot assay indicated that NaHS significantly promoted the phosphorylation of ROCK2 and GFP-ROCK2Y722 but not GFP-ROCK2Y722F. Meanwhile, it was observed that inhibition of NaHS on ROCK2 activity was markedly enhanced in the GFP-ROCK2Y722 group compared to that in empty plasmid or the ROCK2Y722F groups. These results indicated that ROCK2 Tyr722 mediates the H2S-inhibited ROCK2 activity in RHNs.In the present study, by using lentivirus, eukaryotic recombinant ROCK2Y722- pEGFP-N1 plasmid significantly increased the inhibitory effect of NaHS on the H/R injury-decreased cell viability and -released LDH and NSE in RHNs. Therefore, it could be concluded that phosphorylation of ROCK2 at Tyr722 also mediates protective effect of H2S against H/R injury in RHNs. It was well-known that inhibition of the RhoA-ROCK signaling pathway protects cerebral ischemia-reperfusion injury [2S promotes the phosphorylation of ROCK2 at Tyr722 to inhibit ROCK2 activity in RHNs, our results demonstrated that H2S protects RHNs from H/R injury through promoting phosphorylation of ROCK2 at Tyr722 to inhibit ROCK2 activity. Phosphorylation of RhoA is involved in the protection of cerebral ischemic injury . In the n injury ,35. Comb2+-activated K+ channels (KCa) belong to the superfamily of potassium channels [ca is divided into three subseries: big conductance (BKCa), intermediate-conductance (IKCa), and small conductance (SKCa) [Ca channels exist in a variety of non-excitatory and excitatory cells [2S can significantly increase BKCa current in neurons [Ca on vascular reactivity and calcium sensitivity may be mainly through the RhoA-ROCK pathway [2 Tyr722 mediates the NaHS-increased current of BKCa channel in the RHNs. Together with a recent study that NaHS exerts neuroprotective effects via activating the BKCa [2S promotes phosphorylation of ROCK2 at Tyr722 to open BKCa and protects the RHNs from H/R injury. Cachannels . Accordie (SKCa) . BKCa chry cells . They arry cells . Previou neurons ,41. The  pathway . The prethe BKCa , our res2S regulated the phosphorylation of ROCK2 at Tyr722, and this regulation provided significant neuroprotection in rat neurons. There may probably be multiple mechanisms which are responsible for the neuroprotective effects of H2S. However, it is undeniable that the study in vitro cannot fully reflect the situation in vivo. The present study only conducted experiments in vitro, without any in vivo study, which is a limitation of our study. In addition, stroke is an aging-associated disease with comorbidities impacting seriously any drug therapy [2S in the phosphorylation of ROCK2 at Tyr722 resulting in protective effect against cerebral I/R injury, especially in aged animals in our future research. The present study demonstrated that H therapy ; therefo2S protects RHNs from H/R injury through promoting phosphorylation of ROCK2 at Tyr722 to inhibit ROCK2 activity and open BKCa channel. This finding may provide a basis for the use of H2S in the treatment of cerebrovascular diseases such as stroke.In conclusion, the present study provided evidence for the first time to demonstrate that H2 antibody , anti- ROCK2 (phospho Y722) antibody , ATP , recombinant Src  were purchased from Abcam ; 293T cell  was purchased from Beyotime Biotehchnology, Co. . NaHS was obtained from Sigma Chemical ; IPTG , goat anti-rabbit IgG secondary antibody , anti-GAPDH antibody , marker , Coomassie blue R250 , LDH assay kit  were purchased from Servicebio ; NSE assay kit  was obtained from Jiangsu Meimian Industrial, Co., Ltd. ; anti-ROCK2wild-pGEX-6p-1, ROCK2Y722F-pGEX-6p-1 prokaryotic recombin- ant plasmids and the GFP-tag ROCK2Y722-pEGFP-N1, ROCK2Y722F-pEGFP-N1 eukaryotic recombinant plasmids were obtained from Gene Create Biological Engineering, Co.  and constructed from rat genome. ROCK2Y722F, that is Tyr722 of ROCK2 was mutated to a phenylalanine (Phe or F) at the mRNA level. E. coli (BL21) was obtained from Gene Create Biological Engineering, Co. .The GST-tag ROCK2wild-pGEX-6P-1 and ROCK2Y722F-pGEX- 6P-1 were separately transformed into E. coli. Briefly, 20 µL of each plasmid was added to 100 µL of BL21 E. coli and placed on ice for 20 min. E. coli was kept at 42 °C for 90 s, 1 mL Luria-Bertani (LB) liquid medium was then added and the E. coli was cultured at 37 °C for 1 h. The E. coli was inoculated into LB solid medium and cultured at 37 °C for 24 h. The bacteria were inoculated into LB liquid medium. The medium was added with IPTG at a final concentration of 1 mM when the OD value was 0.8 and cultured at 37 °C for 6 h. The bacterial solution was collected and one part of the bacteria was sent to Shanghai Sangong Biological Co., Ltd.  for base sequencing, while the other part was crushed by ultrasonic crushing and RIPA, and total protein in the bacterial homogenate was determined using the BCA method. The homogenate was centrifugated at 10,000 rpm for 10 min. A total of 15 µL of supernatant or resuspended sediment specimen was separated by 8% SDS-PAGE at 120 mV for 40 min. The gel was stained with 200 mL Coomassie Brilliant Blue R-250 dye for 2 h. Then, discarding the staining solution, 200 mL decolorizing solution was added to decolorize the gel for 4 h until the protein band was clearly visible.Prokaryotic recombinant plasmids ROCK2wild in the ROCK2wild-pGEX-6P-1 plasmid-transfected E. coli were TAT, the bases of Tyr. The bases were mutated into TTT, the bases of phenylalanine , in the ROCK2Y722F-pGEX-6P-1 plasmid-transfected E. coli. The sequencing results showed that the bases of site 722 of GST-ROCK2, pH 7.4), and then the mixture was shaken for 30 min at 37 °C. Then, SDS-PAGE loading buffer was added to terminate the phosphoryl transfer reaction. Phosphorylation of GST-ROCK2wild or GST-ROCK2Y722F proteins was examined by Western blotting assay.About 15 μL protein specimen, 100 μmol/L ATP, and 100 ng Src kinase without or with 100 μmol/L NaHS were sequentially added to kinase buffer  three times for 5 min each. The membranes were incubated overnight with anti-ROCK2 (phospho Y722) antibody at 4 °C and secondary antibody for 1 h at room temperature. ECL-Plus reagent was applied to the membrane, and chemiluminescence was visualized using a Fluor-S-max imager.In each lane of the gels, 15 µg GST-ROCK6 cells /mL. The cells were crushed using an ultrasonic crusher with a power of 100 W, and the supernatant was collected after centrifugation of the crushed cells. The protein samples to be measured were added into 96-well plate and the blank wells and standard wells were set up, adjusting the blank wells to zero. The 96-well plate was incubated at 37 °C for 30 min. About 50 μL of termination solution was added to each well and then 50 μL of chromogenic agent A and 50 μL of chromogenic agent B were added. The mixture was shaken gently and mixed well, and allowed to develop for 15 min at 37 °C avoiding light. About 50 μL of termination solution was added to each well to terminate the reaction. The activity of ROCK2 was assayed at 450 nm on a microplate reader. The detection of activity should be completed within 15 min of the addition of the termination solution.Mature RHNs were cultured for 6–8 days and the cells were oval or irregular in shape overall. RHNs were diluted with PBS at pH 7.2–7.4 to a concentration of 1 × 102wild-pEGFP-N1 and ROCK2Y722F-pEGFP-N1 plasmid, lentivirus packaging plasmid pCD/NLBH*DDD, and membrane protein expression plasmid PLTR-G. The RHNs were centrifuged at 10,000 r.p.m for 5 min, resuspended in 1 mL trypsin, and digested at 37 °C for 3 min. Cells were resuspended in 1 mL of fresh media and counted; 1 mL of this suspension (5 × 105/mL cells) was added to each well in a 24-well plate. After adding 40 μL virus solution and 500 μL culture medium, the cells were cultured in an incubator. The medium was replaced with fresh culture medium after 24 h. Post-transfection, fluorescent protein expression was observed under an inverted fluorescence microscope at 36, 72, and 108 h to assess the infection efficiency.The lentivirus vector was a second-generation vector that was cotransfected into 293T cells by empty, ROCK4 cells/well was added to the 96-well plates. The 96-well plates were incubated in an incubator for 24 h . A total of 10 μL of CCK-8 solution was added to each well and the 96-well plate was again incubated in an incubator for 24 h . The absorbance at 450 nm was measured using a microplate reader.Cell viability was determined using a Cell Counting Kit-8 (CCK-8) cell viability assay kit. Mature RHNs cultured for 6–8 days were selected and their cell bodies were relatively full. Most of them are oval or irregular, and axons interweave with each other in a network. After H/R injury, 100 μL of RHNs suspension at a concentration of 2 × 104 cells/well was added to a 96-well plate. The original medium was removed, washed with PBS buffer, and low serum medium was added and incubated for 24 h. After incubation, the original medium was removed, washed again with PBS, and 120 μL of cell lysis working solution was added and incubated in an incubator for 60 min. The cell culture plate was centrifuged for 5 min, and 80 μL of lysis supernatant was added to a new 96-well plate and subsequently assayed. The absorbance of the supernatants was determined at 450 nm using a microplate reader. The results are expressed as U/L.LDH was determined using an LDH Assay Kit. Mature RHNs cultured for 6–8 days were selected and their cell bodies were relatively full. After H/R injury, 100 μL of RHNs suspension at a concentration of 2 × 10g for 20 min and the supernatant was collected. Standard wells and sample wells were set up, and 50 μL of standard at different concentrations was added to each well, and 50 μL of sample to be tested was added to the sample wells. About 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well, and the wells were sealed with sealing film and incubated in an incubator for 60 min. About 50 μL each of substrate A and B was added to each well, and incubated for 15 min at 37 °C, protecting from light. A total of 50 μL of termination solution was added to each well, and the OD value of each well was measured at 450 nm using a microplate reader. The results are expressed as μg/L.NSE was determined using a NSE Assay Kit. Mature RHNs cultured for 6–8 days were selected and their cell bodies were relatively full. After H/R injury, cells were centrifuged at 1000× Ca currents of RHNs were recorded on the recorder. Origin 8.0 software was used to plot concentration–respons curves. Standard extracellular fluid  was adjusted with NaOH to pH 7.4, standard intracellular fluid  was adjusted with KOH to pH 7.0.Recording electrodes were prepared from borosilicate glass pipettes and had a resistance of 5–9 MΩ. The recording electrodes were filled with recording solution. A 35 mm culture dish was prepared and the middle part was coated with an appropriate amount of petroleum jelly. The cultured RHNs crawls were removed, rinsed with PBS to remove dead cells and impurities, and placed in the middle of the culture dish coated with petroleum jelly. About 2 mL of extracellular fluid was added, and then the samples were placed on an inverted microscope. RHNs with smooth surface, large cytosol, and clear edges were found with a high-powered microscope and adjusted to the position so that they were in the center of the field of view. A tiny positive pressure was applied with a syringe before the glass microelectrode is placed in water. Adjust the micromanipulation to let the electrode drop slowly, use the computer software Clampex 10.5 to enter the membrane test window, click bath mode, and click the auto button in the multiClamp 700B window to maintain the entry baseline at about 0. The entry resistance of the electrode should be in the range of 4 to 8 MΩ. Gradually approach the electrode to the target cell first by micromanipulation under low magnification, and then move the electrode slowly toward the cell until contact is made. At this point, remove the positive pressure and apply a small negative pressure to the tip of the electrode, and the resistance will rise to about 1 GΩ if the cell is good. Click patch mode and then compensate the electrode capacitance with the amplifier. After the membrane was broken, the membrane capacitance and series resistance were compensated, and the whole-cell membrane capacitance current appeared, forming the whole-cell recording mode. The clamping voltage was set at −70 mV, I-V stimulation was given in voltage-clamp mode, and pulse stimulation was performed from −40 mV to 70 mV at a step of 10 mV, lasting approximately 150 ms at an interval of 3 s. NaHS was administered using a micropipette. The BK2, 5% CO2) for 8 h. At the end of the incubation, the sugar-free medium was discarded and replaced with complete medium and placed in a normal incubator for 6 h for the next experiment.After the cells were placed in a conventional incubator at 37 °C for 2 h, the medium of the cells was changed to sugar-free medium and placed in a hypoxic incubator , or two-way ANOVA was used to conduct data analysis.


Apostichopus californicus. Here, we sought to determine if heat stress in isolation could trigger wasting symptoms in A. californicus. We exposed sea cucumbers to (i) a simulated marine heatwave (22 °C), (ii) an elevated temperature treatment (17 °C), or (iii) control conditions (12 °C). We measured the presence of skin lesions, mortality, posture maintenance, antipredator defences, spawning, and organ evisceration during the 79-hour thermal exposure, as well as 7-days post-exposure. Both the 22 °C and 17 °C treatments elicited stress responses where individuals exhibited a reduced ability to maintain posture and an increase in stress spawning. The 22 °C heatwave was particularly stressful, as it was the only treatment where mortality was observed. However, none of the treatments induced wasting symptoms as observed in the Nanoose Bay event. This study provides evidence that sea cucumber wasting may not be triggered by heat stress in isolation, leaving the cause of the mass mortality event observed in Nanoose unknown.Oceanic heatwaves have significant impacts on disease dynamics in marine ecosystems. Following an extreme heatwave in Nanoose Bay, British Columbia, Canada, a severe sea cucumber wasting event occurred that resulted in the mass mortality of  Climate change is increasing the intensity, duration, range, and frequency of marine heatwaves across the globe with potentially catastrophic effects on organism fitness, ecosystems, and human economies . In mariMesocentrotus franciscanus) and purple sea urchins (Strongylocentrotus purpuratus) have suffered from “bald sea urchin disease” and “sea urchin wasting disease” along the Pacific coast of British Columbia (B.C.). Pathology of these diseases includes lesions to the body wall and a shortening or loss of spines, and both have been associated with mass mortality events  with wasting symptoms similar to those of sea stars throughout their range in the northeast Pacific Ocean , 50% of observed A. californicus exhibited wasting symptoms. The six to ten week delay between peak near-surface sea temperatures and widespread mortality may be indicative of a potential link between thermal stress and wasting in A. californicus, perhaps mediated by the progression of disease symptoms and immune failure. Temperature is an especially compelling mechanism considering that healthy individuals were abundant below depths of 19 metres where the water was cooler . The effect of temperature-related stressors on wasting in sea cucumbers has yet to be tested.Since 2014, there have been reports of giant California sea cucumbers . The sea cucumbers were initially collected and used in an unrelated short term tagging experiment comparing T-bar and coded wire tags at the end of July 2021, after which they were all tagged with coded wire tags. Tagging had no measurable impact on sea cucumber behaviour or physiology in the short or long term  were randomly divided into one of three temperature treatments: control , warm , or heatwave . Our experimental temperatures reflect average summer temperatures and those recorded in the Strait of Georgia when wasting of A. californicus was observed  with two individuals per container, aside from two smaller containers (33 ×45.7 ×11.4 cm) that housed one individual each. A plastic mesh divider was used to separate sea cucumbers within each container to allow for tracking individuals throughout the experiment. The containers were randomly placed into sea tables which acted as water baths. The target water bath temperatures were achieved by having a constant flow of cooler inlet sea water to act as the control (12 °C), allowed to reach ambient room temperature for the warm treatment (17 °C), or heated using two 800 W aquarium heaters for the heatwave treatment (22 °C). The 22 °C treatment temperature was gradually increased over a 24-hour period in the sea table water baths day 1; . During e organs .We assessed sea cucumbers for wasting symptoms throughout the experiment and counted skin lesions on days 5, 6, 7, and 12. Epidermal damage was classified as either minor lesions or major ulcers based on their size and appearance. We defined minor lesions as regions of epidermal damage at the spine tips without signs of considerable discoloration, where the dermis was not damaged across its full thickness . We clasWe assessed body stiffness using a 3-point ordinal scale to measure heat-stress induced deviations from normal behaviour. To do so, first we removed each individual from their bin and gently palpated them for 10 s to encourage them to stiffen. Then we placed them on an elevated platform to measure their ability to maintain their posture over 5 s. The stiffness testing platform was constructed by cutting a piece of plastic pipe into an eight cm long, eight cm in diameter, and four cm tall section, which we glued onto an eight cm tall block. We assigned a score of 0 if the organism failed to stiffen at all (full droop), a score of 1 if it did not remain stiff for the full 5 s when placed on the platform , and a score of 2 if it maintained its posture for the entire 5 s (no droop). Stiffness was measured on days 1–5 of the experiment (as a baseline and throughout the treatment), on day 7  and day 12 . Frequency of handling individuals occurred equally across treatments.Nbin = 30). We also evaluated whether specimens had eviscerated based on the presence or absence of ejected internal organs during these routine checks.As some sea cucumbers spawned, we recorded whether there was any evidence of spawning every 12 h. Spawning was assigned to each container rather than individual, since some individuals were co-housed  test using the  package .https://github.com/declan-taylor/sea_cucumber_wasting (DOI: 10.5281/zenodo.7259325).All data and annotated code for the analyses described above are publicly available at χ2 = 463.32, df = 2, p < 2.2e−16).The temperature treatments varied slightly from the target temperatures during the experimental heatwaves. During the 79 h treatment, the mean temperature of the control treatment was 12.4 °C and varied from 10.8 - 14.0 °C; the mean of the warm treatment was 16.6 °C and ranged from 14.8 °C to 17.9 °C; the mean of the heatwave treatment was 21.7 °C and varied from 19.6 °C to 23.3 °C. Temperature treatments were significantly different from each other  and heatwave treatments (n = 2). These major ulcers appeared to heal throughout the recovery period and were re-classified as minor lesions on day 12. The maximum number of minor lesions per individual was not significantly explained by treatment, weight, evisceration, or defecation status.Minor skin lesions occurred during the experiment and were not statistically different between treatments (n = 5), while there were no mortalities observed in the control or warm treatments. Based on backwards model selection, mortality was driven by treatment and weight (p = 1.99e−7) and heatwave (p = 2.44e−11) treatments. Structural stiffness values were significantly likely to be lower than day 1 on day 3 (p = 1.37e−5), day 4 (p = 2.50e−5) and day 5 (p = 8.66e−5), but not on day 2 , with spawning occurring in the 17 °C warm (n = 5 bins) and the 22 °C heatwave (n = 4 bins) treatments. However, there was no significant difference in spawning between temperature treatments .Stress spawning was observed during the temperature treatment . Treatment temperature did not explain a significant amount of the variance in the occurrence of evisceration (p = 0.0383) and defecation status (p = 0.0163) were significant drivers increasing likelihood of evisceration , or the isolated wasting events reported along the Pacific coast  and this trend, although statistically insignificant, was expected as stress spawning has been previously reported in other sea cucumber species . We wereperiment . Howeverperiment . Defecatiscerate  or underiscerate . As suchA. californicus. Like wasting, severe cases of SUS are highly transmissible and result in mortality  and non-infectious factors  should both be investigated because widespread environmental degradation and anthropogenic climate change are shifting pathogenic dynamics globally (Since we observed skin lesions under thermal stress that were likely unassociated with wasting, other factors are likely causing recent wasting outbreaks in ortality ; bacteriortality  and sea ortality . A studysymptoms . Avenuesglobally . These iA. californicus to extreme thermal stress as measured by mortality, degraded stiffening behaviour, and the development of skin lesions. Despite this, we found no evidence that wasting is induced by temperature stress alone. Therefore, the August 2021 mass wasting event in Nanoose, British Columbia, was likely not triggered solely by the anomalous heatwave. Determining the factors that cause and exacerbate wasting in A. californicus is essential for predicting and managing mass mortality events. Sea cucumbers are ecologically important benthic detritivores, which break down organic matter, recycle nutrients, and maintain sediment health (A. californicus in the face of global environmental change will require a comprehensive understanding of their stress responses, disease dynamics, and the novel threat of sea cucumber wasting.In this study, we exposed t health . Efforts10.7717/peerj.14548/supp-1Supplemental Information 1Click here for additional data file.


Here we present authenticated  metagenomic marine eukaryote sedaDNA from the Scotia Sea region acquired during IODP Expedition 382. We also provide a marine eukaryote sedaDNA record of ~1 Mio. years and diatom and chlorophyte sedaDNA dating back to ~540 ka . We find evidence of warm phases being associated with high relative diatom abundance, and a marked transition from diatoms comprising <10% of all eukaryotes prior to ~14.5 ka, to ~50% after this time, i.e., following Meltwater Pulse 1A, alongside a composition change from sea-ice to open-ocean species. Our study demonstrates that sedaDNA tools can be expanded to hundreds of thousands of years, opening the pathway to the study of ecosystem-wide marine shifts and paleo-productivity phases throughout multiple glacial-interglacial cycles.Antarctica is one of the most vulnerable regions to climate change on Earth and studying the past and present responses of this polar marine ecosystem to environmental change is a matter of urgency. Sedimentary ancient DNA ( Sedimentary ancient DNA can indicate ecosystem-wide changes. Here, the authors show association between warm phases and high diatom abundance in the Antarctic Scotia Sea, in addition to presenting ancient eukaryote sedimentary DNA spanning the last approximately 1 million years. Antarctica is arguably the most susceptible polar region to climate, evidenced in the fact that West Antarctica has warmed 2.4 ± 1.2 °C between 1958 and 2010, making it one of the fastest-warming regions globally4. Understanding how Southern Ocean organisms respond to climate variability, including throughout past climate shifts, is thus of key importance to predict how the Antarctic marine ecosystem will evolve in the near future.Polar ecosystems are highly vulnerable to ongoing climate change, and rapidly melting ice-sheets and changes in oceanography and in marine ecosystems are expressed on all levels of the food websedaDNA) analysis studies ancient genetic signals preserved in sediments. Because genetic traces of all organisms, fossilising and soft-bodied, can potentially be preserved in sediment records, the analysis of sedaDNA holds enormous potential to go beyond standard environmental proxies and allow reconstruction of entire ecosystems6. Yet, the recovery of sedaDNA is complicated, as only trace-amounts of DNA are preserved and they are fragmented and degraded, which makes sedaDNA prone to contamination from modern environmental DNA7. Recent improvements in sedaDNA techniques, including in anti-contamination measures during field work, laboratory work, and the use of bioinformatic DNA damage analysis, now permit authentication of sedaDNA detected in sediment samples11.Sedimentary ancient DNA  sediments12, and ~650,000-year-old subarctic permafrost deposits13. In polar marine ecosystems, eukaryote sedaDNA has been recovered from up to ~140,000-year-old sediments in the Arctic16 and <25,000-year-old sediments in the Antarctic7. Deep polar marine environments are ideal locations for sedaDNA research because of favourable DNA preservation15. They feature constantly low temperatures (~0 °C) and low oxygen (~5 mL L−1), and UV radiation is absent19.It is yet to be determined, however, how far back in time marine organisms can be detected using 23.In 2019, IODP Expedition 382 ‘Iceberg Alley and Sub-Antarctic Ice and Ocean Dynamics’ set out to investigate the long-term climate and oceanographic history of the Antarctic Ice Sheet (AIS). Five sites (U1534–U1538) were drilled east of the Drake Passage, two shelf sites at 53.2°S, the northern edge of the Scotia Sea , and three abyssal/deep sites at 57.4°–59.4°S in the Scotia Sea   and within the Scotia Sea , and three out of 31 samples from the core centre (<0.64 ng mL−1). All PFMD measurements are provided in Supplementary Table No traces of PFMD were detected at either the periphery (adjacent to core liner) or the centre of the here analysed cores from Sites U1534 and U1536. At Site U1538, PFMD was detected in 21 out of 31 samples from the core periphery  from the mudline samples, maximum number of reads from intermediate depth samples , and few reads in the bottom-most samples . Across all sites, Eukaryota (category not further classified) were the most abundant , followed by Bacillariophyta , Polycystinea , Stramenopiles , Streptophyta (4.6%), Arthropoda (3.5%), Cercozoa (3.4%), Haptista (3.4%), Ascomycota (3.25%), Dinophyceae (3.2%), Foraminifera (3.1%), Chordata (3%), Chlorophyta (3%), Basidiomycota (2.3%), Cnidaria (1.5%), Euglenozoa (1.5%), Annelida (1.5%), PX clade (1.1%), Bilateria (1%), Ciliophora (1%) and Alveolata (1%)  and ~1 Ma (U1536) and acquired few reads  and few taxa, primarily consisting of Fungi , unclassified Eukaryota, and a few rarer groups ; Chlorophyta, Coccomyxa and Micromonas (Chlorophyta) and Synechococcus (Cyanobacteria)  in two samples, including the second-oldest sample analysed at this site  were detected irregularly throughout the core at Site U1538 , after which read numbers decreased again , which may have started sometime after 14.5 ka as increased diatom relative abundances determined by SSU + LSU suggests (see previous section).We retrieved a total of 42 sequences  revealed that, on average across all samples , most diatom reads were assigned on to the broad group of Bacillariophyceae  and related nodes relatively low taxonomic resolution , Mediophyceae (~23%), Fragilariopsis cylindrus (~2.4%), Chaetoceros affinis ~1.9% and Hemiaulus sinensis  are indicators for neritic, open ocean conditions (see Discussion), with their increased influence evident from ~14.5 ka to present , while at Site U1536 the damage was 13 ± 7%, and at Site U1538 it was 11 ± 7% , although this is based on a single read only (classified as passing our stringent default filtering criteria but not showing damage), and U1534 90.95 mbsf (~0.7–2.5 Ma), where only one ancient read was identified , based on a 56-bp-long sequence assigned to a transcribed RNA sequence (NCBI accession no. GBYM01000074). The remaining reads assigned to eukaryotes in this sample passed our default filtering criteria but did not show damage.At Site U1534, 15 ± 6% of the SSU + LSU reads showed psbO read numbers were very low at all sites, sedaDNA damage analysis on the non-subsampled data only achieved results for few taxa and few samples. At Site U1534, U1536 and U1538, 2, 4 and 14 samples showed sedaDNA damage, respectively (Supplementary Data psbO genes showing signs of DNA damage were assigned to Haptista (only detected in the non-subsampled data) and Chlorophyta at 240 ka (Site U1534), and to Bacillariophyta at ~4.4 ka , alkalinity (r = 0.52), phosphate (r = 0.44), δ18O (r = 0.32), temperature (r = 0.31), silicon (r = 0.26), and age of sediments (r = 0.21), very weak positive correlations between sedaDNA damage with pH (r = 0.09), and negative relationships between sedaDNA damage with sulfate (r = −0.42), and salinity  and positive relationships between δ18O and Polycystinea (r = 0.42), Dinophycease (r = 0.39) Choanoflagellata (r = 0.38), Mollusca (r = 0.37) and Annelida (r = 0.35), meaning that diatoms were associated with warm phases and dinoflagellates, radiolarian, choanoflagellates and Annelida (includes Crustacea) with cold phases , these have yet to be replicated and authenticated using, for example, metagenomics, or through paired analysis of diagnostic lipid biomarkers as well as geochemical proxies30. Most of our samples are younger than ~540 ka, and, based on SSU + LSU results, we were able to identify diatom species in samples as old as this (Site U1536), which is, to our knowledge, the oldest diatom record reported from marine sedaDNA. Single-copy psbO genes of this important phytoplankton group were also identified, confirming peak abundance of diatoms between 12.7 and 11.3 ka at Site U1538, i.e., during the Antarctic Cold Reversal of the last glacial termination. Chlorophyte psbO genes were also preserved in samples dating back to ~540 ka (U1536), and, based on SSU + LSU alignments, even in our deepest sample from the early Pleistocene .We investigated marine eukaryote sedaDNA in a sample possibly as old as ~2.5 Ma at Site U1534 is exciting, we consider this result with caution. Firstly, even under ideal conditions  ancient DNA, which degrades over time, is not expected to preserve for much longer than ~1 Ma, although reports of non-replicated/authenticated results of ancient bacterial DNA reach back to many millions of years27 (and references therein). This could mean that this sample’s true age is rather at the younger end of our age-determination, i.e., ~700 ka, given the stratigraphic uncertainties at Site U1534. Secondly, the taxonomic composition of this sample (and that of the 1 Ma sample at U1536) includes especially fungal groups, suggesting that modern contamination might play a role, as living fungi are a common component of sediment records32. Possibly, around this age (~1 Ma) the ancient DNA signal fades from the sediment record and DNA from actively living fungi becomes more pronounced. However, the detection of DNA damage in a fungi sequence from the oldest U1534 sample suggests that at least part of the >1 Ma old DNA is of ancient origin. Our finding of marine eukaryote sedaDNA in precisely dated samples of Site U1536 are significant for paleoecology, as they expand the temporal window of applying sedaDNA analyses as a marine paleo-environmental monitoring tool from ~140 ka to ~1 Ma, i.e., covering multiple glacial-interglacial cycles.While the finding of marine eukaryote sedaDNA data reveal a notable transition from a relatively low proportion of diatoms (<10% of eukaryotes) prior to 14.5 ka to a high proportion (~50%) after this date. This timing coincides with well known interhemispheric ice-sheet deglaciation, associated with sea-level rise following Meltwater Pulse 1A at the Boelling/Alleroed transition (~14.5 ka), the spin up of the AMOC and North Atlantic Deep Water (NADW) production, decrease in sea-ice cover and increase in ocean productivity in the Southern Hemisphere34. Enhanced sea-ice variability and an associated increase in surface marine primary productivity during the Antarctic Cold Reversal  have also been reported from Patriot Hill Blue Ice records35 during this exact same time, and overlap with our finding of high diatom abundance (psbO), a highly productive phytoplankton group, between 12.7 and 11.3 ka. After 11.3 ka, relative diatom abundances remain relatively high while total diatom abundance is reduced, which might indicate a re-calibration of the eukaryote marine food web after the peak-productivity phase triggered by the ACR. While community changes were less obvious in samples older than 14.5 ka (possibly a consequence of higher degradation), δ18O correlation analyses confirmed an association between increased diatom abundance and past warm phases over the last ~1 Ma.At Site U1538, the Chaetoceros affinis and Hemiaulus sinensis increased, at the expense of the sea-ice diatom Fragilariopsis cylindrus. Hemiaulus sinensis is a relatively rare neritic, warm water/temperate species found along the Argentinian coast36, and its detection in sediments as far south as ~59°S in the Scotia Sea is, to our knowledge, a new observation. While it is possible that H. sinensis reads were in fact misidentified Chaetoceros spp. (Results section), complex northward and southward currents in combination with the Antarctic Circumpolar Current may potentially allow the re-distribution and southward displacement of water-column particles and species36, which leaves the possibility of H. sinensis sedaDNA indeed being preserved at our study sites. C. affinis is commonly considered as an upwelling-indicating, open ocean species38, while F. cylindrus is frequently used as sea-ice proxy39. This change in diatom composition is consistent with sea-ice reduction at Site U1538  and Site U1536 , starting around 14.5 ka, i.e., following meltwater Pulse 1A34, and intensifying modern marine conditions ~12.7 ka40. Neither C. affinis nor H. sinensis were previously reported as part of the diatom microfossil record at any of the IODP Exp. 382 sites23 . It has been shown previously that more fragile, in-situ produced water-column diatoms preserve less well than heavily silicified Antarctic diatoms, thus skewing fossil analyses to the latter species41. This underlines the use of sedaDNA techniques in complementing microfossil and other paleo-proxy analyses  for the fine-scale investigation of time periods undergoing environmental change, such as glacial-to-interglacial transitions. Here, the inverse relationship between δ18O and relative diatom abundance over the last ~1 Ma indicates that this phytoplankton group played a consistent role throughout major deglaciations in this time frame, with the most obvious change observed at Exp. 382 Scotia Sea sites for the last deglaciation after 14.5 ka.We showed that, from 14.5 ka to present, the two open ocean diatoms RV Joides Resolution between 20 March and 20 May 2019. Specifically, we collected samples at Site U1534 , U1536 , and Site U1538  23. sedaDNA samples collected at Site U1534 were from Hole C, at Site U1536 from Hole B, and at Site U1538 from Holes C and D , and were stored and extracted at a quarantine approved facility . No ethical approval was required for this study.Samples were collected during IODP Exp. 382 ‘Iceberg Alley and Subantarctic Ice and Ocean Dynamics’ on-board 18O to the LR04 stack43. Wherever present specimens of Uvigerina bifurcata were picked from samples at 10 cm intervals. During warmer periods when U. bifurcata was not present, Melonis affinis and/or Hoeglundina elegans were analysed. Sedimentation rates over the intervals sampled for sedaDNA typically range between 6 and 30 cm/kyr, with rates exceeding 100 cm/kyr during the Last Glacial Maximum ~20,000 years ago (20 ka). For our deepest sample, U1534C-10H-6_115cm (90.95 mbsf), we only have biostratigraphically assigned ages available (shipboard data), which date this sample as early Pleistocene .Age control for Site U1534 is based on tuning of benthic foraminifera δ23. Average sedimentation rates are ~10 cm/kyr for Site U1536, with elevated values (up to 20 cm/kyr) in the upper ~80 mbsf (the last ~400 ka). Site U1538 average sedimentation rates are twice as high, averaging ~20 cm/kyr. Especially in the upper ~430 mbsf (the last 1.8 Ma), rates are up to 40 cm/kyr. Higher resolution age models are based on dust climate couplings, correlating sedimentary dust proxy records such as magnetic susceptibility and sedimentary Ca and Fe records to ice-core dust proxy records over the last 800 ka45 and to a benthic isotopic stack26 before that. These age models were established for Site U1537 (adjacent to Site U1536) and provide orbital to millennial scale resolution. For this study we correlated sedimentary cycles of Sites U1536 and U1538 to U1537 to achieve similar resolution and to be able to determine if a sample originates from a glacial or interglacial period  at each cutting point. Core cutting tools were sterilised before each cut (3% bleach and 80% ethanol) of the core in 1 m sections. The outer ~3 mm of surface material were removed from the bottom of each core section to be sampled, using sterilised scrapers . A cylindrical sample was taken from the core centre using a sterile (autoclaved) 10 mL cut-tip syringe, providing ~5 cm3 of sediment material. The syringe was placed in a sterile plastic bag (Whirl-Pak) and immediately frozen at −80 °C. The mudline (sediment/seawater interface) was transferred from the core liner into a sterile bucket (3% bleach treated), and 10 mL sample was retained in a sterile 15 mL centrifuge tube  and frozen at −80 °C. Samples were collected at various depth intervals depending on the site to span the Holocene up to ~1 million years , the infusion rate was doubled prior to sedaDNA sampling at Site U1538 to ensure low PFMD concentrations represent low contamination and not delivery failure of PFMD to the core. At each sedaDNA sampling depth, one PFMD sample was taken from the periphery of the core , and one next to the sedaDNA sample in the centre of the core . We transferred ~3 cm3 of sediment using a disposable, autoclaved 5 mL cut-tip syringe into a 20 mL headspace vial with metal caps and Teflon seals. We also collected a sample of the tracer-infused drill fluid at each site, by transferring ~10 mL of the fluid collected at the injection pipe on the rig floor via a sterile plastic bottle into a 15 mL centrifuge tube (inside a sterile plastic bag) and freezing it at −80 °C. These drill fluid controls were processed and analysed in the same way as the sedaDNA samples including sequencing. Samples were analysed using gas chromatography .As part of the APC process, drill fluid  is pumped into the borehole to trigger the hydraulic coring system, therefore, the potential for contamination exists due to drill fluid making contact with the core liner. To assess the latter, we added the non-toxic chemical tracer perfluoromethyldecalin (PFMD) to the drill fluid at a rate of ~0.55 mL minsedaDNA samples but that are not part of this study. PFMD is categorised as the stereoisomers of PFMD (C11F20), which add up to 87-88% . We exclusively refer to the first and measurable PFMD category, calibrating for the 88% in bottle concentrations during concentration calculations. Each GC analysis run included the measurement of duplicate blanks and duplicate PFMD standards. Due to a large sample number, PFMD at Site U1538 was measured in three separate runs, with the first and last run including triplicate blank and triplicate PFMD standards (duplicates in the second run), and the last run also containing a drill-fluid sample. To blank-correct PFMD concentrations, we subtracted the average PFMD concentration of all blanks per run from PFMD measurements in that run. To determine the detection limit of PFMD, we used three times the standard deviation of the average blank PFMD values per run; due to all blank values for the U1538 runs being 0, we used three times the standard deviation of the lowest PFMD standard for this site in this calculation. This provided us with a PFMD detection limit of 0.2338 ng mL−1. Any PFMD measurements of samples below this limit were rejected.A detailed description of the PFMD GC measurements is provided in ref. sedaDNA extracts and metagenomic shotgun libraries , and the last batch including mudline and PFMD samples to avoid contamination of the sedaDNA samples. In brief, we used 20 µL sedaDNA extracts in a repair reaction , then purified the sedaDNA , ligated adaptors  barcodes were attached to the double-stranded DNA; 60 min, 22 °C), purified the sedaDNA again , and then added a fill-in reaction with adaptor sequences . We amplified the barcoded libraries using IS7/IS8 primers50 , purified  and quantified them . We amplified the libraries  using IS4 and GAII Indexing Primers50, purified , quantified and quality-checked using Qubit  and TapeStation . We combined the libraries into an equimolar pool , diluted this pool with nuclease-free H2O to 100 µL, and performed a ‘reverse’ AxyPrep clean-up to retain only the small DNA fragments typical for ancient DNA . We added one more AxyPrep clean-up to remove primer-dimer (library:beads ratio of 1:1.05) and checked sedaDNA quantity and quality via TapeStation and qPCR . The libraries sequenced at the Garvan Institute for Medical Research, Sydney, Australia (Illumina NovaSeq 2 × 100 bp).A total of 80 es Table  were pre52,–threshold 0.55) and duplicate reads (‘dedupe’ tool in BBMap v.37.36.), and quality control after each step .The sequencing data was processed and filtered as described in detail in refs. 54), we ran our data against different curated reference databases that are commonly used to identify marine eukaryotes, including the SILVA small  and large subunit  ribosomal RNA database , as well as a combined SSU + LSU database. For the latter, we merged the initial SSU and LSU databases (using the ‘cat’ command) into one database prior to the alignments, with the intention to maximise taxonomic resolution and number of reads , we avoided normalising (rarefying) our SSU/LSU data and worked with relative abundances. For an estimate of total abundance of phytoplankton, we also ran our data against a recently developed database for the single-copy photosynthetic gene psbO, which is present in both prokaryotes and eukaryotes, mainly in one copy per genome55. The latter was initially performed with non-subsampled (non-rarefied) data to be able to determine an adequate subsampling depth56 (representative of the diversity in the data) for subsequent quantitative analyses . We determined 1.1 Mio reads as an adequate subsampling depth  were converted to .rma6 files using the Blast2RMA tool in MEGAN (version 6_18_9), with the default settings except for a minimum support percent of zero (‘off’) and a minimum percent identity of 95%.Using MALT . The filtered read counts per taxon  for the psbO dataset) and site were exported for downstream analysis. All taxa determined in the controls post-SSU + LSU alignments are listed in Supplementary Data psbO dataset. We used the default settings for analysing the taxonomic data in MEGAN CE, i.e., following the NCBI taxonomy and using a LCA (lowest common ancestor) approach, where every read is assigned to a taxon and if a read has significant matches to two different taxa a and b, where a is an ancestor of b, then only the more specific match to b will be used57. Where a taxon was determined multiple times with different reference-groups  then they were grouped . For visualisation purposes of the large SSU-LSU phylum-level dataset, we kept all taxa that were abundant with at least 1% on average across all samples as separate taxa , while taxa less abundant than that were grouped  was conducted with MEGAN CE version .21.12. TsedaDNA, we tested sedaDNA damage using the ‘MALTExtract’ and ‘Postprocessing’ tools of the HOPS v0.33-2 pipeline25. We used the same configurations as in Armbrecht et al. (2021), using the taxalist ‘Eukaryota’  for the SSU + LSU dataset, and a taxalist containing all taxa identified via the non-subsampled psbO data, see psbO dataset. We used the non-subsampled psbO data because the subsampled data provided too few reads  for damage analysis. MaltExtract outputs a read summary, with reads are categorised as ancient (showing damage) or default (passing stringent filtering criteria but not showing damage) for the taxalist-specified taxa25. Based on the latter (ancient vs. default reads), the proportion of sedaDNA damage per taxon was determined for each dataset (SSU + LSU and non-subsampled psbO). A summary of reads classified as ancient and default are provided with the Supplementary Data As a means of authenticating our 58; and with results provided in Supplementary Information Fig. To ensure that our new combined SSU + LSU database generates comparable taxonomic composition to SSU and LSU alone, we performed regression and correlation analyses on the average relative abundance per taxon (phylum-level) acquired when using the SSU, the LSU and the combined SSU + LSU database , relative abundances of eukaryotes post SSU + LSU alignment (phylum-level) and downcore temperature and porewater geochemical parameters, which were measured as part of the shipboard sampling during IODP Exp. 382. Downhole formation temperatures were calculated based on the temperature gradient obtained at each Site with the Advanced Piston Corer Temperature Tool (APCT)24. Key geochemical parameters for the assessment of organic matter degradation rates , and total silicon (µM), were routinely determined for each site for the characterisation regarding the intensity of redox zonation in each core24. To investigate relationships between taxonomic composition and cold and warm climate phases, we added benthic δ18O data from26 corresponding to the ages assigned to our samples to this correlation analysis. Mudline samples, as well as the two samples 23162 (U1438 57.55 mbsf) and 23165 (U1538 62.85 mbsf), were removed from the following analyses as no eukaryotes were determined in these samples. Pearson correlation analysis was performed in PAST v.4.0358.Lastly, we investigated potential relationships between Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1 -6Reporting Summary


After curing, the samples were tested to determine their water-resistant properties, uniaxial compressive strength, and pH value and a micro scanning electron microscope was used. The results indicated that the carbonation reaction can quickly improve the water resistance and compressive strength of imitation site soil, and reduced the water absorption by 16.67% compared to the specimens conditioned at 0.03% CO2 concentration. The UCS of specimens at 5%, 10%, and 15% CO2 concentrations increased by 72.22%, 131.19%, and 219.27%, respectively, compared with those at 0.03% CO2 concentration after the specimens were environmentally maintained in the carbonation chamber at 0.03%, 5%, 10%, and 15% CO2 concentrations for 120 h, respectively. The internal particle gradation of the imitation site soil improved after carbonation. These results provide a basis for improving the restoration technology of earthen sites.In this study, sodium methylsilicate and lime were selected to prepare the same proportion of Imitation Site Soil, and according to the principle of carbonation reaction of restoration materials, the effect of carbonation reaction on the performance of restoration soil of earthen sites was studied. The study has good significance for the conservation and restoration of earthen sites. The samples were cured with CO Earthen sites are an important carrier of civilization transmission, and they provide rich historical and cultural connotations and contain non-renewable information. Some earthen sites collapse and disappear due to weathering by wind, sun, rain, and other environmental effects. In such situations, many scholars have performed reinforcement research on earthen archaeological sites ,2,3,4. T2 to produce Ca(OH)2 and CaCO3 to enhance soil cohesion between particles [2 conditions had a better performance than naturally cured materials [3, thereby resulting in surface cracking, peeling off, and alkali flashes in varying degrees due to the influence of the surrounding environment and efflorescence. As shown in 3Si(OH)2ONa) is an inorganic chemical waterproof material with excellent waterproof performance. It is widely used in waterproof, hydrophobic, material protection, and other fields [In ancient times, lime, glutinous rice slurry, river sand, pottery fragments, or other materials were widely used and mixed with soil to form a triad to improve the strength and durability of the earthen archaeological site. Lime can be employed as a remediation material ,8 becausarticles ,10,11. Harticles . Faster aterials ,14. After fields ,22.2 concentration and curing time as variables. A disintegration test, uniaxial compression test, pH value, and micro scanning electron microscope test were performed on the cured samples to explore the effects of different curing conditions on the restoration and protection of the imitation earthen archaeological site in order to provide technical support for the restoration of the earthen archaeological site.This paper considers the Kaifeng City Wall soil as the object of study and configures the imitation earthen archaeological site accordingly. The imitation earthen archaeological site is reinforced with sodium methylsilicate and lime, and the sample is cured with COThe soil samples were obtained from the vicinity of the Kaifeng City Wall. The physical properties are listed in 3, and the optimal water content is 14.32%. The maximum dry density of the imitation ruins mixed with 7% lime is 1.608 g·cm3, and the optimal water content is 17.55%.The collected silty clay was dried, crushed, and screened for later use. The compaction test was performed for the soil mixed with 7% lime. The maximum dry density of the silty clay is 1.68 g·cm2 concentrations for carbonation curing and cured for 6 h, 12 h, 24 h, 48 h, 72 h, and 120 h. The different curing conditions are listed in The silty clay, 2% sodium methylsilicate, 7% lime, and pure water were mixed evenly and sealed. The mixture was allowed to stand for 24 h to promote the complete mixing of the silty clay, sodium methylsilicate, lime, and pure water. After standing, samples of size 39.1 mm × 80 mm were placed in an environment with different COWater-resistance test: in accordance with the standard for geotechnical test methods (GB/T50123-2019), a double-cup disintegration instrument was adopted  and the sample quality was weighed during the test. The outer layer of the instrument was made of plexiglass, and the inner layer was composed of a beaker, wire mesh (10 cm diameter), ring knife (6.18 cm diameter), and steel wire. The sample was placed on a hanging net and then immediately placed into the beaker. The sample wetting at each stage was recorded in real-time using a recording device for 24 h.Unconfined compressive strength (UCS) test: The specimens were dried in an oven at 105 °C for 48 h after curing and then subjected to the UCS test. The full-automatic geotechnical triaxial compression test system (TSZ-10) was used with a loading rate of 0.5 mm/min. When the pressure displacement curve in the test reached the peak value, the pressure reduced continuously for a period of time, after which the test was stopped, the peak pressure was recorded, test data were calculated, and a stress–strain relationship curve of the improved, repaired soil was obtained.Soil Sample pH value test: A Shanghai Sanxin PH5S soil pH sensor was used to test the pH value under different curing conditions and different curing times. The changes in the pH of the soil were observed, and the influence of the carbonation reaction on the performance of the imitation earthen archaeological site was analyzed.SEM test: SEM tests were performed using FEI Quanta 250 , USA. The crushed specimens were dried and cut into 0.5 cm × 0.5 cm × 0.5 cm soil blocks. The development of the pore space in the soil with was observed after the gold spray treatment to analyze the causes of macroscopic property changes from a microscopic perspective.2 concentrations were maintained for 120 h, The water-resistance test was performed after drying, and the influence of the carbonation reaction on the performance of the imitation earthen archaeological site was compared and analyzed. The pure soil group ) underwent complete wetting and disintegration within 5 min and could not be weighed for mass. For the specimens maintained in 0.03% CO2 concentration (consistent with the CO2 concentration in air) environment ), during the initial stage of the water-resistance test (1)), the specimens exhibited no obvious wetting disintegration phenomenon, and tiny bubbles on the surface were observed along with a small amount of water absorption. After 24 h of the water-resistance test, (2)), the color of the specimen changed because sodium methylsilicate did not fully react with water and CO2, resulting in uneven water resistance of the specimen. For the specimens of carbonization conservation in a 5% CO2 concentration environment ), during the initial stage of the water-resistance test, (1)) no bubble generation and no obvious water absorption phenomenon were observed, and there was a better water-resistance effect. After 24 h of the water-resistance test, (2)) the color of the outer surface was uniform, and there was no trace of water absorption. For the specimens ) of intensive carbonization conservation in a 10% CO2 and 15% CO2 concentration environment, the water-resistance test resulted in a change in the appearance of the specimens, similar to that observed in the 5% CO2 concentration environment, with relatively uniform surface color and no obvious traces of water absorption. This is because, under a high CO2 concentration and an intensive carbonation environment, sodium methylsilicate can fully react with water and CO2 to generate a polysiloxane film that can enhance the water resistance of the specimen. The specific chemical reaction is as shown in Equations (1) and (2). Moreover, it can be initially concluded that the increasing CO2 concentration is beneficial for improving the water-resistance effect of the imitation site soil.To further observe the water-resistant performance of the imitation earthen archaeological site in depth, the pure soil and the imitation earthen archaeological site maintained in an environment with different CO2 concentrations were 22.59%, 5.92%, 5.49%, and 5.37%, respectively, after 24 h of specimen water absorption. The water absorption rates of the specimens maintained at 5%, 10%, and 15% CO2 concentrations were similar and decreased by 16.67%, 17.1%, and 17.22%, respectively, compared with those maintained at 0.03% CO2 concentration. This indicates that the water absorption of the imitation site soil does not decrease linearly with the increase in the CO2 concentration, and the water resistance of the imitation site soil can be improved by carbonization under 5% CO2 concentration. Maintaining proper carbonation can rapidly improve the water resistance of the imitation site soil and prevent the imitation site soil from collapsing when it is washed by rain.The effect of water absorption with time on the water absorption rate of the specimens is shown in 2 concentrations under the same maintenance time was reduced to a greater extent than that of the specimens at 0.03% CO2 concentration. The water absorption of the specimens maintained at 5%, 10%, and 15% CO2 concentrations did not differ much under the same maintenance time. The imitation site soil could achieve better water resistance after 12 h of maintenance at 5% CO2 concentration because the addition of sodium methylsilicate forms a polysiloxane film on the surface of the specimen [2 concentration  had little effect on the water absorption of the specimens when the specimens were cured for more than 48 h. This may be because the carbonation reaction of sodium methylsilicate is completed after the specimens are maintained for 48 h at a 5% CO2 concentration. The increase in the CO2 concentration at this point has a limited influence on the water-resistance performance.The effect of time conservation on the water absorption of the specimens is shown in specimen , and the2 concentration in the conservation environment. After the specimens were maintained in the carbonation chamber at 0.03%, 5%, 10%, and 15% CO2 concentrations for 120 h, the UCS of the imitation site soil increased by 34.24%, 131.56%, 211.93%, and 329.29%, respectively, as compared to that of the plain soil. The UCS of the specimens at 5%, 10%, and 15% CO2 concentrations increased by 72.22%, 131.19%, and 219.27%, respectively, as compared to that of the specimens at 0.03% CO2 concentration.The effect of curing time on the UCS of the specimens is shown in 2 concentration environment. 2 concentration environments. The compressive strength of the 24 h-cured specimen increased by 44.21%, 37.68%, and 6.7%, respectively, compared with the 12-h specimen. The compressive strength of the cured 48-h specimen increased by 4.9%, 11.36%, and 9.49%, respectively, compared with the cured 24-h specimen. The compressive strength of the cured 72 h specimen increased by 3.59%, 3.58%, and 20.38%, respectively, compared with the cured 48-h specimen and 11.94%, 4.27%, and 6.09% for the specimens maintained for 120 h as compared to those maintained for 72 h. It can be seen that the UCS growth rate tends to increase and then decrease with the increase in the curing time. Moreover, the UCS growth rate is the highest at 24 h of curing time and then starts to decrease slowly.The UCS increased with the increase in the curing time under the same CO2 concentration and curing time have important effects on the UCS of the imitation site soil. A sufficient amount of CO2 accelerates the carbonation reaction inside the imitation site soil, and CaCO3 precipitation and Na2CO3 can be produced rapidly. The carbonate fills between soil particles, thereby enhancing the inter-particle bonding and increasing the strength of the imitation site soil. This law provides an important basis for optimizing the restoration process of the imitation site soil.Lime and sodium methylsilicate can effectively improve the compressive strength of powdered clay, and CO2 concentration, the pH of the specimens gradually decreased and stabilized. Moreover, at the same CO2 concentration, the UCS and pH of the specimens exhibited opposite trends with the increase in the curing time, that is, the smaller the pH value, the larger the corresponding UCS. This was due to the conversion of strongly alkaline Ca(OH)2 to neutral CaCO3 deposition, during which the pH of the imitation site soil gradually decreased. Changes in pH values can reflect the state of CaCO3 deposition from the side and provide a basis for analyzing its mechanical properties [operties . The str2 concentrations, the pH of the imitation site soil decreased significantly before 24 h and 12 h, and the growth rate of the compressive strength was also larger than that of other maintenance periods. However, after 24 h and 12 h, the pH of the imitation site soil and the growth rate of compressive strength decreased slowly. When Ca(OH)2 was partially converted to CaCO3 in the imitation site soil, the pH value of the imitation site soil was stabilized. This shows that increasing the CO2 concentration in the maintenance environment of the imitation site soil can effectively improve the carbonation reaction and the compressive strength of the imitation site soil. Moreover, it can accelerate the stabilization of the pH value of the soil, thereby improving the stability of the imitation site soil.Under 5%, 10%, and 15% CO2 for 120 h to further investigate the mechanism of improving the water-resistant performance and unconfined compressive strength of the imitation site soil under different maintenance conditions.To investigate the microscopic changes inside the mock site soils, the internal particle morphology and pore characteristics of the imitation site soil samples at different magnifications were analyzed using SEM. Soil specimens were analyzed in the pure soil group. Moreover, the soil specimens were maintained at 0.03% and 15% CO2 and CaCO3 crystals generated from lime undergo a chemical reaction in the soil to make the dispersed soil particles cohesive into a whole. The deposition of CaCO3 can enhance cementation between soil particles, to improve the overall stability of the earthen archaeological site. The site imitation earthen archaeological site cured at 15% CO2 concentration can quickly generate CaCO3 crystals in 12 h curing time, further improving the disintegration resistance and unconfined compressive strength of the site imitation earthen archaeological site.2 concentration with the same maintenance time.The effect of carbonization maintenance on improving the water resistance of the imitation site soil was significant. Moreover, the imitation site soil can achieve better water resistance through carbonization maintenance under 5% CO2 concentration environments increased with the increase in CO2 concentration. Under the same CO2 concentration environment, the compressive strength increased with increasing curing time. The growth rate of the unconfined compressive strength was large with the curing time of 24 h, and then it decreased slowly.Carbonation curing has an obvious effect on improving the compressive strength of imitation earthen archaeological sites. The compressive strength of the samples cured in different CO2 concentration. The compressive strength showed a negative correlation with the pH value, that is, the smaller the pH value, the greater was the corresponding compressive strength. The interior of the soil sample was observed through SEM, which showed that the improved soil was densely filled among soil particles, the particle gradation was improved, and the adhesion between the particles was good. Moreover, we found that the porosity of the soil sample was reduced, the voids were less and dense, and CaCO3 precipitates were formed, which effectively improved the overall stability of the earthen archaeological site.Under the same curing conditions, the pH value of the imitation earthen archaeological site first decreased with increasing curing time and then gradually stabilized. Under the same carbonization curing time, the pH value of the imitation earthen archaeological site decreased with increasing COIn this study, the Kaifeng City Wall was selected as the research object, and lime and sodium methylsilicate were selected as enhanced materials to prepare the Kaifeng City Wall imitation earthen archaeological site. The properties of the imitation earthen archaeological site were analyzed with test indices such as the specimen water absorption rate, compressive strength, microanalysis, and pH change of the imitation site soil. The following results were obtained: 2 concentration in the maintenance environment can make the carbonation reaction of the imitation earthen archaeological site uniform and rapid, promote CaCO3 precipitation, and effectively improve the specimen water absorption rate and compressive strength of the imitation site soil. Moreover, it can enhance the overall stability of the imitation earthen archaeological site.Increasing the CO


Pneumothorax is a rare surgical complication in spinal surgery with thoracic pedicle screws. The penetrating endplate screw (PES) technique has been developed as a strong alternative spinal anchor to conventional pedicle screws for diffuse idiopathic skeletal hyperostosis (DISH). We present an intraoperative pneumothorax without deviation to the thoracic during the maneuver of the PES.A 56-year-old male who presented with non-union of DISH-related T12 vertebral fracture underwent T12 kyphoplasty and T10-L2 posterior fixation using the PES technique. The left pneumothorax was developed postoperatively without screw deviation to the thorax throughout screw insertion. Postoperative CT suggested that a displaced rib head by the lateral misposition of the screw at the inserting point and the pedicle level might injure the pleura. Spine surgeons should know that the lateral insertion of PES has a potential risk for pneumothorax by the displacement of the rib head because of screw trajectory from caudal to cranial apart from conventional pedicle screw. The pedicle screw is a wide-spread spinal anchor that enables control of the instrumented segments and rigid internal immobilization in cases of spinal fixation surgery . The pedDiffuse idiopathic skeletal hyperostosis (DISH) is a non-inflammatory skeletal disease characterized by calcification and ossification of spinal ligaments and entheses . VertebrA 56-year-old male under a prednisolone prescription of 15 mg per day for pemphigoid fell to the floor. T12 vertebral fracture was not diagnosed by the initial radiological examinations, x-rays and computed tomography (CT), but was diagnosed by magnetic resonance imaging (MRI) Figure . DISH waT12 kyphoplasty and T10-L2 posterior fixation using the PES technique  was performed percutaneously under single fluoroscopy without the navigation system Figure . All scr2 indicated 90% even under a simple oxygen mask with a flow rate of 4 L/min. Pneumothorax of the left lung was noticed by chest x-ray (Figure The patient complained of slight dyspnea two days after surgery. SpOy Figure  and CT (y Figure . CT alsoy Figure . There wy Figure , circle.Chest drainage was performed for the pneumothorax immediately by the general surgeon. The left lung was re-expanded after chest drainage. The drainage tube was removed after five days. The patient was discharged after three weeks after the surgery without any other complications and recurrent pneumothorax. Bone union of T12 was confirmed at six months after surgery without any instrument failure.Pneumothorax is occurred by injuring both the parietal and visceral pleura at the surgery. Under mechanical ventilation, it easily develops tension pneumothorax which is a life-threatening complication . Among pPneumothorax has been reported as one of the surgical complications of the conventional thoracic pedicle screw , howeverThe actual cause of pneumothorax in this case remains unclear. The maneuver of percutaneous screw insertion was performed in one-shot placement for all screws percutaneously and there was no screw deviation to the thorax in the postoperative CT. One possible reason was injury of the pleura and lung by the displaced rib head due to lateral extrapedicular placement of the PES. Postoperative CT showed rib head of the left T10 was laterally and anteriorly displaced Figure , circle,Although the percutaneous screw placement technique using fluoroscopy was reported as an acceptable technique with a lower deviation/complication rate than the conventional open free-hand technique, the direction of the screw misplacement in the percutaneous technique was reportedly more lateral than medial pedicle violation . It arisPneumothorax is a rare complication of the conventional thoracic pedicle screw. However, the PES which trajectory from the caudal to the cranial of the pedicle has a greater risk of pneumothorax than the conventional screw in the case of lateral deviation even without direct injury of the pleura by the instrument. Because pneumothorax under ventilation is a life-threatening complication, spine surgeons should prevent lateral deviation in cases using the PES technique.


We report the complete genome sequences of two strains of JP-1 genotype (GI-18) infectious bronchitis virus (IBV) isolated from the kidneys of dead chickens in Japan in 2000 and 2017. This information will help researchers better understand the evolution and epidemiology of IBV in Japan. Gammacoronavirus in the family Coronaviridae of the order Nidovirales (–Avian infectious bronchitis virus (IBV) belongs to the genus ovirales . IBV hasovirales , 3. Addiovirales –6.In Japan, seven genotypes of IBV  have been confirmed based on the partial nucleotide sequence of the S1 gene , 8. AmonThe JP/Toyama/2000 strain was isolated from the kidney of a dead chicken in Toyama Prefecture, located in central Japan, in 2000. The JP/Yamagata/2017 strain was isolated from the kidney of a dead chicken in Yamagata Prefecture, located in eastern Japan, in 2017.These viruses were grown in embryonated chicken eggs and used for genetic analysis at the fourth passage. Viral RNA was extracted from the infected allantoic fluids using a QIAamp viral RNA minikit (Qiagen), and cDNA was synthesized using random hexamer primers . As desc9–The sequenced fragments were trimmed and assembled using ATGC-Mac v5 . The lengths of the complete genomes of the JP/Toyama/2000 and JP/Yamagata/2017 strains, excluding the poly(A) tail, were 27,675 nucleotides (nt), with a G+C content of 38.19%, and 27,667 nt, with a G+C content of 38.15%, respectively. When the open reading frame (ORF) lengths of three JP-I strains in Japan were compared, differences in the lengths of the S, E, M, 4c, and 6b genes were observed .Based on the complete nucleotide sequences of S1, IBV was recently classified into 32 genotypes . PhylogeLC683779 and LC683780.The genome sequences were deposited in GenBank under the accession numbers


In 7 to 15-year-old operated syndromic craniosynostosis patients, we have shown the presence of microstructural anomalies in brain white matter by using DTI. To learn more about the cause of these anomalies, the aim of the study is to determine diffusivity values in white matter tracts in non-operated syndromic craniosynostosis patients aged 0–2 years compared to healthy controls.DTI datasets of 51 non-operated patients with syndromic craniosynostosis with a median [IQR] age of 0.40 [0.25] years were compared with 17 control subjects with a median of 1.20 [0.85] years. Major white matter tract pathways were reconstructed with ExploreDTI from MRI brain datasets acquired on a 1.5 T MRI system. Eigenvalues of these tract data were examined, with subsequent assessment of the affected tracts. Having syndromic craniosynostosis (versus control), gender, age, frontal occipital horn ratio (FOHR), and tract volume were treated as independent variables.ʎ2 and ʎ3 of the tracts genu of the corpus callosum and the hippocampal segment of the cingulum bundle show a ƞ2 > 0.14 in the comparison of patients vs controls, which indicates a large effect on radial diffusivity. Subsequent linear regressions on radial diffusivity of these tracts show that age and FOHR are significantly associated interacting factors on radial diffusivity (p < 0.025).Syndromic craniosynostosis shows not to be a significant factor influencing the major white matter tracts. Enlargement of the ventricles show to be a significant factor on radial diffusivity in the tracts corpus callosum genu and the hippocampal segment of the cingulate bundle.Clinical trial registration: MEC-2014-461The online version contains supplementary material available at 10.1007/s00234-022-02997-8. FGFR)—which are expressed during early embryonic development—are known to be responsible for the pattern of abnormal skull development in sCS  years . The effect of age was significant for both tracts (p < 0.025). A rise of 0.1 FOHR gives a rise of 0.15 × 10−3 mm2/s in RD for the corpus callosum genu  and a rise of 0.07 × 10−3 mm2/s for the hippocampal segment of the left cingulate bundle . The effect of gender and the effect of tract volume were not significant on the RD values in the two assessed tracts.By linear regression, we found no significant effect of having syndromic craniosynostosis on RD in the corpus callosum genu and the hippocampal segment of the left cingulate bundle Below is the link to the electronic supplementary material.


The results showed that EGC exhibited the inhibition of α-glucosidase and α-amylase in a non-competitive manner, showing a continuous upward trend as EGC’s concentration increased. There was a fluorescence quenching effect of EGC on α-glucosidase and α-amylase. Molecular docking confirmed that EGC can bind to amino acid residues in the enzyme through intermolecular hydrogen bonds and hydrophobic interactions, resulting in the changed chemical structure and spatial conformation of the enzymes. This decreased enzyme activity. This result suggested that EGC has the potential to inhibit two key glycoside hydrolases, and it would be beneficial to incorporate EGC into functional foods for diabetics.In this study, the inhibition and mechanism of epigallocatechin (EGC) on two key glycoside hydrolases  were explored from the molecular structure level. The chemical structure of EGC was characterized by X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and proton nuclear magnetic resonance spectroscopy. EGC’s inhibition on these enzymes was colorimetrically determined. The effects of EGC on the chemical structure and spatial configuration of the enzymes were explored  A sharpoglibose . Althougoglibose . Increasoglibose –9. Therevia catechin monomer EGC in vitro, and the mechanism of action affecting it is not clear. Therefore, it is a necessary aid in systematically exploring the inhibitory mechanism of polyphenols against glycoside hydrolases by studying the molecular structure changes and interaction mechanism between polyphenols and enzymes.Epigallocatechin (EGC), natural hydrolyzed catechin monomers from tea, is one of the most abundant, bioactive, and non-toxic polyphenols in tea . Modern via Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy. The molecular mechanism of EGC’s inhibition interacting with α-glucosidase and α-amylase was preliminarily elucidated. Completion of this study gives a better understanding of the possible effect and mechanism of tea polyphenol and catechin for II-DM’s possible prevention.In this work, EGC’s chemical structure and types of inhibitory effects on α-glucosidase and α-amylase were analyzed. EGC changes in the chemical and spatial structures of α-glucosidase and α-amylase were observed Saccharomyces cerevisiae), α-amylase , 4-Nitrophenyl α-D-galacto-pyran-oside (PNPG) (≥ 99% purity), and acarbose (≥ 98% purity) were purchased from Macklyn Bio-Technology Co., Ltd. . KBr  and starch soluble were purchased from Aladdin Industrial Co., Ltd. . DNS (Ghose method) was purchased from Beijing Solarbio Science & Technology Co., Ltd . All other chemical reagents used were of analytical grade.Epigallocatechin (≥ 95% purity) was obtained from Shennong Bio-Technology Co., Ltd. . α-glucosidase . Cu–Kα radiation from a sealed tube was used. An appropriate sample amount was applied to the groove on the slide, and data were collected in the 2θ range of 5°–90° with a step of 10°/min . FTIR data was obtained by collecting data in the range of 400–4,000 cm–1. KBr was the background during the measurement. The spectral resolution was set to 4 cm–1 at a rate of 16 nm/s . Tetramethylsilane (TMS) was the relative internal standard, and all chemical shifts were in ppm  solvent was added to dissolve the compound e in ppm .The assay of inhibition activity on α-glucosidase and α-amylase was performed according to the method of literature , 21 with2CO3  was added to terminate the reaction. The reaction solution’s absorbance was measured at 405 nm via the Synergy H4 Hybrid microplate reader . Triplicate samples for each test were created. The samples’ inhibitory activity was described by the inhibition rate and half-maximal inhibitory concentration (IC50) values. The α-glucosidase solution  and different concentrations of EGC solution  were cultured at 37°C for 10 min to determine α-glucosidase inhibition activity, and PNPG  was added. Solutions containing PNPG were incubated at 37°C for 30 min. Following this, Navia the Synergy H4 Hybrid microplate reader . Triplicate samples for each test were created. The samples’ inhibitory activity was described via the inhibition rate and IC50 values. The α-amylase solutions  and EGC solutions  of different concentrations were incubated at 37°C for 10 min to determine α-amylase inhibition activity. The 1% starch solution (250 μL) was added and incubated at 37°C for 10 min. The DNS reagent (200 μL) was added to the reaction solution after incubation. The mixed solution was incubated in water at 100°C for 15 min and cooled to 20°C. Finally, 200 μL distilled water diluted the reaction solution. The absorbance of the reaction solution was measured at 540 nm S] as the X-axis and the 1/v as the Y-axis. The inhibition mode was determined via the kinetic Cornish-Bowden (Eq. 2).Epigallocatechin’s inhibition kinetics against α-glucosidase and α-amylase were investigated according to the method of literature . The assCornish-Bowden equation:V (mol/L/min) is the enzymatic reaction rate. Vmax (mol/L/min) is the maximum reaction rate. Km (mg/ml) is the Michaelis constant. [C] (mg/ml) is the concentration of EGC. Kic is the competitive inhibition constant. Kiu is the non-competitive inhibition constant. [S] (mg/ml) is the concentrations of starch or PNPG solution.where -buffered saline (PBS) . The EGC solution was thoroughly mixed with the α-glucosidase and α-amylase solutions (V/V = 1/1) and was incubated at 37°C for 10 min before its fluorescence absorbance was measured. The excitation wavelength was 280 nm, and the scanning range was 290–500 nm, with a scanning interval of 5 nm  tested the fluorescence spectrum of the sample. Different concentrations of EGC solution, α-glucosidase solution (0.2 mol/L), and α-amylase solution (0.5 mg/ml) were prepared in phosphate of 5 nm .via the Autodock Vina software , FTIR, and ibration . The ranormation . Additioormation . The strtion  under the same conditions. This also indicated that the acarbose presented a greater potent inhibitory effect on α-glucosidase and α-amylase compared to EGC. Conversely, compared with the inhibitory effect of other tea extracts and catechins monomers on α-glucosidase and α-amylase activities. For example, the IC50 value of the ethanolic extract of partridge tea on α-amylase was 2.56 ± 0.35 mg/m  at the Y-axis of the enzymatic reaction rate line of EGC to α-glucosidase (Vmax) when decreased. At all EGC concentrations tested, the intercepts of the line on the X-axis (-1/Km) remained unchanged. The Michaelis constant (Km) remained constant regardless of EGC’s concentration. This suggests that EGC’s inhibition on the enzyme is independent of the substrate concentration, so EGC was a non-competitive inhibitor on α-glucosidase and α-amylase (The Lineweaver–Burk plots based on the kinetic Cornish-Bowden equation (Eq. 2) were established to define the inhibition mode. cosidase  and α-amcosidase  increase-amylase . Therefo–1 was mainly due to the -OH stretching vibration. However, after EGC’s introduction, the characteristic peaks of -OH in the two EGC-enzyme complexes showed a slight blue shift, indicating the existence of hydrogen bonds in the structure. The amide I  originated from the C=O stretching vibration, amide II  originated from N-H, C-N stretching vibration, and amide III  originated from the C-O-C stretching vibration , tyrosine (Tyr), and phenylalanine (Phe) can cause fluorescent properties in α-glucosidase and α-amylase . Trp andTo further investigate EGC’s interaction with α-glucosidase and α-amylase at the molecular level, molecular docking technology was used. in vitro experiments, making it difficult to reflect EGC’s mechanism on enzymes in vivo. Animal experiments should be performed to gain insight into the complex role of EGC in vivo in the future. Additionally, individual cases of acute hepato-toxicity from a large amount of green tea consumption have been reported in the literature, but the diverse composition and coingestants make it difficult to establish a clear causal relationship between green tea extract and reported cases of hepato-toxicity (In this study, spectroscopic techniques and molecular docking were used to investigate EGC’s interaction with two key glycoside hydrolases. The results showed that EGC had a non-competitive inhibitory effect on α-glucosidase and α-amylase, and the inhibition was mainly achieved by changing the enzyme’s chemical structure and spatial conformation through hydrogen bonding and hydrophobic interactions. The -OH in EGC could be combined with elements N, H, and O in the two enzymes through hydrogen bonds and combined with the non-polar groups in the enzyme through hydrophobic interactions to form more stable complexes. Therefore, the realization of hypoglycemic function may be related to the number of phenolic hydroxyl groups in EGC and the number and ratio of non-polar amino acids in α-glucosidase and α-amylase. These interactions do not simultaneously change during EGC’s metabolism, nor do they affect its inhibition of α-glucosidase and α-amylase. Thus, this research can provide a theoretical basis for EGC as a novel food additive or glycoside hydrolase inhibitor to develop functional foods or hypoglycemic drugs. However, in this research, EGC’s hypoglycemic activity was only explored through toxicity –50. Mosttoxicity , 51, butThe original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.XW, ZM, JX, and HY planned for the research. ZM, YF, and XW performed the experiments. All authors have contributed in the research work and the write-up of this article.