Low-dose lipopolysaccharide (LPS) exacerbated liver injury in CCl4-induced mice. Significant apoptosis, HNF1α upregulation, and nuclear factor kappa B (NF-κB) activation were observed in human-derived hepatocytes during ER stress. Knockdown of Rela, NF-κB p65, inhibited the HNF1α upregulation. Following CCl4 treatment ER stress, apoptosis, HNF1α expression and RelA phosphorylation were significantly increased in mice. HNF1α knockdown reduced activating transcription factor 4 (ATF4) expression, and aggravated ER stress as well as hepatocyte apoptosis in vivo and in vitro. The double fluorescent reporter gene assay confirmed that HNF1α regulated the transcription of ATF4 promoter. LPS aggravated CCl4-induced liver injury and reduced HNF1α, and ATF4 expression. Therefore, in combination, HNF1α and ER stress could be mutually regulated forming a feedback loop, which helps in protecting the injured liver by down-regulating hepatocyte apoptosis. Low-dose LPS aggravates hepatocyte apoptosis and promotes the SAE of liver injury by interfering with the feedback regulation of HNF1α and ER stress in acute liver injury.Hepatocyte nuclear factor alpha (HNF1α), endoplasmic reticulum (ER) stress, and hepatocyte apoptosis contribute to severe acute exacerbation (SAE) of liver injury. Here, we explore HNF1α–ER stress-hepatocyte apoptosis interaction in liver injury. LO2, HepG2 and SK-Hep1 cells were treated with thapsigargin (TG) or tunicamycin (TM) to induce ER stress. Carbon tetrachloride (CCl Despite advances in treatment strategies, patients with liver failure have high morbidity and mortality rates due to late diagnosis, severe complications in the advanced disease stage, and the limited availability of donor organs3. Therefore, understanding the mechanism of SAE in liver injury and its early symptoms could result in a timely diagnosis that could improve the patient prognosis. The SAE of liver diseases is usually attributed to various risk factors that include underlying medical conditions, bacterial or fungal infection, and alcohol consumption5. In addition, the aggravation of hepatocyte damage is correlated to the severity of liver injury6. Hepatocyte death controls the development and outcome of most liver diseases7. Therefore, improving hepatocyte resistance to apoptotic signals could be crucial for maintaining liver function and delaying the SAE of liver injury. Hepatocyte nuclear factor 1α (HNF1α) and endoplasmic reticulum (ER) stress contribute to a protective response in hepatocytes9, which might influence the SAE of liver injury.Severe acute exacerbation (SAE) of liver diseases is an early stage of the acute or chronic form of liver failure that often results from cirrhosis following viral infection, excessive alcohol consumption, or exposure to toxins10. ER stress involves the unfolded protein response (UPR) that is triggered by the aggregation of unfolded or misfolded proteins in the ER lumen11. Similarly, ER stress promotes an inflammatory response, such as the activation of nuclear factor kappa B (NF-κB)13. Activated transcription factor 6 (ATF6), inositol requiring enzyme 1 (IRE1), and protein kinase R-like kinase (PERK) are crucial for the regulation of UPR14. On ATF6 activation, X-box binding protein-1 (XBP1) is upregulated15. Subsequently, XBP1 mRNA is alternatively spliced by the active IRE1 that results in the translation of the spliced XBP1 form (XBPls), which then promotes ER stress-related gene expression16. PERK activation phosphorylates the eukaryotic translational initiation factor 2 alpha (eIF2α), which attenuates the overall protein translation and decreases the ER burden17. In addition, eIF2α selectively initiates the expression of activated transcription factor 4 (ATF4), which induces glucose-regulated protein 78 (GRP78) expression. Under physiological conditions, GRP78 inhibits the activation of IRE1, PERK, and ATF6 signaling pathways19. Previously, it has been reported that ER stress increases multidrug-resistance protein 2 (MRP2) expression by activating NF-κB signaling20. The upregulation of MRP2 controls acute liver injury through a negative feedback mechanism that reduces ER stress. In addition, the inhibition of eIF2α dephosphorylation reduced hepatocyte apoptosis by alleviating ER stress in acute liver injury21. In combination, the regulation of ER stress could alleviate the pathological progression of liver injury. Thapsigargin (TG) and tunicamycin (TM) are known ER stress inducers that interrupt the intracellular calcium balance and inhibit protein glycosylation in the ER cavity, respectively22.ER stress is induced by various injury factors, and it is correlated to liver disease pathology23. Following acute inflammation, HNF1α participates in the regulation and repair of acute liver inflammation by promoting the expression of C-reactive protein24. To date, the impact of HNF1α on ER stress in liver injury remains unclear. Therefore, the impact of HNF1α on ER stress and apoptosis in human-derived hepatocytes and mice will be investigated.HNF1, which is a transcription factor enriched in the liver, is crucial for glucose metabolism, detoxification, and plasma protein synthesisp < 0.05). In addition, it increased the expression of HNF1α, UPR signaling proteins , cleaved caspase-3, and RelA phosphorylation in LO2 cells at 12, 24, and 48 h . Treatment of LO2 cells with different TG concentrations  for 24 h revealed that the TG significantly reduced the viability of the LO2 cells in a dose-dependent manner . In addition, the treatment of LO2 cells with 1.0 μg/mL TM, another ER stress inducer, reduced LO2 viability  and significantly upregulated HNF1α, ATF4, and cleaved caspase-3 expression and RelA phosphorylation . Similarly, treatment of HepG2 cells with 1.0 μmol/L TG significantly decreased HepG2 viability  and increased HNF1α, ATF4, and cleaved caspase-3 expression and RelA phosphorylation . Similar results were observed in SK-Hep1 cells after treatment with 0.25 μmol/L TG .In vitro, the addition of 1.0 μmol/L TG significantly decreased the viability of LO2 cells at 24 and 48 h  and related protein expression was analyzed 48 h later. Transfection of HNF1A shRNA significantly reduced the expression of HNF1α protein . HNF1A shRNA reduced the viability of LO2 cells , which was more obvious after 36-h TG treatment. In addition, the expression of HNF1α, ATF4, and GRP78 proteins decreased with or without TG . However, it significantly increased the expression of ATF6, XBP1s, and cleaved caspase-3, and RelA phosphorylation.To determine the role of HNF1α in hepatocyte apoptosis and ER stress in vitro, LO2 cells were transfected with ATF4 promoter. Compared with the control group, HNF1α had an enhanced transcriptional regulation on either the wild-type ATF4 promoter or mutant ATF4 promoter (p < 0.01). Of interest, the transcriptional activity of HNF1α on wild-type ATF4 promoter was stronger than that on mutant ATF4 promoter . The knockdown of ATF4 significantly reduced LO2 viability , downregulated the expression of ATF4, GRP78 proteins, and increased the expression of cleaved caspase-3 in LO2 cells with or without TG . However, knockdown of ATF4 did not alter HNF1α expression and RelA phosphorylation.The downregulation of ATF4 protein was confirmed 48 h post-ATF6 expression in LO2 cells by ATF6 shRNA did not alter the expression of HNF1α protein . However, knockdown of RELA reduced the expression of HNF1α protein . Of note, knockdown of RELA reduced the viability of LO2 cells with or without TG treatment , and decreased TG-induced the expression of HNF1α, ATF4, and GRP78 proteins, but increased the expression of cleaved caspase-3 . In addition, bioinformatic analysis using JASPAR (http://jaspar.genereg.net/) predicted the presence of fifteen binding sites in HNF1A promoter for human RELA, based on the relative profile score of ≥ 80% (Table Knockdown of 4) had significantly increased levels of serum alanine aminotransferase (ALT) and total bilirubin (TBil) at different time points . The area of necrotic tissue in the liver increased significantly at 12, 24, and 48 h . Compared with the control group, HNF1α expression, protein levels in the UPR pathway , cleaved caspase-3 expression, and the phosphorylation of RelA significantly increased at 12, 24, and 48 h after CCl4 injection . The peak expression of HNF1α was observed at 48 h, and the peak expression of proteins involved in the ER stress pathway was observed 24 h after CCl4 injection. Similarly, the apoptotic index was significantly elevated at 12, 24, and 48 h after CCl4 injection, peaking at 24 h .Compared with the control group (olive oil), mice that were injected with 1.0 mL/kg carbon tetrachloride  was analyzed at 24 h post-CCl4 injection. Compared with the control group, serum ALT and TBil levels as well as the necrotic tissue area significantly increased in a dose-dependent manner . The protein expression of HNF1α, UPR signaling , cleaved caspase-3, and RelA phosphorylation increased in a dose-dependent manner in liver tissue . Similarly, the apoptotic index was significantly elevated after CCl4 injection . The expression of HNF1α and GRP78 was significantly upregulated in the injured liver tissue . Compared with the CCl4 group (control shRNA + CCl4), Hnf1a downregulation resulted in a significant reduction in the serum ALT level , increased the serum TBil level  and the necrotic liver tissue area  in Hnf1a shRNA + CCl4 group. In addition, Hnf1a knockdown before CCl4 injection decreased the expression of HNF1α, ATF4, and GRP78, but significantly increased the expression of caspase-12, cleaved caspase-3, and p-RelA , as well as the elevated apoptotic index .Mice were transfected with p > 0.05), the necrotic liver tissue area , the expression of ATF4, GRP78, caspase-12, cleaved caspase-3, and p-RelA , and the apoptotic index  in mice. However, 2.5 and 5.0 mg/kg LPS significantly increased the serum levels of ALT and TBil post-injection . In addition, the necrotic liver tissue area significantly increased in the 2.5 or 5.0 mg/kg LPS . Similarly, the same LPS doses increased the expression of ATF4, GRP78, caspase-12, cleaved caspase-3, and RelA phosphorylation , and the apoptotic index , but decreased the expression of HNF1α in the liver.The dose-dependent impact of the intraperitoneal administration of lipopolysaccharide  on hepatic injury was examined. The low dosage of LPS (0.1 and 0.5 mg/kg) did not significantly alter the serum levels of ALT and TBil , a significant increase in the serum TBil level , and the necrotic liver tissue area  in the LPS + CCl4 group. This combination significantly downregulated the expression of HNF1α, ATF4, and GRP78 increased the expression of caspase-12 and cleaved caspase-3, the phosphorylation of RelA , and the apoptotic index in model mice .Compared with the CCl4. In addition, knockdown of RelA significantly inhibited the upregulation of HNF1α in vitro. Taken together, these results suggest that ER stress enhanced HNF1α expression in liver injury and might be involved in activating NF-κB signaling. Furthermore, the downregulation of HNF1α in vitro showed that apoptosis was aggravated. Of interest, the proteins involved in ER stress signaling were differentially expressed: the expression of ATF4 and GRP78 was significantly downregulated while ATF6 and XBP1 expression was upregulated. Similar results were observed in vivo, as well as enhanced ER stress-related apoptosis. In addition, the double fluorescent reporter gene assay confirmed that HNF1α regulated the transcription of ATF4 promoter; knockdown of ATF4 decreased GRP78 expression and aggravated apoptosis in TG-treated LO2 cells. This interesting result implied potential crosstalk between HNF1α and ER stress through a feedback loop to alleviate hepatocyte apoptosis in liver injury.In this study, we examined the regulatory mechanism of HNF1α and ER stress and their impact on apoptosis in SAE of liver injury. Our results show that ER stress inducer TG or TM treatment induced apoptosis, and increased HNF1α expression in LO2, HepG2, and SK-Hep1 cells. Similarly, the upregulation of HNF1α expression, ER stress, and hepatocyte apoptosis were observed in the liver injury mouse model induced by CCl4-induced HNF1α, ATF4, and GRP78 expression and further aggravated ER stress-related hepatocyte apoptosis and liver injury. The ability of low-dosage LPS in inducing apoptosis and hence promoting the SAE of liver injury could be possibly attributed to the interference with the feedback loop between HNF1α and ER stress.Spontaneous peritonitis aggravated liver injury by LPS was partially simulated. The results demonstrated that LPS induced liver injury, hepatocyte apoptosis, and ER stress, but inhibited the expression of HNF1α in a dose-dependent manner. Treatment with low dosage LPS (0.1 mg/kg) selectively decreased CCl26. In this study, liver injury was induced by CCl4 or LPS. Liver injury was evaluated by the levels of serum ALT and TBil, and the necrotic area of the liver tissue. In the liver, CCl4 is converted into carbon trichloride that causes oxidative stress, inflammation, and ER stress28. LPS causes liver injury by activating inflammatory pathways or directly damaging hepatocytes29. In this study, CCl4 and LPS increased serum ALT and TBil levels, as well as the area of liver tissue necrosis, in a dose-dependent manner. This suggests the successful establishment of different severity degrees of liver injury models.Hepatocyte degeneration and necrosis are fundamental in the pathogenesis of various liver diseases. This can cause the leakage or release of ALT, and irregular bilirubin metabolism in hepatocytes30. Caspase plays a crucial role in apoptosis signal transduction31. Caspase-12 is related to ER-stress-mediated apoptosis32. Degradation of DNA into fragments of about 180–200 bp is a prominent morphological change marking apoptosis. In this study, hepatocyte apoptosis was comprehensively evaluated by analyzing the protein expression of caspase-12 and cleaved caspase-3. The apoptosis index was measured by TUNEL staining. Our results demonstrated that hepatocyte apoptosis significantly increased in CCl4-induced liver injury. Aggravation of liver injury by LPS increased hepatocyte apoptosis. This agreed with previous reports, where hepatocyte apoptosis was associated with the severity of liver injury33. Therefore, enhancing the resistance of hepatocytes towards apoptosis might be a feasible strategy to prevent liver injury.Hepatocyte apoptosis is closely related to liver injury34. Simultaneously, ER stress inhibits the overall protein synthesis through PERK/eIF2α signaling and upregulates the expression of molecular chaperones, such as GRP78 through ER stress-related transcription factors, such as ATF6, ATF4, and XBP121. In this study, the expression of ATF4, ATF6, XBP1s, GRP78, and caspase-12 were analyzed to monitor ER stress. Results demonstrated that ER stress was associated with hepatocyte apoptosis and liver injury that was induced by CCl4 or LPS. In addition, TG or TM induced ER stress and apoptosis in LO2, HepG2, and SK-Hep1 cells. Accumulating evidence suggests that targeted regulation of ER stress may change the progression of liver injury by altering hepatocyte apoptosis35.ER stress promotes intracellular homeostasis through UPR response, but excessive ER stress activates apoptosis signaling pathways36. In rats, downregulating HNF1α promoted the development of liver fibrosis and the overexpression of HNF1α significantly reduced liver fibrosis in rats37. Following acute inflammation, HNF1α regulates the repair of acute liver inflammation by promoting C-reactive protein expression39. In this study, it was demonstrated that HNF1α expression increased in TG-induced ER stress. Similarly, the upregulation of HNF1α expression in CCl4-induced liver injury in mice and HNF1α expression was positively correlated with the severity of liver injury. Immunohistochemistry indicated elevated HNF1α expression and ER stress. These results imply that ER stress can induce HNF1α expression, as well as hepatocyte apoptosis, in liver injury both in vivo and in vitro.HNF1α is a transcription regulator that is essential for normal liver function19. ATF4 plays an essential role in stress signaling, which includes ER stress, hypoxia, amino acid deletion, and oxidative stress40. In particular, ATF4 is involved in the transcriptional regulation of amino acid synthesis, protein folding and degradation, redox balance, autophagy, and apoptosis42. Mutations in ATF4 significantly altered glucose homeostasis and energy consumption43 .The activation of ATF6 upregulates the expression of ER stress-related proteins, which include XBP1 and GRP78, and enhances the ability of cells to eliminate misfolded proteins45. NF-κB signaling refers to a family of nuclear transcription factors, which includes RelA (NF-κB p65), RelB, c-Rel, NF-κB1/p50, and NF-κB2/p529. In this study, ATF4, ATF6 and RelA were knocked down to analyze the impact of ER stress on HNF1α. The knockdown of RelA reduced the expression of HNF1α in TG-treated LO2 cells; however, ATF4 and ATF6 knockdown did not downregulate HNF1α expression. These results suggest that the activation of NF-κB might be one of the ways by which ER stress mediates the upregulation of HNF1α.In ER stress, IRE1, PERK/eIF2α/ATF4, and ATF6 signaling are activated which promotes the downstream signaling cascades. On the other hand, HNF1α knockdown aggravated the CCl4-induced liver damage as indicated by increased TBil levels, apoptosis, and necrotic liver area. Of note, serum ALT levels did not increase, which might be attributed to the regulatory role of HNF1α on numerous liver-specific genes46. ALT is a metabolic enzyme; therefore, knocking down HNF1α might decrease liver ALT levels and decrease serum ALT These results suggest that HNF1α upregulation mitigates liver injury by reducing hepatocyte apoptosis. Further, knockdown of HNF1α differentially affects the expression of ER stress-related proteins. Specifically, it significantly downregulated the expression of ATF4 and GRP78, but increased ATF6, XBP1s, and caspase-12 expression in vivo and in vitro. In addition, this confirmed that HNF1α regulated the transcription of ATF4 promoter. These results suggest that HNF1α may reduce hepatocyte apoptosis through mitigating ER stress during acute phase liver injury.However, to the best of our knowledge, the regulatory role of HNF1α in ER stress and apoptosis in hepatocytes is unknown. In this study, the knockdown of HNF1α increased TG-induced apoptosis in vivo50. These results suggest that HNF1α may reduce ER stress-mediated hepatocyte apoptosis through upregulating the expression of ATF4 and GRP78.The knockdown of ATF4 reduced GRP78 levels and increased apoptosis in TG-treated LO2 cells. This suggested that ATF4-mediated GRP78 expression in ER stress is beneficial to alleviate apoptosis, which agrees with previous research52. Endotoxins, which are the main toxic effect component is LPS, are a cell wall component of Gram-negative bacteria. Under normal physiological conditions, a small amount of LPS produced by Gram-negative bacteria in the intestine can reach the liver via the portal circulation53. Most LPS is cleared by Kupffer cells without damaging hepatocytes. Compromised intestinal barrier function increases LPS leakage and this eventually leads to hepatocyte damage54. LPS causes liver inflammation, hepatocyte apoptosis, and aggravates liver cell injury56. Blood endotoxin levels are positively correlated with the degree of hepatocyte apoptosis58. In this study, the effect of spontaneous peritonitis aggravated liver injury by LPS injection was partially simulated. Our results demonstrated that LPS dose-dependently induced liver injury, hepatocyte apoptosis, and ER stress, but inhibited the expression of HNF1α during liver injury. In addition, low-dose LPS did not cause liver damage, and its preintervention could alleviate the liver injury59. However, low-dose LPS aggravates the liver injury, hepatocyte apoptosis and ER stress, inhibits the mutual regulation of HNF1α and ATF4, and reduces GRP78 expression in the CCl4-induced liver injury model. Therefore, LPS treatment can have different outcomes depending on the baseline status of the liver. Under physiological conditions, low-dose LPS is not enough to cause liver damage but it significantly increases the hepatocyte sensitivity towards apoptotic signaling mediated by ER stress after liver damage has occurred. Intestinal endotoxemia is common in severe liver injury60. However, the role of low-level LPS in promoting the SAE of the liver in future studies needs to be determined.Viral and bacterial infections are common risk factors that are associated with severe liver injury61. The results of this study agree with previous research and support the existence of multiple regulatory modes of HNF1α in liver injury. The expression level of HNF1α might not correlate with the degree of injury, but the effective upregulation of HNF1α in liver injury could be beneficial in controlling liver injury.It has been demonstrated that HNF1α expression was not correlated with chronic liver failureIn conclusion, following liver injury, hepatocyte HNF1α and ER stress are mutually regulated to form a feedback loop, which can help reduce the severity of liver injury by regulating hepatocyte apoptosis. Whereas ER stress upregulates the expression of HNF1α by activating NF-κB signaling and the upregulated HNF1α reduces ER stress through selective regulation of ATF4 increasing GRP78 expression. In addition, high-dose LPS can directly cause liver injury, hepatocyte apoptosis as well as ER stress and inhibit the expression of HNF1α. Low-dose LPS alone cannot cause liver damage; however, it reduces the tolerance of hepatocytes to apoptosis in liver injury and leads to the deterioration of liver injury through interfering with the feedback regulation of HNF1α and ER stress Fig. .Figure 962.Human-derived hepatocytes LO2, HepG2, and SK-Hep1 cells were purchased from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences  and were maintained in RPMI-1640 that was complemented with 10% fetal bovine serum and 1% antibiotics. To induce UPR, LO2, HepG2, or SK-Hep1 cells were treated for 12, 24, and 48 h with either dimethyl sulfoxide , phosphate buffer saline , TG (Sigma), or TM (Sigma). Alternatively, LO2 cells were treated with different concentrations of TG  and samples were analyzed after 24 h. To analyze the impact of HNF1α downregulation in vitro, LO2 cells were transfected with the target shRNA or control shRNA using a plasmid vector according to the manufacturer protocols and protein expression was analyzed 48 h later Table . Similar4–105 cells/mL) were seeded in the inner wells of a 96 well plate. Each experimental condition was seeded in quadruplicate and wells around the edges were filled with sterile PBS to maintain humidity in the 96-well plate. The following day, cells underwent different interventions. At the end of the experiment, MTS assay solution was added to each well and incubated at 37 °C for 4 h. Finally, the colorimetric signal was observed at 490 nm on a microplate reader . The activity of the experimental control group was set to100%.The percentage of living cells was assessed using an MTS assay, [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay  according to the manufacturer protocol. Briefly, 100 μL of LO2, HepG2, or SK-Hep1 cells  were obtained from the Animal Center of Zunyi Medical University . Mice were maintained under temperature and humidity-controlled conditions  with food and water available ad libitum. Following acclimatization, mice were randomly assigned to different experimental groups . The experimental protocol was reviewed and approved by the Animal Experimental Ethics Committee, Zunyi Medical University (ZMC-LS [2020] No. 2-321) according to the animal care and research guidelines4 dissolved in olive oil (CCl4: olive oil ratio = 1:4). Mice in the CCl4 model group received 1.0 mL/kg CCl4 injection for 12, 24, or 48 h whereas mice in the control group received the same volume of olive oil. Otherwise, mice in the CCl4 model group received either 0.5, 1.0, or 2.0 mL/kg CCl4 injections and the control group mice received the same dose of olive oil. Mice were not allowed to consume food or water 6 h before the injection. Alternatively, acute liver injury was induced by LPS; whereas mice in the model group were given an intraperitoneal injection of LPS  dissolved in PBS for 24 h. Mice in the solvent control group received the same volume of PBS and mice in the normal control group were untreated. Mice in the combination group (LPS and CCl4) received LPS (0.1 mg/kg) and CCl4 (1.0 mL/kg) for 24 h. Mice were not allowed to consume food or water 6 h before injection. The degree of liver injury was evaluated by detecting biochemical liver function indices, and histopathological changes in liver tissues. Further, liver injury was monitored at 12, 24, and 48 h. Mice were sacrificed by CO2 euthanasia. Mice were placed in a 4000-mL euthanasia box and the box chamber air was replaced with 100% CO2 at a rate of 30% of chamber volume/min64. Following the loss of consciousness, tissue and blood were harvested when blood circulation was maintained.Acute liver injury was induced by a single intraperitoneal injection of CClHnf1a shRNA , control shRNA group (n = 12), and Hnf1a shRNA group (n = 12). Then, the role of HNF1α in liver injury was explored. Mice were randomly assigned to a control , Hnf1a shRNA , CCl4 , Hnf1a shRNA + CCl4 group (n = 12). Mice in the control and Hnf1a shRNA groups received olive oil injections (same volume of CCl4 as previously); whereas mice in the control shRNA + CCl4 and Hnf1a shRNA + CCl4 groups received 1.0 mL/kg CCl4 injection for 36 h.Recombinant serotype 8 adeno-associated virus (rAAV8) that carried LO2, HepG2, SK-Hep1 cells, or liver tissues were homogenized in immunoprecipitation assay lysis buffer . Liver lysates (40 µg) were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene fluoride membranes . Following blocking, membranes were probed with mouse monoclonal antibodies against ATF6 , eIF2α , GAPDH , HNF1α , p-RelA , or RelA , rabbit monoclonal antibodies against ATF4 , cleaved caspase-3 , GRP78 , XBP1s , or p-eIF2α , or rabbit polyclonal antibody against caspase-12 , protein bands were detected with enhanced chemiluminescent and images were processed using Quantity One software . Densitometric analysis was used to detect the level of each protein relative to the control.Following fixation, liver tissues were dehydrated, paraffin-embedded and sliced at 4 μm thickness. Tissue samples were stained with hematoxylin–eosin (H&E) according to standard protocols and scanned on a sliced Panoramic scanner  and CaseViewer2.2 software .65.Otherwise, sections were immunostained using monoclonal antibodies against HNF1α  and GRP78 . Then, sections were visualized under a light microscope (OLYMPUS CX31). Each H&E section was independently scored by two experienced pathologists using the Histology Activity Index–Knodell score as detailed previously66. Positive staining was examined using a fluorescent microscope with 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. The apoptotic index was calculated from six randomly selected fields according to the following formula: apoptotic index = number of positive cells/total number of cells × 100%.Hepatocyte apoptosis was detected using a TUNEL kit . Deoxyribonucleotide terminal transferase (TdT) and deoxyribonucleotide derivative digoxigenin [(digoxigenin)-11-dUTP] were mixed at 2:28, added to the paraffin-embedded liver sections and incubated in a humidified chamber for 2 h according to standard protocols67.Serum ALT and TBil levels were measured using the rate method and diazonium, respectively according to the standard protocols ATF4 (https://genome.ucsc.edu/). The binding site of HNF1α to the ATF4 promoter sequence was predicted through JASPAR (http://jaspar.genereg.net/). Then, the ATF4 promoter sequence was synthesized. Next and cloning it on the firefly luciferase gene, constructing as an ATF4-promoter-wt plasmid. In addition, the HNF1α binding site on the ATF4 promoter sequence was mutated to serve as the ATF4-promoter-mt plasmid. The nucleotide sequence that corresponded to HNF1A was cloned into the pcDNA3.1 vector. The pcDNA3.1 empty vector was used as a control. HEK 293FT cells (ATCC) were cultured and seeded into 24-well plates and grown for 10–24 h (80% confluence). Cells were cotransfected with the reporter gene plasmid, and the transcription factor expression plasmid, and the Renilla luciferase plasmid . Following cell lysis and protein extraction, luciferase activity was detected by firefly luciferase and Renilla luciferase detection reagents  to determine the relative light unit (RLU). The RLU value (Fluc) obtained by the firefly luciferase assay was divided by the RLU (Rluc) value obtained by the Renilla luciferase assay as an internal reference. According to the obtained ratio, the activation degree of the target reporter gene between the different groups was compared68.The search engine of the University of California, Santa Cruz Genomics Institute was used to find the promoter sequence of p-value < 0.05 was statistically significant.One sample Kolmogorov-Smirnow test was used to test whether the continuous variables satisfied a normal distribution. Quantitative data that satisfied a normal distribution were shown as mean ± standard deviation  according to the animal care and research guidelines. All procedures were performed following the relevant guidelines and regulations.Supplementary Information 1.Supplementary Information 2.


The frequency of remaining nystagmus during walking was further modulated in a manner that depended on the specific phase of the gait cycle (p = 0.015). These attenuating effects on nystagmus intensity during walking suggest that ocular-motor control disturbances are selectively suppressed during locomotion in DBN. This suppression is potentially mediated by locomotor efference copies that have been shown to selectively govern gaze stabilization during stereotyped locomotion in animal models.Downbeat nystagmus (DBN) is a common form of acquired fixation nystagmus related to vestibulo-cerebellar impairments and associated with impaired vision and postural imbalance. DBN intensity becomes modulated by various factors such as gaze direction, head position, daytime, and resting conditions. Further evidence suggests that locomotion attenuates postural symptoms in DBN. Here, we examined whether walking might analogously influence ocular-motor deficits in DBN. Gaze stabilization mechanisms and nystagmus frequency were examined in 10 patients with DBN and 10 age-matched healthy controls with visual fixation during standing vs. walking on a motorized treadmill. Despite their central ocular-motor deficits, linear and angular gaze stabilization in the vertical plane were functional during walking in DBN patients and comparable to controls. Notably, nystagmus frequency in patients was considerably reduced during walking compared to standing ( Downbeat nystagmus (DBN), a frequent form of acquired fixation nystagmus, is characterized by a spontaneous upward drift of the eyes compensated by fast resetting saccades directed downwards. Patients suffer from visual disturbance due to vertical oscillopsia, to-and-fro vertigo, postural ataxia, and an increased risk of falling , 24, 29.The intensity of DBN-related symptoms is known to be modulated by a variety of factors. Accordingly, nystagmus intensity depends on the direction of gaze, the orientation of the head relative to gravity, and illumination , 18, 25.It is unknown whether and how locomotion might also affect ocular-motor symptoms in DBN. Symptoms could either improve in a manner analogous to postural stability or even be further aggravated due to the hypothesized central VOR deficits in DBN. Since visual disturbance during walking presents a major risk factor for falling , the preN = 2) or episodic ataxia type 2 (N = 1). Ten healthy, age-matched subjects  without any auditory, vestibular, neurologic, cardio-vascular or orthopedic disorders served as controls. All participants had normal or corrected-to-normal vision. Ten patients with DBN  participated in the study . DBN patients were secured with a safety belt. Subjects were asked to visually fixate on the red point at all times and to minimize eye blinks during recordings. Recordings during standing and walking were performed in randomized order to control for potential habituation effects.Horizontal and vertical eye movements during standing and walking were captured at a sampling rate of 220 Hz using a monocular head-mounted video-oculography system  as described previously . The monWalking performance of each participant was estimated by calculating the following gait cycle parameters: gait velocity, the mean and the coefficient of variation (CV) of stride length, stride time, base of support as well as the percentage of double support and swing phases with respect to the total gait cycle duration.Eye movement recordings were initially screened for potential motion artifacts . Eye blivs head velocity; see [For the VOR gain calculation, eye velocity was low-pass filtered using a fourth-order Butterworth filter with a cutoff frequency of 10 Hz to remove fast phase eye movements, such as during nystagmus. Angular head velocity and linear head acceleration were filtered analogously. For each locomotion trial, the eye velocity and head angular/linear velocity traces were segmented for successive gait cycles and resampled to the average gait cycle duration of the recording. To quantify VOR responses during walking, the angular and linear VOR gain  were caDBN intensity is usually quantified as the mean velocity of the slow upward drift of the eyes . Since during locomotion the DBN-related slow phase eye motions are superimposed by slow, VOR-driven eye movements, it is impossible to determine the exact slow phase velocity of DBN during walking. Thus, to quantify the intensity of DBN symptoms during locomotion, we focused on the mean frequency of DBN quick phases  . As a fit-test. Activity-dependent differences of nystagmus frequency in patients were analyzed using Student's paired two-sample t-test. Statistical analysis was performed using IBM SPSS . Results were considered significant at p < 0.05.Descriptive statistics are reported as mean ± standard deviation (SD). Differences in gait performance, VOR gains, and nystagmus frequency were analyzed using Student's independent two-sample p = 0.041) and increased spatiotemporal gait variability . Other spatiotemporal gait characteristics were comparable between patients and controls .Patients and controls showed comparable self-chosen speeds (0.8 ± 0.24 m/s vs. 0.84 ± 0.18 m/s) and step frequencies (107.4 ± 18.8 steps/min vs. 99.0 ± 14.1 steps/min) while walking on the treadmill. Patients exhibited walking alterations typically observed in (sensory)-ataxic gait disorders, with an increased base of support   Fig. B. In heap = 0.015). This phase-dependent modulation of DBN frequency resulted in frequency peaks across the gait cycle that closely matched the timing of vertical aVOR gain peaks  compared to the double support phase (DSP) of the gait cycle Fig. A–C (p = see Fig. C, D.Fig.Here we studied the impact of locomotion on general gaze stabilizing mechanisms and nystagmus characteristics in patients with DBN. During walking at preferred pace and velocity, patients exhibited a broad-based, staggering stepping pattern compatible with a sensory and/or cerebellar gait disorder . In contSpontaneous, downwards directed fixation nystagmus is the primary source for visual disturbance and oscillopsia in DBN. Nystagmus intensity is known to be influenced by static changes in head position relative to gravity , 26. So Alternatively, walking-induced attenuation of DBN symptoms could directly arise from locomotor-activity itself. In a previous study we could demonstrate that postural imbalance in DBN becomes attenuated during locomotion in particular during fast stereotyped walking . AnalogoA detailed investigation of the timing of remaining nystagmus occurrence during walking further demonstrated that the degree of DBN attenuation during locomotion depends on the specific gait cycle phase. These phasic changes in nystagmus intensity across the gait cycle parallel the phasic modulation of the VOR Fig. C and vesIn conclusion, the present findings demonstrate that while DBN has profound effects on patients walking ability, it does not impair general gaze stabilizing mechanisms during locomotion. However, walking in turn has an attenuating effect on nystagmus intensity in DBN. This observation parallels previous reports of a locomotion-induced mitigation of sensory ocular-motor and balance deficits during walking. We propose that a common mechanism based on a predictive feed-forward regulation of posture and gaze might explain this attenuation of peripheral and central sensorimotor balance and ocular-motor deficits. Subsequent studies should examine whether, in analogy to postural stability, walking-related attenuation of DBN becomes even more pronounced at non-preferred, fast walking modes. Besides, further investigations are required to explore the functional consequences of the present findings in particular related to the dynamic visual acuity and risk of falling in patients with DBN.


The effects of air oxidation and ammonia solution heat treatment on the pore structure and surface chemistry of the carbon spheres were studied for catalytic oxidation of CH3SH. The structure property and surface chemistry of the obtained carbon spheres were characterized by N2 adsorption–desorption, FTIR, scanning electron microscopy, XRD, elemental analysis, X-ray photoelectron spectroscopy and Boehm titration, and then thermal analysis and gas chromatography-mass spectrometry were applied to investigate the catalytic oxidation product. Results show that the as-prepared microporous carbon spheres through direct ammonia treatment have a high surface area value of 1710 m2 g−1 and a total pore volume of 0.83 cm3 g−1. Moreover, the preoxidation-assisted nitrogen enrichment strategy not only increases the surface area and total pore volume of the carbon spheres, but also introduces more active nitrogen species such as pyridinic nitrogen and quaternary nitrogen, leading to the highest nitrogen content of 7.13 wt% and the highest CH3SH capacity of 622.8 mg g−1 due to the pyridinic nitrogen and quaternary nitrogen as function of catalysts. In addition, water and oxygen have a beneficial effect on CH3SH oxidation over the nitrogen modified carbon spheres, and the basic oxidation product is CH3SSCH3 that can be further oxidized into CH3SO2SCH3 according to DTG and GC/MS analysis. The great recycling stability after ten cycles with a reserved CH3SH capacity of 97% demonstrates that the porous carbon spheres obtained by preoxidation-assisted enriched nitrogen strategy are promising for catalytic oxidation of CH3SH.Porous carbon spheres with high surface area and microporous structure were synthesized from alkyl phenols and formaldehyde  via suspension polymerization and steam activation.Porous carbon spheres with high surface area and microporous structure were synthesized from alkyl phenols and formaldehyde  More research activities have been developed to remove CH3SH efficiently. For instance, the mature commercial technology is hydrodesulfurization that has been widely used in the traditional refineries with good removal results, but it has serious drawbacks such as high hydrogen consumption and energy requirements.8,9 Therefore, extensive research has been carried out to propose alternative technologies focused on convenient conditions without hydrogen.It is well known that methyl mercaptan  is beneficial for the adsorption of S compounds through either polar or acid–base interactions, and pyridinic N and pyrrolic N improve the effects of S interaction with CNT and the capacitance, respectively. Liu23et al. prepared nitrogen-doped coconut shell activated carbon catalysts for the removal of CH3SH by impregnating urea or melamine with subsequent heat treatment. The results showed that the CH3SH removal capacity increased with the nitrogen content, especially pyridinic nitrogen and quaternary nitrogen. Moreover, the exhausted nitrogen-rich activated carbon can be easily regenerated with a reserved CH3SH capacity of 88.33% after four cycles.These alternative technologies are related to adsorption, decomposition, catalytic adsorption/oxidation, and photocatalytic oxidation.24,25 leading to the widespread technological applications in chemical protective clothing, blood purification, catalyst support and adsorption processes.26–28 Various polymers such as petroleum pitch, sulfonated poly(styrene-divinylbenzene) and phenolic resins have been mainly adopted to prepare porous carbon spheres by carbonization and activation. Romero-Anaya29et al. obtained the carbon spheres from petroleum pitch by CO2 or steam activation with high microporosity and low surface oxygen groups content that show a positive influence on the adsorption application of non-polar volatile organic compounds (VOCs) at low concentrations, and thus leading to toluene adsorption capacities as high as 46 g toluene/100 g carbon spheres. Wickramaratne30et al. synthesised phenolic resin spheres with diameters from 200 to 420 nm by the one-pot modified Stöber method, and the high CO2 adsorption capacities reached 4.55 and 8.05 mmol g−1 on these AC spheres at 1 bar and two temperatures, 25 and 0 °C, respectively. Sun31et al. prepared activated carbon beads with desirable spherical forms from phenolic resins using a novel hydrothermal process coupled with a range of post-preparation treatments, and found that the well-developed porous system is important to obtain a higher CO2 capture capacity at both ambient and elevated pressures. Sulfur-doped microporous carbon spheres were synthesized from poly(styrene-divinylbenzene) through the sulfonation, carbonization, and KOH activation for CO2 adsorption, and the CO2 uptake presents 4.21 mmol g−1 and 2.54 mmol g−1 at both 25 °C and 50 °C under ambient pressure, respectively. The high capacity has been attributed to abundant ultramicropores and high contents of oxidized sulfur functional groups.32Porous carbon spheres as one kind of the carbon-based materials show many charming advantages over powdered and granular activated carbon, such as suitable ball size, smoother surface, better fluidity, high mechanical strength, and high adsorption capacity,33,34 Resin based carbon spheres with high surface area and different porous structure were prepared by adjusting two organic additives such as ethylene glycol and polyethylene glycol for dibenzothiophene adsorption in our previous works,24 and the carbon spheres were proposed as promising adsorbents for deep desulfurization of liquid hydrocarbons. In the present work, we firstly prepared resin spheres from mixture of isopropylphenol and formaldehyde by suspension polymerization, and then obtained carbon spheres with high surface area and microporous structure from these resin spheres via steam activation. Furthermore, the effects of air oxidation and ammonia solution heat treatment on the pore structure and surface chemistry of carbon spheres were investigated for efficient catalytic oxidation of CH3SH. Moreover, N2 adsorption–desorption, Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometer (XRD), scanning electron microscope (SEM), elemental analysis, X-ray photoelectron spectroscopy (XPS) and Boehm titration were applied to characterize samples, and then thermal analysis (DTG) and gas chromatography-mass spectrometry (GC/MS) were carried out to investigate the created oxidation product in the process of catalytic oxidation. In addition, the stability and regeneration activity were evaluated by cycling tests. Finally, the possible mechanism of adsorption/oxidation of CH3SH over modified carbon spheres was discussed according to the experiment and characterization results.What's more, phenolic resins are considered as promising polymeric precursors to prepare high surface area and desired pore structure of carbon spheres for their low level of inorganic impurities and negligible ash content.2.2.1Isopropylphenol (99%), formaldehyde (37 wt% in water), triethanolamine, poly, hexamethylene tetraamine and ammonia absolute (25 wt% in water) were purchased from Tianjin Tianli Chemical Corp. All solvents and other chemicals were AR grade and utilized without further purification.2.224,25 Firstly, resin spheres were prepared from isopropylphenol and formaldehyde by suspension polymerization. In a typical synthesis, the polymerization reaction was carried out in a 1000 mL round-bottomed four-neck reaction vessel with a Teflon stirrer, a reflux condenser, and a thermocouple. The isopropylphenol was polymerized with an aqueous solution  of formaldehyde in a molar ratio of 1 : 5 in the presence of triethylamine , followed by dispersing the resulting mixture into 300 mL of deionized water via stirring at 600 rpm. Then, poly  was added to the above mixture at 96 °C by stirring at 600 rpm for 30 min. Afterwards, hexamethylene tetraamine  was added to reaction vessel and polymerization reaction was carried out at the same temperature and fixed agitation rate (600 rpm) for 4 h. After cooling to room temperature, resin spheres were obtained via solid–liquid separation. And then they were further carbonized at 850 °C for 45 min under nitrogen atmosphere and activated at the same temperature with steam for 1.5 h. The obtained carbon spheres will be denoted as ACS. Furthermore, the prepared ACS was oxidized by dry air with a flow rate of 60 mL min−1 for 2 h at 350 °C, and denoted as ACSO.Series of millimeter porous carbon spheres were prepared from alkyl phenol and formaldehyde according to the recipe reported previously.−1, the temperature was increased with a ramp rate of 3 °C min−1. When the temperature attained 800 °C, the ammonia solution was introduced at a flow rate of 10 mL min−1 for 1 h, and then cooled down to room temperature. The obtained samples were named as ACSN and ACSON, respectively. In addition, the commercialized activated carbon spheres (CACS) was purchased from Taiyuan New Process Technology Corp  for comparison.The ACS and ACSO were separately further functionalized by heat treatment with ammonia solution. In a typical run, the sample was placed in a vertical cylindrical furnace under nitrogen flow rate of 60 mL min2.326,27 The textural parameters of all the studied ACSs were carried out by N2 adsorption/desorption at −196 °C using a ASAP 2020 instrument. The samples were separately degassed at 250 °C in a vacuum environment for a period of at least 4 h prior to measurements. Experimental adsorption data at the relative pressure (P/P0) less than 0.3 was used to calculate surface area values using the standard Brunauer, Emmett, and Teller (BET) equation. The pore size distribution (PSD) was determined by applying density functional theory (DFT) method based on nitrogen adsorption data. FT-IR spectra of activated carbon samples were obtained utilizing a PerkinElmer Spectrum 100 FT-IR spectrometer. A disk made of pure KBr was used as a reference sample for background measurements. The carbon spheres–KBr mixtures at a ratio of 1 : 300 were ground in an agate mortar and then pressed under vacuum conditions in a hydraulic press. Before the spectrum of a sample was recorded, the background line obtained was arbitrarily and automatically subtracted. The spectra were recorded from 4000 to 400 cm−1 at a scan rate of 0.2 cm s−1, and the number of interferograms with a nominal resolution of 4 cm−1 was fixed at 100. The spectra were recorded in the range of 400–4000 cm−1 at a resolution of 4 cm−1. The surface morphology of the samples was observed on a Hitachi S-4800 field emission scanning electron microscope (SEM) operating at 3 kV. The crystallinity information on the surface of samples was determined by a X-ray diffraction-meter  using a Cu Kα radiation source . Surface chemistry was evaluated using the Boehm titration method. For the purpose of this research, 1 g of carbon sample was placed in 50 mL of 0.05 N sodium hydroxide or hydrochloric acid. The vials are sealed and shaken for 24 h and then 10 mL of each filtrate was pipetted and the excess of base or acid was titrated with HCl or NaOH. The numbers of acidic sites were calculated under the assumption that NaOH neutralizes acidic groups and HCl neutralizes basic groups. The chemical composition of the activated carbon samples was measured by an Elementar Vario Macro EL Cube microanalyzer. XPS was measured on a PHI5300 X-ray photoelectron spectrometer. Monochromatic Al Kα source (1486.6 eV) was used at a power of 210 W. The resolution of the instrument is 0.55 and 0.70 eV for Ag 3d and Au 4f peaks, respectively. Survey scans were collected for binding energy ranging from 1100 eV to 0 with an analyzer pass energy of 160 eV with a step of 0.6 eV for a dwell time of 150 ms. For the high-resolution spectra, the pass-energy was 20 eV with a step of 0.1 eV and dwell time of 200 ms. 0.4 g carbon powder was placed in 20 mL of water and equilibrated during the night. Then the pH of the suspension was measured by a pH meter  for comparison. For exhausted samples, the pH is denoted as pHE. 1 mL methanol and 0.5 mL carbon powder were mixed in a flask and warmed at 60 °C for 1 h. Then, the suspension liquid was analyzed by gas chromatography-mass spectrometry (GC/MS) experiments using a PerkinElmer gas chromatograph/mass spectrometer.Materials characterizations were carried out reference to our previous publications.2.43SH removal under wet conditions.18 Adsorbent samples were ground and packed into a glass column, and prehumidified with moist air (relative humidity 80% at 25 °C) for 1 h. The amount of water adsorbed was estimated from the increase in the sample weight. Moist air (relative humidity 80% at 25 °C) containing 0.3% (3000 ppm) CH3SH was then passed through the column of adsorbent at 0.5 L min−1. The breakthrough of CH3SH was monitored using a Micromax monitoring system with an electrochemical sensor calibrated with CH3SH. The test was stopped at the breakthrough concentration of 20 ppm. After the adsorption test, the exhausted samples were designated with the letter E. To study the roles of water and oxygen in the removal of CH3SH, contrast experiments were also tested using dry air and dry nitrogen as carrier gases for ACS and ACSON, and denoted as ACS-A, ACS-N, ACSON-A, and ACSON-N, where A and N represent dry air and dry nitrogen, respectively. The adsorption capacities of each sorbent in terms of mg of CH3SH per gram of carbon were calculated by integration of the area above the breakthrough curves, and from the CH3SH concentration in the inlet gas, flow rate, breakthrough time, and mass of sorbent. In addition, the process of regeneration of exhausted samples was carried out as follows, 6 mL exhausted sample was mixed with 300 mL of ethanol for 5 h at 50 °C and then filtered. Ten cycles were performed, and then the carbon sample was heated at 500 °C for 1 h under a nitrogen atmosphere. Finally denoted as ACSON-10 ,25,26 Furthermore, all of the carbon spheres show pore size distribution around 4 nm in Fig. S1(b),2 adsorption/desorption isotherms.The N2 and NH under high temperature (800 °C).35,36 Moreover, partial gasification reaction between the carbon spheres and the generated free radicals from the above thermal decomposition also makes a contribution to the porous development. Therefore, the highest surface area, total pore volume and micropore volume can attain 1710 m2 g−1, 0.83 cm3 g−1 and 0.74 cm3 g−1 for the direct ammonia treated sample ACSN, respectively. It can also been observed that the ammonia thermal treatment shows an evident contribution to the increase of micropore volume compared to the virgin carbon spheres, which can be ascribed to the above partial gasification reaction between the carbon spheres and the generated free radicals that exerts an active influence on the major microporosity domain by producing new micropore sites.37,38It is well known that the porous structure parameters such as BET surface area, total pore volume and micropore volume are favourable implements to study the pore characteristics, and the corresponding results are listed in 2, and eventually some micropores or/and mesopores became expanded or collapsed in different extent, resulting in the decrease of micropore volume.39,40 However, when the preoxidized carbon spheres are subjected to the ammonia thermal treatment under 800 °C, the oxygen surface groups firstly give rise to decomposition that creates the vacant sites in the pores, which provide more opportunities to increase reaction probability with created free radicals under the simultaneous ammonia treatment at 800 °C. It has been also reported that gasification with ammonia has a profitable to recover its porosity blocked by preoxidization, and increase the porous structure parameters over that of the oxidized sample.41,42 Moreover, the carbon spheres (ACSON) obtained by the preoxidation-assisted ammonia thermal treatment show bigger BET surface, total pore volume and microporous volume than those of the direct oxidized carbon spheres.It can be seen that the BET surface are, total pore volume and micropore volume decrease in some degree after thermal air oxidization compared to the virgin carbon spheres. The results can be probably attributed to the partial collapse of some pore walls or the recession of some pore channels by the reaction related to O2 g−1, 0.64 cm3 g−1 and 0.46 cm3 g−1 after the oxidation, respectively. On the contrary, the BET surface area, total pore volume and microporous volume considerably become bigger after subsequent ammonia thermal treatment than those of the oxidized carbon spheres, and present 1592 m2 g−1, 0.71 cm3 g−1 and 0.61 cm3 g−1, respectively. Moreover, the carbon spheres have a higher micropore ratio (≥70%) except for CACS. In addition, the average pore size of all carbon spheres is less than 2 nm, which is in accordance with the characteristic of N2 adsorption/desorption. Therefore, the performance of these carbon spheres in catalytic oxidation of CH3SH may be primarily ascribed to the different surface chemical properties.It can be observed from the data in 3.2−1 appeared in the FTIR spectrum of virgin carbon spheres, which were related to O–H stretching vibration, –CH2– stretching vibration, C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O groups, CC groups, aromatic ring, C–O stretching vibrations and C–H groups, respectively.37,43 It can be found that a sharp peak is located at 1716 cm−1 for oxidized carbon spheres ACSO compared to the ACS and other samples, which can be attributed to the carboxylic structure induced by thermal air oxidization.25 These oxygen surface groups can have a contribution to introduce the nitrogen containing functional groups into the carbon spheres during the subsequent ammonia treatment.The carbon spheres were subjected to the FTIR measurements in order to investigate the nature of the functional groups, and the FTIR analysis results of virgin carbon spheres and modified samples are shown in −1, which can be corresponding to CN stretching vibrations, C–N stretching vibrations and N–H stretching vibrations for all ammonia modified carbon spheres, respectively.44,45 Furthermore, another two obvious peaks at 881 and 753 cm−1 related to amine or amides functionalities can be found only in the in the spectra of ACSN and ACSON, which can be due to the formation of hydrogen bonds between the free radicals (such as NH2 and NH) and surface groups.46,47 These bands indicate that nitrogen containing functional groups can be effectively incorporated on the surface of carbon spheres by facile ammonia thermal treatment. It is noticeable that intensities of these bands related to the nitrogen containing functional groups are increased by the preoxidization-assisted ammonia thermal treatment compared to the direct ammonia modification. For instance, oxygen chemisorption, carboxylic species and cyclic anhydrides generated from preoxidization appear on the surface of carbon spheres, when the oxidized carbon spheres are subjected to the ammonia thermal treatment, the generated free radicals such as NH2 and NH can easily react with these oxygen functional groups to form amine or amide structures by dehydration reaction in the process of ammonia treatment, making a great contribution to the increase of bands intensity corresponding to these nitrogen containing functional groups.37 In addition, it can be seen that the peak related to carboxylic structure at 1716 cm−1 considerably diminished after ammonia thermal treatment, which can be due to the reaction that the generated free radicals give rise to attack to the surface oxides and active sites on the surface of carbon spheres to form nitrogen containing functional groups. And or decomposition of oxygen containing surface groups during higher temperature.35 Therefore, the band intensities related to nitrogen containing species exhibited an evident increase for oxidized carbon spheres following ammonia treatment compared to the direct ammonia treated carbon spheres, indicating that preoxidation-assisted ammonia treatment under high temperature significantly incorporates nitrogen containing species onto the surface of carbon spheres, which will be favorable for enhancement of CH3SH adsorption.It can be observed that some changes have been created after ammonia thermal treatment in the spectra of ACSN and ACSON. Compared to the ACS and CACS, the new peaks can be seen at 1458, 1225 and 1104 cm3.32 departure, the pores expansion or collapse, and the formation of new oxygen containing groups.25,40 These consequences are consistent with the FTIR analysis and N2 adsorption. Furthermore, it can be observed that the surface of carbon spheres obtained by direct ammonia thermal treatment shows much more rough with stable sphericity than that of oxidized carbon spheres in 3 molecules are smaller and easier to diffuse into the pore channels, and the created free radicals  gradually reacted with the carbon surface or pore walls under high temperature, resulting in the surface etching and the expandation of pore structure or formation of new microporosity.37 As a result, the higher BET surface area and pore volume were shown for ACSN, and also generated obvious nitrogen containing groups in the FTIR spectrum. It is noticeable that the exterior surface of ACSON obtained by preoxidation-assistant ammonia thermal treatment shows considerable roughness and much big pores in 37,39 in which the surface oxygen functional groups to create vacant sites through thermal decomposition, and free radicals  react with these vacant sites or surface oxygen functional groups to develop pore structure, leading to the increase of BET surface area and pore volume and simultaneously the introduction of nitrogen containing functional groups in the ACSON. On the contrary, CACS from commercial activated carbon is very different, the exterior surface shows some big cracks with roughness in The surface morphology of the obtained activated carbon spheres were measured through SEM, and the SEM images are shown in 3.4θ = 26.58 °and 44.21° for all the prepared carbon spheres from the above XRD patterns, and no other obvious peaks appear in the spectra, which can be ascribe to the characteristics of porous carbon materials.48,49 In our work, the suspension polymerization is used to prepare the resin spheres precursor, afterwards, the resin spheres were carbonized and activated under high temperature with steam as a function of active agent that can react with the carbon. The primary reaction equations in the activation process are as follows to create the main carbon containing product, leading to the formation of the carbon network structure and the development of pore channel.The as-obtained porous carbon spheres were subjected to the XRD measurement to investigate the crystal structure information, and the corresponding XRD analysis spectra were carefully recorded and shown in 2O reaction.50 In our study, the primary component is carbon with small other composition, although the virgin carbon spheres were modified by different procedures. Therefore, the characteristics of XRD analysis spectra is highly consistent with reported porous carbon materials.Furthermore, it has been proposed that the etching action of steam is generally completed accompanied by the decomposition of volatile components and the formation of new pores in the process of continuous C–H3.53SH removal of porous carbon materials, and the Boehm titration method is widely used to evaluated the surface chemistry of activated carbon. Therefore, the chemical analysis of all the porous carbon spheres was completed by means of the Boehm titration, and the corresponding surface pH values and amounts of acidic and basic groups related to the surface of the obtained porous carbon spheres are summarized in −1 among of all samples. On the contrary, the amount of basic groups nearly decreases to zero (0.02 mmol g−1) after thermal air oxidation compared to the virgin carbon spheres ACS. Our previous study has proposed that the reaction between thermal air and the surface of activated carbon spheres was created in the process of air oxidation under 400 °C by means of the surface and pore channels related to the diffusion and etching action, finally resulting in the increase of oxygen containing groups, especially the surface oxygen groups of carbonyl group with strong acidity.25 As a result, the larger the number of acidic groups the smaller the pH of the carbon spheres surface. However, when the carbon spheres were subjected to the ammonia heat treatment, no matter direct treatment or preoxidation-assistant modification made a significant contribution to the amount of basic groups for ACSN and ACSON, which can be attributed to the incorporation of basic nitrogen-containing groups by means of decomposition of oxygen containing surface groups and the reaction between the generated free radicals and the surface oxides or active sites.37,43 As expected, the pH values and number of basic groups considerably increase compared with ACS and ACSO, and such basic nitrogen-containing on the surface of porous carbon spheres could accounted for the dissociation of CH3SH or oxidation of CH3S− ions to disulfides. Moreover, these basic groups may take advantage of weak acid–base interactions to attract CH3SH. In addition, the amount of acidic groups is almost equivalent to the number of basic groups for CASC with the pH of 7.09.It has been proposed that the surface chemistry is an important role in the CH3.62 departure, pores expansion or collapse, and the formation of new oxygen containing groups, finally leading to the increase of oxygen containing groups, especially the oxygen content of the oxidized carbon spheres.25,39 Furthermore, the virgin and oxidized carbon spheres were separately treated by ammonia thermal treatment, and the reactions between the created free radicals during ammonia decomposition and the surface of carbon spheres considerably introduced nitrogen into the carbon structure. For example, the nitrogen content increased to 5.21 wt% and 7.13 wt% for ACSN and ACSON compared with the ACS (0 wt%), respectively. Moreover, it can be observed that the ACSON shows higher nitrogen content than that of ACSN, which may be ascribe to the method of incorporating nitrogen at high temperatures. The nitrogen content differences between the ACSN and ACSON samples suggested that the oxygen functional groups that existed on the surface of carbon spheres played an important role in the process of ammonia treatment by controlling the amount of nitrogen incorporation to the carbon surface, and preoxidation treatment obviously improved the degree of nitrogen incorporation in the process of ammonia treatment.In order to investigate the element contents and the species of the surface functional groups, the prepared carbon spheres were subjected to the Elemental and XPS analysis. As can be seen in 41,51In the process of subsequent ammonia thermal treatment, these oxygen containing groups can give rise to thermal decomposition, resulting in the formation of active sites that react with ammonia molecules or free radicals to introduce the nitrogen containing functionalities. For instance, the ammonia molecules react with carboxylic acid sites on the surface of carbon spheres, and ammonium salts are firstly formed. Furthermore, the created ammonium salts transform into amides and/or nitriles groups by dehydration reactions. On the other hand, the reaction of ammonia molecules by the substitution of OH groups can easily create amines on the carbon spheres surface. In addition, the surface oxygen containing groups like ether can be replaced by –NH– in the process of ammonia thermal treatment under high temperature, and imine and finally the pyridine functional groups incorporate on the carbon surface by dehydrogenation reactions. The related reaction equations are as follows.52,53 Furthermore, 23 It can be also found that the relative amounts of N-6 and N-Q in the ACSN obtained by direct ammonia treatment show a little higher than those of ACSON prepared by preoxidation-assistant ammonia modification. However, the preoxidation-assistant ammonia modification introduces more nitrogen on the ACSON. As a result, the overall contents of N-6 and N-Q in the ACSON increase when the preoxidation-assistant ammonia modification is adopted for incorporation nitrogen.In order to investigate the nature of the functional groups created by the above reactions, the elemental speciation and surface binding of the prepared carbon spheres were analyzed by XPS. 3.73SH over the virgin and modified carbon spheres were evaluated in a fixed bed reactor, and the corresponding breakthrough curves were shown in 3SH breakthrough capacities shown in 3SH capacity is 181.1 mg g−1 and 132.2 mg g−1 for the virgin carbon spheres and commercial carbon spheres, respectively. However, the CH3SH breakthrough capacity of ACSO is only 87.7 mg g−1, which can be due to the lower surface area and pore volume caused by the air oxidation, especially the negative influence of acidic oxygen species on the surface of ACSO. Furthermore, the higher amounts of nitrogen, the higher CH3SH capacities for ammonia modified carbon spheres, and the CH3SH capacity is 534.3 mg g−1 and 622.8 mg g−1 for ACSN and ACSON, respectively. The ACSON shows the highest CH3SH capacity with the highest nitrogen content of 7.13 wt%, and the CH3SH breakthrough capacity is higher or similar to other reported adsorbents. Therefore, it is critical to introduce the basic nitrogen containing functional groups for enhancement of the CH3SH removal.The adsorption/oxidation performances of CH21,54 As can be seen from the XPS analysis, the envelope N 1s peak of the ACSN and ACSON were composed of four different nitrogen containing functional groups, and they presented different relative contents. Thus, the correlation between the CH3SH capacities of the ACSN and ACSON and the contents of N-6 and N-Q according to XPS results is also discussed to investigate the effects of the two nitrogen containing species on the CH3SH removal as illustrated in the reported literatures. It can be observed that the relative amounts of N-6 and N-Q in the ACSON prepared by preoxidation-assistant ammonia modification show a little smaller than those of ACSN obtained by direct ammonia treatment. However, the preoxidation-assistant ammonia modification introduces more nitrogen on the ACSON with a high nitrogen content of 8.02 at%. As a result, the overall contents of N-6 and N-Q in the ACSON are bigger than those of ASCN, and the CH3SH capacity increases with the contents of pyridinic nitrogen and quaternary nitrogen as shown in 3SH oxidation. Therefore, we conclude that the contents of N-6 and N-Q play an important role on the catalytic oxidation of CH3SH over carbon spheres.It has been reported that the two nitrogen containing groups of pyridinic nitrogen and quaternary nitrogen with strong electron transfer ability have a profit for the oxidation of sulfurs species.3SH, thus, the absence of water and oxygen in the mixed gas is implemented for ACS and ACSON, and the results are shown in 3SH breakthrough capacity of ACS decreases by around two times under the absence of moisture compared with the presence of moisture. Furthermore, it is noticeable that oxygen has a different effect on the adsorption/oxidation of CH3SH for ACS and ACSON. For instance, the CH3SH breakthrough capacity is 53.8 mg g−1 for ACS-A without moisture, while the ACS-N shows the similary CH3SH breakthrough capacity of 51.2 mg g−1 in the absence of oxygen. The two conditions indicate that moisture and oxygen have no obvious discrepancy for ACS. On the contrary, the CH3SH breakthrough capacity is 313.9 mg g−1 for ACSON-A in the presence of dry air, while the ACSON-N has the CH3SH breakthrough capacity of 203.6 mg g−1 without oxygen. It can be seen that a considerable discrepancy exits in the ACSON. On the one hand, the moisture can create a thin water film on the surface of carbon spheres while the fed gas is in the presence of water, and the adsorbed CH3SH can dissolve into the water film, and further dissociate to the thiolate ion that can be easily oxidized to CH3SSCH3. On the other hand, when the fed gas has no water, the gaseous CH3SH is firstly adsorbed onto the surface of carbon spheres, afterwards the adsorbed CH3SH is also further oxidized to CH3SSCH3 in absence of moisture. According to the above results, it can be observed that the CH3SH capacity of ACS in dry air and dry nitrogen show little difference, indicating that no oxygen participates in the CH3SH removal under air condition, which can be ascribe to the lack catalytic activity sites like nitrogen containing species. However, the CH3SH capacity under dry air is bigger than that in dry nitrogen for ACSON, which demonstrates the nitrogen containing functional groups  give rise to the strong catalytic oxidation performance on the surface of ammonia modified carbon spheres. As a result, it can be deduced that water and oxygen have a profit for the CH3SH removal over the nitrogen-enriched carbon spheres.It has been also essential to study the effect of various gas conditions on the removal of CH23,55 The corresponding DTG curves are shown in 2O. It has been proposed that the desorption of CH3SSCH3 created by the oxidation of CH3SH is completed in range of 100 °C and 300 °C. Therefore, the sharp peak located at about 280 °C is ascribe to removal of CH3SSCH3 for the exhausted samples. In addition, another mild peak located between 300 °C and 400 °C is related to the deeper oxidation product, and the peak becomes obvious accompanied by the nitrogen content of the samples.In order to investigate the created product on the surface of the exhausted carbon spheres, the exhausted samples were subjected to the thermal analysis (TA) experiments, and it can provide the information of related sulfur species through the weight loss in range of certain temperatures.3SSCH3 and methyl methane thiosulfonate appear in the spectra. Combining with the above DTG results, the sharp peak around 280 °C may be also created by the desorption of methyl methane thiosulfonate. It has been observed that further oxidation of sulfur species took place over the ammonia modified carbon spheres under wet air conditions, and the decrease of pH in the exhausted carbon spheres also indicated the formation of acidic oxidation products compared with the fresh samples in 3SH can be catalytically oxidized by the nitrogen containing active species of ACSON, resulting in the highest CH3SH breakthrough capacity. In addition, no peak related to the water is found, indicating that the surface or pore channel of carbon spheres were easily covered by more created CH3SSCH3 other than water.The GC/MS spectra for species extracted from ACSON-E is measured by gas chromatography-mass spectrometry (GC/MS) experiments, and the GC/MS results are shown in 3SH removal, since it can reflects the economy and practicability of porous carbon adsorbents. It is well known that CH3SSCH3 is the basic oxidation product of CH3SH, and it can be easily removed from the surface of carbon adsorbents. Furthermore, the exhausted carbon spheres was regenerated by means of ethanol scrubbing and heat treatment, and 3SH removal. It can be observed that no noticeable loss of the CH3SH capacity appears for ACSON even after ten cycles, and the CH3SH capacity of ACSON reserved as high as 97%. Furthermore, the discrepancies of pore structure and chemical properties were investigated to analysis the cause for fresh ACSON and regenerated ACSON-10. According to the pore structural parameters shown in 2 g−1, 0.69 cm3 g−1 and 0.56 cm3 g−1 compared with the fresh ACSON, respectively. And ACSON-10 also keeps a high micropore rate of 81.15%. Moreover, it can be seen that the nitrogen content of ACSON-10 reserves as high as 7.87 at%, and the relative contents of N-6 and N-Q slightly decrease to 47.82% and 14.05%, respectively. Only a slight decrease is shown compared to those of fresh ACSON in 3SH adsorption/oxidation performance.Another important factor is stable cyclic operation performance relation to the actual application of an adsorbent for CH3.83SH adsorption/oxidation over the nitrogen enriched porous carbon spheres. Firstly, a thin water film is formed on the surface of carbon spheres with the introduction of a moist gas, and then CH3SH molecules are adsorbed on the surface and dissolve into the water film. Hereafter, they dissociate into protons and thiolate ions. As suggested by Bandosz and Liu, the N-6 (pyridinic nitrogen) and N-Q (quaternary nitrogen) act as the Lewis basic sites and promote the electrons transfer in the adsorption/oxidation of CH3SH for the nitrogen doped carbon materials.21,23 Afterwards, the dissociation of CH3SH to thiolate ions become more easier after the introduction of N-6 with a lone electron pair that play an important role on Lewis basic sites. Later, the extra electrons in the functional groups of N-6 and N-Q, give rise to the transmission between the thiolate ion and the adsorbed oxygen, leading to the formation of thiolate radicals and superoxide ions. Simultaneously, the imported water reacts with the created superoxide ions, and thus hydroxyl radicals are formed in the intermediate process. Next, as the created actives species such as oxygen radicals and hydroxyl radicals also provide benefit conditions for oxidation, the oxidation product of CH3SSCH3 is further oxidized into CH3SO2SCH3, which is consistent with the results of DTG and GC/MS and other reported literatures,21,23 and the oxidation process attains balance states until the oxidation products occupy all the pores with nitrogen active species. Finally, the exhausted carbon spheres are regenerated through ethanol exaction and heat treatment, and the CH3SH capacity shows a reserved CH3SH capacity of 97% for ten cycles after the slight decrease in BET surface area, total pore volume and nitrogen content of carbon spheres.Based on the above results, the possible mechanism is proposed to depict the CH4.via suspension polymerization and steam activation, and then the effects of air oxidation and ammonia solution heat treatment on the pore structure and surface chemistry of carbon spheres were investigated for efficient catalytic oxidation of CH3SH. The results showed that the obtained porous carbon spheres were mainly microporous, and the carbon spheres showed a high surface area value of 1710 m2 g−1 and a total pore volume of 0.83 cm3 g−1 after the direct ammonia treatment. However, the surface area and total pore volume of carbon spheres after air oxidation decreased from 1636 m2 g−1 to 1397 m2 g−1 and 0.77 cm3 g−1 to 0.64 cm3 g−1 compared with virgin carbon spheres, respectively. Moreover, the preoxidation followed by enriched nitrogen strategy not only increased the surface area and total pore volume of carbon spheres, but also incorporated more active nitrogen species of pyridinic nitrogen and quaternary nitrogen, which demonstrated in this study that the two nitrogen containing functional groups promoted a better catalytic oxidation of CH3SH as function of catalysts. Furthermore, the carbon spheres obtained from preoxidation-assisted enriched nitrogen presented the highest CH3SH capacity of 622.8 mg g−1 with the highest nitrogen content of 7.13 wt%, and the CH3SH capacity increased with the contents of pyridinic nitrogen and quaternary nitrogen. In addition, water and oxygen have a profit for the CH3SH removal over the nitrogen-enriched carbon spheres. The results of DTG and GC/MS demonstrated that the basic oxidation product is CH3SSCH3 that can be further oxidized into CH3SO2SCH3. Finally, the exhausted carbon spheres showed a great recycling stability after ten cycles without obvious decrease of the CH3SH capacity, which demonstrates that the preoxidation-assisted nitrogen enriched porous carbon spheres are promising for catalytic adsorption/oxidation of CH3SH.In summary, millimeter resin-based porous carbon spheres with high surface area and microporous structure were firstly prepared from 3-isopropylphenol and formaldehyde There are no conflicts to declare.RA-010-D0RA07375J-s001


Blautia and Coprococcus were lower in the HLA-B27+ offspring, while Faecalibacterium prausnitzii was higher. HLA-B27+ offspring without arthritis were compared to children with treatment-naïve HLA-B27+ SpA. After adjustments, clustering by diagnosis was present. A total of 21 OTUs were significantly associated with diagnosis state, including Bacteroides (higher in SpA patients) and F. prausnitzii (higher in controls). Thus, our data confirmed associations with B. fragilis and F. prausnitzii with juvenile SpA, and also suggest that the mechanism by which HLA-B27 is associated with SpA may not involve alterations of the microbiota.Multiple studies have shown the microbiota to be abnormal in patients with spondyloarthritis (SpA). The purpose of this study was to explore the genetic contributions of these microbiota abnormalities. We analyzed the impact of HLA-B27 on the microbiota of children at risk for SpA and compared the microbiota of HLA-B27+ pediatric offspring of ankylosing spondylitis (AS) patients with that of HLA-B27+ children with SpA. Human DNA was obtained from the offspring for determination of HLA-B27 status and polygenic risk score (PRS). Fecal specimens were collected from both groups for sequencing of the V4 region of the 16S ribosomal RNA gene. Among the offspring of AS patients, there was slight clustering by HLA-B27 status. After adjusting for multiple comparisons, five operational taxonomic units (OTUs) representing three unique taxa distinguished the HLA-B27+ from negative children:  Spondyloarthritis (SpA) has a prevalence of about 1–2% of the adult population  comparedIt has been hypothesized that HLA-B27 itself mediates disease, at least in part, by acting upon the microbiota . To testThis was a cross-sectional study that first compared the contents of the fecal microbiota among HLA-B27+ versus HLA-B27- offspring of patients with AS, and subsequently compared the microbiota of treatment-naïve children with HLA-B27+ SpA with that of the healthy HLA-B27+ offspring.Controls were a sample of children of patients with AS identified by a single rheumatologist (LG) as per the modified New York criteria . ChildreJuvenile SpA subjects were either children with enthesitis-related arthritis as per the International League of Associations for Rheumatology criteria  or who mThis was conducted as previously reported . Briefly2 fold change between the two groups, while correcting for multiple comparisons with the Benjamini–Hochberg false discovery rate (FDR) test [The purified DNA (~100 ng) underwent PCR amplification using primers designed to amplify the conserved region flanking the V4 region from the 16S ribosomal RNA (rRNA) gene, as described previously ,17. ResuDR) test  with a cAs a complementary tool to tease out which taxa were most essential in distinguishing the two groups of subjects, we used the random forest algorithm , which iDifferences in total read counts and in alpha diversity were evaluated using the Student’s t-test, with an alpha of 0.05. Differences in the PRS by HLA-B27 status were assessed with the nonparametric Kruskal–Wallis test.HLA-B27 imputation was performed with SNP2HLA [Calculation of the PRS was performed as recently described by our group , using t SNP2HLA  against  SNP2HLA .A total of 56 offspring of AS patients met inclusion criteria for the current study and submitted a fecal specimen. The PRS was obtained on 55 of the offspring, although we were still able to impute HLA-B27 status on the subject without a PRS. One of the subjects was diagnosed with SpA at the research exam on the basis of arthritis, enthesitis, and clinical sacroiliitis. That subject was found to be HLA-B27+ by genotyping, subsequently confirmed through clinical testing, and was, therefore, included in the SpA group. Of the 56 offspring, 32 (57%) were found to be HLA-B27 positive. A description of the probands with AS is included in p = 0.022). There likewise was association between the PRS and the structure of the microbiota . However, a major driver of the PRS is HLA-B27 itself; the PRS of the HLA-B27- offspring was −0.25 , while that of HLA-B27+ offspring was 0.22 . There was no association between the PRS and the structure of the microbiota when the analyses were performed separately on HLA-B27+ and HLA-B27- offspring. For HLA-B27+ subjects, the F-score was 1.5 (p = 0.081) and, for their HLA-B27- counterparts, the F score was 1.1 (p = 0.345).There were no differences in alpha diversity of raw read counts between HLA-B27+ and HLA-B27- offspring . OrdinatF. prausnitzii had higher abundance in the HLA-B27+ subjects, while Coprococcus and three OTUs from the Blautia genus were all higher in the HLA-B27- subjects.Next, we used the DeSeq2 program  to perfoF. prausnitzii , which remained significant following adjustment for NSAID use, age, and sex . DeSeq2 analysis demonstrated 21 OTUs that distinguished the patients from controls  was higher in the SpA patients versus the HLA-B27+ siblings, while the other (Coprococcus) was lower in the SpA patients. As discussed above with respect to the random forest plot, several organisms appear more than once, due to multiple entries representing species or strains that cannot be distinguished at the 16S level.Alpha diversity analyses demonstrated that the SpA patients had decreased richness (Chao1 test) and evenness (Shannon test) as compared to the controls, although a second test of evenness (inverse Simpson) did not demonstrate any differences between the two groups . Ordinatcontrols . ConsistThis is the first study to compare the contents of the fecal microbiota of HLA-B27+ versus HLA-B27- offspring of AS patients and is also the first to compare that of HLA-B27+ pediatric subjects with and without SpA. Several important messages emerged from this work.Blautia among the HLA-B27+ subjects. In the current study, a key organism distinguishing HLA-B27+ from negative subjects was F. prausnitzii, which, despite being depleted in juvenile SpA patients  but not limited to SpA [Finally, this study appears to underscore the importance of age or disease state in the nature of the microbial abnormalities. We have previously demonstrated that the fecal abundance of nile SpA , while id to SpA ,11,37,38d to SpA ,34,39,40d to SpA .This study has limitations, including the potential for differences due to the age differences between the HLA-B27+ offspring and the SpA patients. We do not suspect this difference to be problematic, as the microbiota appears to stabilize after age 3 . Age didA novel aspect of this work is that of comparing both HLA-B27+ and HLA-B27- offspring who are well-balanced for age, sex, and geographic factors, and also comparing HLA-B27+ SpA patients to HLA-B27+ controls. Prior studies of SpA patients ,31,34,39


The correct data is 13.4 ± 12.5 instead of 413.4 ± 12.5.In the paper by Oh The publisher apologizes for this error.


Cities and urban environments can do peculiar things to biodiversity that shares them with us. How cities affect their invited and uninvited inhabitants has become an increasingly important question. More than half of the world's population dwells in urban areas, and these environments will keep expanding considerably. Understanding how this relatively recent, rapid, and pervasive form of landscape modification influences the ecology and evolution of organisms that cannot escape, or may benefit from it, is an emerging field of biology. Although we are aware of how some birds, mammals or plants respond to urban environments, less is known about insects and invertebrates in general. In this issue of Molecular Ecology, Blumenfeld et al. (2022) bring new remarkable insights into how a common ant species adjusts to urban settings across the United States by changing its social structure and behaviour. Using a large‐scale molecular, chemical and behavioural dataset, they document how the odorous house ant Tapinoma sessile differs in its colony organisation and dispersal strategy between rural and urban habitats. In each of the study regions and continent‐wide, rural and urban colonies are genetically and chemically differentiated, suggesting that urban settings act as potent agents of selection and isolation. The novelty and importance of this study are that it documents multiple independent transitions toward the same social organisation and the apparent effect of habitat on the life history of a eusocial insect species. Traits which make ants successful invaders are mainly known and often shared across unrelated species. They are linked to reproduction and dispersal strategies such as polygyny (multiple reproductive queens), dependent colony foundation (budding) or parthenogenesis such as thelytoky. Others consist of behavioural shifts, such as loss of aggression among non‐nestmates, increased hostility towards other species or changes in foraging activity. Finally, ecology‐related features can involve shifts in habitat or food preferences. Many of these traits facilitate easy dispersal and high competitiveness. When combined with polydomy, in which a single colony inhabits multiple nests, polygyny may lead to unicoloniality. Unicolonial populations form a network of interconnected nests among which workers move freely and lack aggression, despite having low genetic relatedness. In their extreme form, called supercolonies, they can reach extraordinary densities, span hundreds of kilometres and effectively outcompete other invertebrates. In ant invaders, some of these traits are linked to reduced genetic variability, or inbreeding or reduced nestmate discrimination capacity caused by their introduction.T. sessile populations. Named the odorous house ant, this species native to North America is highly adaptable and found across many habitats, including cities. The authors knew from earlier research that the urban colonies are larger, polygynous and potentially polydomous, unlike most colonies in natural settings.Encountering some of these links was what Blumenfeld et al.  expectedT. sessile. They confirmed with microsatellite markers that the colony structure is indeed flexible and linked to a habitat. The polygynous colonies were much more common in urban settings, while monogynous colonies were prevalent in natural habitats. Surprisingly, polydomy was relatively rare and detected only on a few occasions. And perhaps more remarkable is that polygyny has not only independently and repeatedly arisen under the urban settings on such a large scale but also that both types of colonies were consistently highly distinct genetically and chemically (in their cuticular hydrocarbons), confirming that habitat is a strong driver of divergence.Using a large‐scale sampling and smartly integrating phylogeographical, population genetic, chemical and behavioural methods, Blumenfeld et al. disentangled the social structure of Formica). While these nonurban species are often monogynous and monodomous, they can be polygynous and polydomous in highly fragmented environments, such as small forest patches in agricultural areas (Seifert et al., A similar polymorphism has been documented, for example, in European wood ants (T. sessile is probably a result of the adoption of daughter queens to the maternal colony. Instead of flying off and founding colonies independently, the daughter queens of T. sessile use budding—a dependent colony foundation with the help of the worker ants of the maternal colony. To prevent inbreeding, which this reproduction mode may lead to, T. sessile probably favours male‐biased dispersal to avoid sib‐mating. Such strategies were proposed by theoretical models and also documented in ants on islands or in patchy, isolated habitats. Whether this is a response to dispersal limitation, the high unpredictability of nesting sites or the patchiness of food sources in urban settings is a pending question.What Blumenfeld et al.  found inBy performing a detailed mapping of social and population structures as done by Blumenfeld et al., we can better assess the effect of ecological conditions on various life‐history traits. The authors placed their results into a broader perspective on how disturbance and urban environments can affect different taxa. They also propose reliable scenarios of how particular conditions in cities could lead to the specific traits and strategies encountered in urban odorous ant populations. Their work provides another excellent example of urban‐mediated shifts or adaptations and opens an array of questions to be addressed with follow‐up studies.Lasius niger, workers in urban colonies had more diverse cytochrome gene families and DNA repair systems, while the olfactory systems were reduced (Konorov et al., For example, genomic comparisons could map if there is selection towards specific sets of traits at the level of individuals. In the ant T. sessile in urban vs. natural settings also deserves further attention. While another study of tiny acorn ant Temnothorax nylanderi did not reveal signatures of urbanization on population demography and structure (Khimoun et al., T. sessile, we can look into the mechanisms reinforcing the urban and natural habitat barriers and the chances for human‐mediated dispersals. Robust markers should assess the population divergence among more habitats and spatial scales. Isolation by environment can regularly produce considerable population structuring; therefore, comparing the differentiation levels across diverse habitats would help us assess the contribution of urban‐specific factors to population divergence in T. sessile.The substantial differentiation found among populations of Tapinoma and across unrelated clades with similar life features would help assess the contribution of different factors for succeeding in disturbed environments.Finally, a macroevolutionary perspective may provide helpful insights into what traits are primary preadaptations for exploiting urban environments or becoming invasive. Ancestral shifts in preference to open and disturbed habitats in several South Pacific ants increased their macroevolutionary dispersal rates. These lineages then successfully colonized remote islands and broadened their distribution considerably, including giving rise to several invasive species (Matos‐Maraví, Clouse, et al., Organisms may respond to many different aspects of the urban environments, and these phenotypic and genotypic differences do not always imply adaptation (McDonnell & Hahs, CONACYT DICB 282471.None.The author declare no conflict of interest.None.