Gut microbiota composition and changes in patients with sepsis: potential markers for predicting survival.

BACKGROUND: Sepsis can cause immune dysregulation and multiple organ failure in patients and eventually lead to death. The gut microbiota has demonstrated its precise therapeutic potential in the treatment of various diseases. This study aimed to discuss the structural changes of the gut microbiota in patients with sepsis and to analyze the differences in the gut microbiota of patients with different prognoses. METHODS: We conducted a multicenter study in which rectal swab specimens were collected on the first and third days of sepsis diagnosis. A total of 70 specimens were collected, and gut microbiota information was obtained by 16S rRNA analysis. RESULTS: The relative abundance of Enterococcus decreased in rectal swab specimens during the first three days of diagnosis in patients with sepsis, while the relative abundance of inflammation-associated Bacillus species such as Escherichia coli, Enterobacteriaceae, and Bacteroidetes increased. By comparing the differences in the flora of the survival group and the death group, we found that the abundance of Veillonella and Ruminococcus in the death group showed an increasing trend (p < 0.05), while the abundance of Prevotella_6 and Prevotella_sp_S4_BM14 was increased in surviving patients (p < 0.05). CONCLUSIONS: The Firmicutes/Bacteroidetes ratio, reflecting overall gut microbial composition, was significantly lower on day three of sepsis diagnosis. Changes in the abundance of specific gut microbiota may serve as prognostic markers in patients with sepsis.

IKK1 aggravates ischemia-reperfusion kidney injury by promoting the differentiation of effector T cells.

Ischemia-reperfusion injury (IRI) is one of the major causes of acute kidney injury (AKI), and experimental work has revealed detailed insight into the inflammatory response in the kidney. T cells and NFκB pathway play an important role in IRI. Therefore, we examined the regulatory role and mechanisms of IkappaB kinase 1 (IKK1) in CD4+T lymphocytes in an experimental model of IRI. IRI was induced in CD4cre and CD4IKK1Δ mice. Compared to control mice, conditional deficiency of IKK1 in CD4+T lymphocyte significantly decreased serum creatinine, blood urea nitrogen (BUN) level, and renal tubular injury score. Mechanistically, lack in IKK1 in CD4+T lymphocytes reduced the ability of CD4 lymphocytes to differentiate into Th1/Th17 cells. Similar to IKK1 gene ablation, pharmacological inhibition of IKK also protected mice from IRI. Together, lymphocyte IKK1 plays a pivotal role in IRI by promoting T cells differentiation into Th1/Th17 and targeting lymphocyte IKK1 may be a novel therapeutic strategy for IRI.

Early gut microbiological changes and metabolomic changes in patients with sepsis: a preliminary study.

The gut microbiota is closely related to the development of sepsis. The aim of this study was to explore changes in the gut microbiota and gut metabolism, as well as potential relationships between the gut microbiota and environmental factors in the early stages of sepsis. Fecal samples were collected from 10 septic patients on the first and third days following diagnosis in this study. The results showed that in the early stages of sepsis, the gut microbiota is dominated by microorganisms that are tightly associated with inflammation, such as Escherichia-Shigella, Enterococcus, Enterobacteriaceae, and Streptococcus. On sepsis day 3 compared to day 1, there was a significant decrease in Lactobacillus and Bacteroides and a significant increase in Enterobacteriaceae, Streptococcus, and Parabacteroides. Culturomica_massiliensis, Prevotella_7 spp., Prevotellaceae, and Pediococcus showed significant differences in abundance on sepsis day 1, but not on sepsis day 3. Additionally, 2-keto-isovaleric acid 1 and 4-hydroxy-6-methyl-2-pyrone metabolites significantly increased on sepsis day 3 compared to day 1. Prevotella_7 spp. was positively correlated with phosphate and negatively correlated with 2-keto-isovaleric acid 1 and 3-hydroxypropionic acid 1, while Prevotella_9 spp. was positively correlated with sequential organ failure assessment score, procalcitonin and intensive care unit stay time. In conclusion, the gut microbiota and metabolites are altered during sepsis, with some beneficial microorganisms decreasing and some pathogenic microorganisms increasing. Furthermore, Prevotellaceae members may play different roles in the intestinal tract, with Prevotella_7 spp. potentially possessing beneficial health properties and Prevotella_9 spp. potentially playing a promoting role in sepsis.

Machine-learning screening of luminogens with aggregation-induced emission characteristics for fluorescence imaging.

Due to the excellent biocompatible physicochemical performance, luminogens with aggregation-induced emission (AIEgens) characteristics have played a significant role in biomedical fluorescence imaging recently. However, screening AIEgens for special applications takes a lot of time and efforts by using conventional chemical synthesis route. Fortunately, artificial intelligence techniques that could predict the properties of AIEgen molecules would be helpful and valuable for novel AIEgens design and synthesis. In this work, we applied machine learning (ML) techniques to screen AIEgens with expected excitation and emission wavelength for biomedical deep fluorescence imaging. First, a database of various AIEgens collected from the literature was established. Then, by extracting key features using molecular descriptors and training various state-of-the-art ML models, a multi-modal molecular descriptors strategy has been proposed to extract the structure-property relationships of AIEgens and predict molecular absorption and emission wavelength peaks. Compared to the first principles calculations, the proposed strategy provided greater accuracy at a lower computational cost. Finally, three newly predicted AIEgens with desired absorption and emission wavelength peaks were synthesized successfully and applied for cellular fluorescence imaging and deep penetration imaging. All the results were consistent successfully with our expectations, which demonstrated the above ML has a great potential for screening AIEgens with suitable wavelengths, which could boost the design and development of novel organic fluorescent materials.

circ-Katnal1 Enhances Inflammatory Pyroptosis in Sepsis-Induced Liver Injury through the miR-31-5p/GSDMD Axis.

Background: Sepsis is a systemic inflammatory response that can elicit organ dysfunction as well as circulatory diseases in serious cases. When inflammatory responses are especially dysregulated, severe complications can arise, including sepsis-induced liver injury. Various microRNAs along with circular (circ) RNAs are involved in inflammatory responses; nevertheless, their functions in regulating sepsis-induced liver injury remain unknown. The cecal ligation and puncture (CLP) procedure can induce liver injury as well as polymicrobial sepsis. Methods: In this study, CLP was used to induce liver injury as well as polymicrobial sepsis. Then, liver function, inflammatory cytokine expression, and hepatic histopathology were evaluated. High-throughput sequencing was employed to investigate the abnormal hepatic circRNA expression after CLP. Raw264.7 cells were utilized to simulation an in vitro sepsis inflammation model with LPS induce. The relative mRNA as well as protein levels of TNF-α, IL-1ß, and IL-6 was explored by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays. We explored functional connections among circRNAs, miR-31-5p, and gasdermin D (GSDMD) using dual-luciferase reporter assays. Western blot was employed to test GSDMD, caspase-1, and NLRP3 expression in mice and cell models. Results: Our results showed that CLP-induced sepsis promoted liver injury via increasing inflammatory pyroptosis. The abnormal expression of circ-Katnal1 played an important role in CLP-induced sepsis. Downregulating circ-Katnal1 suppressed LPS-induced inflammatory pyroptosis in Raw264.7 cells. Bioinformatics and luciferase reporter results confirmed that miR-31-5p and GSDMD were downstream targets of circ-Katnal1. Inhibiting miR-31-5p or upregulating GSDMD reversed the protective effects of silencing circ-Katnal1. Conclusion: Taken together, circ-Katnal1 enhanced inflammatory pyroptosis in sepsis-induced liver injury through the miR-31-5p/GSDMD axis.

Identification of distinct clinical phenotypes of acute respiratory distress syndrome with differential responses to treatment.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome, and the identification of homogeneous subgroups and phenotypes is the first step toward precision critical care. We aimed to explore whether ARDS phenotypes can be identified using clinical data, are reproducible and are associated with clinical outcomes and treatment response. METHODS: This study is based on a retrospective analysis of data from the telehealth intensive care unit (eICU) collaborative research database and three ARDS randomized controlled trials (RCTs) (ALVEOLI, FACTT and SAILS trials). We derived phenotypes in the eICU by cluster analysis based on clinical data and compared the clinical characteristics and outcomes of each phenotype. The reproducibility of the derived phenotypes was tested using the data from three RCTs, and treatment effects were evaluated. RESULTS: Three clinical phenotypes were identified in the training cohort of 3875 ARDS patients. Of the three phenotypes identified, phenotype I (n = 1565; 40%) was associated with fewer laboratory abnormalities, less organ dysfunction and the lowest in-hospital mortality rate (8%). Phenotype II (n = 1232; 32%) was correlated with more inflammation and shock and had a higher mortality rate (18%). Phenotype III (n = 1078; 28%) was strongly correlated with renal dysfunction and acidosis and had the highest mortality rate (22%). These results were validated using the data from the validation cohort (n = 3670) and three RCTs (n = 2289) and had reproducibility. Patients with these ARDS phenotypes had different treatment responses to randomized interventions. Specifically, in the ALVEOLI cohort, the effects of ventilation strategy (high PEEP vs low PEEP) on ventilator-free days differed by phenotype (p = 0.001); in the FACTT cohort, there was a significant interaction between phenotype and fluid-management strategy for 60-day mortality (p = 0.01). The fluid-conservative strategy was associated with improved mortality in phenotype II but had the opposite effect in phenotype III. CONCLUSION: Three clinical phenotypes of ARDS were identified and had different clinical characteristics and outcomes. The analysis shows evidence of a phenotype-specific treatment benefit in the ALVEOLI and FACTT trials. These findings may improve the identification of distinct subsets of ARDS patients for exploration in future RCTs.

Individualized resuscitation strategy for septic shock formalized by finite mixture modeling and dynamic treatment regimen.

BACKGROUND: Septic shock comprises a heterogeneous population, and individualized resuscitation strategy is of vital importance. The study aimed to identify subclasses of septic shock with non-supervised learning algorithms, so as to tailor resuscitation strategy for each class. METHODS: Patients with septic shock in 25 tertiary care teaching hospitals in China from January 2016 to December 2017 were enrolled in the study. Clinical and laboratory variables were collected on days 0, 1, 2, 3 and 7 after ICU admission. Subclasses of septic shock were identified by both finite mixture modeling and K-means clustering. Individualized fluid volume and norepinephrine dose were estimated using dynamic treatment regime (DTR) model to optimize the final mortality outcome. DTR models were validated in the eICU Collaborative Research Database (eICU-CRD) dataset. RESULTS: A total of 1437 patients with a mortality rate of 29% were included for analysis. The finite mixture modeling and K-means clustering robustly identified five classes of septic shock. Class 1 (baseline class) accounted for the majority of patients over all days; class 2 (critical class) had the highest severity of illness; class 3 (renal dysfunction) was characterized by renal dysfunction; class 4 (respiratory failure class) was characterized by respiratory failure; and class 5 (mild class) was characterized by the lowest mortality rate (21%). The optimal fluid infusion followed the resuscitation/de-resuscitation phases with initial large volume infusion and late restricted volume infusion. While class 1 transitioned to de-resuscitation phase on day 3, class 3 transitioned on day 1. Classes 1 and 3 might benefit from early use of norepinephrine, and class 2 can benefit from delayed use of norepinephrine while waiting for adequate fluid infusion. CONCLUSIONS: Septic shock comprises a heterogeneous population that can be robustly classified into five phenotypes. These classes can be easily identified with routine clinical variables and can help to tailor resuscitation strategy in the context of precise medicine.

Utilizing reclassification to explore characteristics and prognosis of KDIGOSCr AKI subgroups: a retrospective analysis of a multicenter prospective cohort study.

BACKGROUND: Acute kidney injury (AKI) is widespread in the intensive care unit (ICU) and affects patient prognosis. According to Kidney Disease: Improving Global Outcomes (KDIGO) guidelines, the absolute and relative increases of serum creatinine (Scr) are classified into the same stage. Whether the prognosis of the two types of patients is similar in the ICU remains unclear. METHODS: According to the absolute and relative increase of Scr, AKI stage 1 and stage 3 patients were divided into stage 1a and 1b, stage 3a and 3b groups, respectively. Their demographics, laboratory results, clinical characteristics, and outcomes were analyzed retrospectively. RESULTS: Of the 345 eligible cases, we analyzed stage 1 because stage 3a group had only one patient. Using 53 or 61.88 µmol/L as the reference Scr (Scrref), no significant differences were observed in ICU mortality (P53=0.076, P61.88=0.070) or renal replacement therapy (RRT) ratio, (P53=0.356, P61.88=0.471) between stage 1a and 1b, but stage 1b had longer ICU length of stay (LOS) than stage 1a (P53<0.001, P61.88=0.032). In the Kaplan-Meier survival analysis, no differences were observed in ICU mortality between stage 1a and 1b (P53=0.378, P61.88=0.255). In a multivariate analysis, respiratory failure [HR = 4.462 (95% CI 1.144-17.401), p = 0.031] and vasoactive drug therapy [HR = 4.023 (95% CI 1.584-10.216), p = 0.003] were found to be independently associated with increased risk of death. CONCLUSION: ICU LOS benefit was more prominent in KDIGOSCr AKI stage 1a patients than in stage 1 b. Further prospective studies with a larger sample size are necessary to confirm the effectiveness of reclassification.

Risk Factors and Molecular Epidemiology of Complicated Intra-Abdominal Infections With Carbapenem-Resistant Enterobacteriaceae: A Multicenter Study in China.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) infections are associated with poor patient outcomes. Data on risk factors and molecular epidemiology of CRE in complicated intra-abdominal infections (cIAI) in China are limited. This study examined the risk factors of cIAI with CRE and the associated mortality based on carbapenem resistance mechanisms. METHODS: In this retrospective analysis, we identified 1024 cIAI patients hospitalized from January 1, 2013 to October 31, 2018 in 14 intensive care units in China. Thirty CRE isolates were genotyped to identify ß-lactamase-encoding genes. RESULTS: Escherichia coli (34.5%) and Klebsiella pneumoniae (21.2%) were the leading pathogens. Patients with hospital-acquired cIAI had a lower rate of E coli (26.0% vs 49.1%; P < .001) and higher rate of carbapenem-resistant Gram-negative bacteria (31.7% vs 18.8%; P = .002) than those with community-acquired cIAI. Of the isolates, 16.0% and 23.4% of Enterobacteriaceae and K pneumoniae, respectively, were resistant to carbapenem. Most carbapenemase-producing (CP)-CRE isolates carried blaKPC (80.9%), followed by blaNMD (19.1%). The 28-day mortality was 31.1% and 9.0% in patients with CRE vs non-CRE (P < .001). In-hospital mortality was 4.7-fold higher for CP-CRE vs non-CP-CRE infection (P = .049). Carbapenem-containing combinations did not significantly influence in-hospital mortality of CP and non-CP-CRE. The risk factors for 28-day mortality in CRE-cIAI included septic shock, antibiotic exposure during the preceding 30 days, and comorbidities. CONCLUSIONS: Klebsiella pneumoniae had the highest prevalence in CRE. Infection with CRE, especially CP-CRE, was associated with increased mortality in cIAI.

Acute right ventricular dysfunction in severe COVID-19 pneumonia.

To investigate the right heart function in coronavirus disease 2019 (COVID-19) patients with acute respiratory distress syndrome (ARDS), a retrospective analysis of 49 COVID-19 patients with ARDS was performed. Patients were divided into severe group and critically-severe group according to the severity of illness. Age-matched healthy volunteers were recruited as a control group. The cardiac cavity diameters, tricuspid annular plane systolic excursion (TAPSE), tricuspid valve regurgitation pressure gradient biggest (TRPG), pulmonary arterial systolic pressure (PASP), maximum inferior vena cava diameter (IVCmax) and minimum diameter (IVCmin), and inferior vena cava collapse index (ICV-CI) were measured using echocardiography. We found that the TAPSE was significantly decreased in pneumonia patients compared to healthy subjects (P < 0.0001), and it was significantly lower in critically-severe patients (P = 0.0068). The TAPSE was less than 17 mm in three (8.6%) severe and five (35.7%) critically-severe patients. In addition, the TAPSE was significantly decreased in severe ARDS patients than in mild ARDS patients. The IVCmax and IVCmin were significantly increased in critically-severe patients compared to healthy subjects and severe patients (P < 0.01), whereas the ICV-CI was significantly decreased (P < 0.05). COVID-19 patients had significantly larger right atrium and ventricle than healthy controls (P < 0.01). The left ventricular ejection fraction (LVEF) in critically-severe patients was significantly lower than that in severe patients and healthy controls (P < 0.05). Right ventricular function was impaired in critically-severe COVID-19 patients. The assessment and protection of the right heart function in COVID-19 patients should be strengthened.

GYY4137 attenuates LPS-induced acute lung injury via heme oxygenase-1 modulation.

GYY4137, a slow-releasing hydrogen sulfide (H2S) donor, has been reported to exert anti-inflammatory activity and protect against sepsis. Heme oxygenase-1 (HO-1) is an important anti-inflammatory heat shock protein and plays a similar effect on sepsis. This study investigated the role of GYY4137 in acute lung injury (ALI) via HO-1 regulation. Lung injury was assessed in mice challenged with intratracheal lipopolysaccharide (LPS) and the mechanism of anti-inflammatory effects of GYY4137 was investigated in mice and RAW264.7 cells. GYY4137 reduced the LPS-mediated pulmonary injury and neutrophil infiltration, and inhibited the LPS-induced production of proinflammatory cytokines, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Moreover, GYY4137 suppressed the LPS-evoked NF-κB activation in RAW264.7 cells. GYY4137, not time-expired GYY4137 significantly induced HO-1 expression compared with the LPS group. The beneficial effects of GYY4137 above were reversed by the HO-1 inhibitor tin protoporphyrin (SnPP). These results suggest an anti-inflammatory effect and a therapeutic role of GYY4137 in LPS-induced ALI via HO-1 regulation.

Angptl2 deficiency attenuates paraquat (PQ)-induced lung injury in mice by altering inflammation, oxidative stress and fibrosis through NF-κB pathway.

Paraquat (PQ) is one of the most extensively used herbicides, possessing high toxicity for humans and animals. The lung is the main target organ by the poisoning of PQ resulting in acute lung injury. Nonetheless, molecular mechanisms underlying PQ-induced lung injury remain unclear. Here, we ask if angiopoietin-like protein 2 (Angptl2), a pro-inflammatory protein, contributes to inflammation that accelerates acute lung injury. The results indicated that abundant Angptl2 expression was observed in lung tissues of PQ-treated mice. Histological analysis revealed that PQ-induced histological changes were alleviated by Angptl2 knockout (Angptl2-/-). Angptl2-/- in PQ-treated mice attenuated acute lung injury progression by reducing the number of total cells, total leukocytes, neutrophils and macrophages in bronchoalveolar lavage fluid (BALF) and reducing inflammatory response through the inactivation of nuclear factor kappa B (NF-κB) pathway. Angptl2-/- reduced oxidative stress in PQ-treated mice, as evidenced by the enhanced superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) levels in serum or lung tissue samples, which was accompanied with increased expressions of nuclear respiratory factor 2 (Nrf-2), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1). PQ-induced fibrosis was also improved in Angptl2-/- mice by decreasing pulmonary transforming growth factor (TGF)-ß1 expressions. In vitro, we found that Angptl2 knockdown-suppressed inflammation, oxidative stress and fibrosis was restored by increasing NF-κB activation in PQ-incubated A549 cells; however, the results above were significantly reversed by inactivating NF-κB using its inhibitor, Bay 11-7085 or LY2409881. Therefore, Angptl2 could provide therapeutic effects on PQ-induced acute lung injury through inhibiting inflammation, oxidative stress and fibrosis by regulating NF-κB pathway.

Does training improve diagnostic accuracy and inter-rater agreement in applying the Berlin radiographic definition of acute respiratory distress syndrome? A multicenter prospective study.

BACKGROUND: Poor inter-rater reliability in chest radiograph interpretation has been reported in the context of acute respiratory distress syndrome (ARDS), although not for the Berlin definition of ARDS. We sought to examine the effect of training material on the accuracy and consistency of intensivists' chest radiograph interpretations for ARDS diagnosis. METHODS: We conducted a rater agreement study in which 286 intensivists (residents 41.3%, junior attending physicians 35.3%, and senior attending physician 23.4%) independently reviewed the same 12 chest radiographs developed by the ARDS Definition Task Force ("the panel") before and after training. Radiographic diagnoses by the panel were classified into the consistent (n = 4), equivocal (n = 4), and inconsistent (n = 4) categories and were used as a reference. The 1.5-hour training course attended by all 286 intensivists included introduction of the diagnostic rationale, and a subsequent in-depth discussion to reach consensus for all 12 radiographs. RESULTS: Overall diagnostic accuracy, which was defined as the percentage of chest radiographs that were interpreted correctly, improved but remained poor after training (42.0 ± 14.8% before training vs. 55.3 ± 23.4% after training, p < 0.001). Diagnostic sensitivity and specificity improved after training for all diagnostic categories (p < 0.001), with the exception of specificity for the equivocal category (p = 0.883). Diagnostic accuracy was higher for the consistent category than for the inconsistent and equivocal categories (p < 0.001). Comparisons of pre-training and post-training results revealed that inter-rater agreement was poor and did not improve after training, as assessed by overall agreement (0.450 ± 0.406 vs. 0.461 ± 0.575, p = 0.792), Fleiss's kappa (0.133 ± 0.575 vs. 0.178 ± 0.710, p = 0.405), and intraclass correlation coefficient (ICC; 0.219 vs. 0.276, p = 0.470). CONCLUSIONS: The radiographic diagnostic accuracy and inter-rater agreement were poor when the Berlin radiographic definition was used, and were not significantly improved by the training set of chest radiographs developed by the ARDS Definition Task Force. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov (registration number NCT01704066 ) on 6 October 2012.

CORM-2 inhibits TXNIP/NLRP3 inflammasome pathway in LPS-induced acute lung injury.

OBJECTIVE: Accumulated studies suggest that exogenously administered carbon monoxide is beneficial for the resolution of acute lung inflammation. The present study aimed to examine the effects and the underlying mechanisms of CORM-2 on thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome pathway in lipopolysaccharide (LPS)-induced acute lung injury (ALI). METHODS: ALI was intratracheally induced by LPS in C57BL6 mice. CORM-2 or iCORM-2 (30mg/kg i.p.) was administered immediately before LPS instillation. 6 h later, lung bronchoalveolar lavage (BAL) fluids were acquired for IL-18, IL-1ß, and cell measurement, and lung issues were collected for histologic examination, wet/dry weight ratio, and determination of TXNIP/NLRP3 inflammasome expression, NLRP3 inflammasome and NF-ΚB activity, and reactive oxygen species (ROS) production. RESULTS: LPS triggered significant lung edema, lung injury, and leukocyte infiltration, and elevated the levels of IL-1ß and IL-18 in lung BAL fluids. CORM-2 pretreatment resulted in a marked amelioration of lung injury and reduced IL-1ß and IL-18 secretion in BAL fluids. In lung tissues; CORM-2 down-regulated mRNA and protein level of TXNIP, NLRP3, ASC, and caspase-1. Furthermore, CORM-2 reduced ROS production, inhibited NLRP3 inflammasome and NF-κB activity, and interaction of TXNIP-NLRP3. However, no significant differences were detected between the LPS and iCORM-2 (an inactive variant of CORM-2) group. CONCLUSION: CORM-2 suppresses TXNIP/NLRP3 inflammasome pathway and protects against LPS-induced lung injury.

Crystal structure of the FTO protein reveals basis for its substrate specificity.

Recent studies have unequivocally associated the fat mass and obesity-associated (FTO) gene with the risk of obesity. In vitro FTO protein is an AlkB-like DNA/RNA demethylase with a strong preference for 3-methylthymidine (3-meT) in single-stranded DNA or 3-methyluracil (3-meU) in single-stranded RNA. Here we report the crystal structure of FTO in complex with the mononucleotide 3-meT. FTO comprises an amino-terminal AlkB-like domain and a carboxy-terminal domain with a novel fold. Biochemical assays show that these two domains interact with each other, which is required for FTO catalytic activity. In contrast with the structures of other AlkB members, FTO possesses an extra loop covering one side of the conserved jelly-roll motif. Structural comparison shows that this loop selectively competes with the unmethylated strand of the DNA duplex for binding to FTO, suggesting that it has an important role in FTO selection against double-stranded nucleic acids. The ability of FTO to distinguish 3-meT or 3-meU from other nucleotides is conferred by its hydrogen-bonding interaction with the two carbonyl oxygen atoms in 3-meT or 3-meU. Taken together, these results provide a structural basis for understanding FTO substrate-specificity, and serve as a foundation for the rational design of FTO inhibitors.

Improved stability and cell response by intrinsic cross-linking of multilayers from collagen I and oxidized glycosaminoglycans.

Stability of surface coatings against environmental stress, such as pH, high ionic strength, mechanical forces, and so forth, is crucial for biomedical application of implants. Here, a novel extracellular-matrix-like polyelectrolyte multilayer (PEM) system composed of collagen I (Col I) and oxidized glycosaminoglycans (oGAGs) was stabilized by intrinsic cross-linking due to formation of imine bonds between aldehydes of oxidized chondroitin sulfate (oCS) or hyaluronan (oHA) and amino groups of Col I. It was also found that Col I contributed significantly more to overall mass in CS-Col I than in HA-Col I multilayer systems and fibrillized particularly in the presence of native and oxidized CS. Adhesion and proliferation studies with murine C3H10T1/2 embryonic fibroblasts demonstrated that covalent cross-linking of oGAG with Col I had no adverse effects on cell behavior. By contrast, it was found that cell size and polarization was more pronounced on oGAG-based multilayer systems, which corresponded also to the higher stiffness of cross-linked multilayers as observed by studies with quartz crystal microbalance (QCM). Overall, PEMs prepared from oGAG and Col I give rise to stable PEM constructs due to intrinsic cross-linking that may be useful for making bioactive coatings of implants and tissue engineering scaffolds.