In-situ growth of a covalent organic framework-based matrix-compatible microextraction coating for sensitive extraction of multiple pesticides.

The potential pesticide hazard to non-target organisms is a global concern. It is critical to develop the sensitive detection methods of multiple pesticides in various complex matrices. Here, benzene-1,3,5-tricarbaldehyde (BTCA) and 1,3,5-Tri (4-aminophenyl) benzene (TAPB) were employed as precursors for the in-situ growth of COFTAPB-BTCA on the surface of amino-functionalized stainless steel wire (SS) via a solvothermal method. The successful COFTAPB-BTCA bonded fiber exhibited significant enrichment capability of pyrethroids insecticides (PYs), organophosphorus (OPPs), and organochlorine (OCPs), with enrichment factors (EFs) ranging from 1133-7762, 1319-7291, and 734.1-2882, respectively. X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) calculations indicated that various interactions contributed to its high enrichment capacity. Automated detection of PYs, OPPs, and OCPs in water, foods, and biological samples was realized by coupling this fiber with gas chromatography-mass spectrometry (GC-MS). The detection limits were as low as 0.0370-0.657 ng/L, 0.0128-0.400 ng/L, and 0.0329-0.202 ng/L for PYs, OPPs, and OCPs, respectively. In addition, the environmental risks of these samples were assessed based on the above data. This work not only provided a straightforward technique for sensitive monitoring of pesticides in complex matrices but also presented a novel approach for the in-situ controlled growth of versatile adsorbents with broad-spectrum properties.

Blocking ß2-AR and Inhibiting COX-2: A Promising Approach to Suppress OSCC Development.

OBJECTIVES: ß2-adrenergic receptor (ß2-AR) and cyclooxygenase-2 (COX-2) are overexpressed in various malignant tumours including oral squamous cell carcinoma (OSCC), suggesting that they may contribute to the development of OSCC. This study aims to investigate the potential synergistic effect of ß2-AR blockade and COX-2 inhibition on suppressing the development of OSCC. METHODS: Effects of blocking ß2-AR and inhibiting COX-2 on migration and invasion of OSCC cells were detected by wound-healing assay and transwell invasion assay. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of genes related to the progression of OSCC. In vivo, OSCC xenograft models were established to evaluate the effect of combined treatment on survival time, tumour size, and submandibular lymph node metastasis. Immunohistochemistry, Western blot, and ELISA were used to detect the expression of invasion and metastasis relative genes. RESULTS: In vitro, blocking ß2-AR or inhibiting COX-2 alone could suppress invasion and metastasis of OSCC cells, and suppression with combined treatment was more significant. Expression of genes related to invasion and metastasis, including EGFR, TGF-ß1, IL-1ß, MMP2, and VEGFA, were downregulated significantly, especially in the combined treatment group. In vivo, the combined treatment could significantly prolong survival time in tumour-bearing mice and inhibit the growth of tumours. Furthermore, submandibular lymph node metastasis was less in the combined treatment group, and expression of the abovementioned genes was also downregulated. CONCLUSIONS: The combination of ß2-AR blockade and COX-2 inhibition can significantly suppress the development of OSCC via downregulating EGFR, TGF-ß1, IL-1ß, MMP2, and VEGFA. Findings suggest that the combined use of a ß2-AR blocker and a COX-2 inhibitor could be a promising adjuvant therapy in OSCC. Both drugs are commonly prescribed, and their safety and efficacy are well established. Their use in adjuvants in OSCC should therefore be promoted in clinical practice.

Atomically Precise Regulation of the N-Heterocyclic Microenvironment in Triazine Covalent Organic Frameworks for Coenzyme Photocatalytic Regeneration.

Artificial photosynthesis represents a sustainable strategy for accessing high-value chemicals; however, the conversion efficiency is significantly limited by its difficulty in the cycle of coenzymes such as NADH. In this study, we report a series of isostructural triazine covalent organic frameworks (COFs) and explore their N-substituted microenvironment-dependent photocatalytic activity for NADH regeneration. We discovered that the rational alteration of N-heterocyclic species, which are linked to the triazine center through an imine linkage, can significantly regulate both the electron band structure and planarity of a COF layer. This results in different separation efficiencies of the photoinduced electron-hole pairs and electron transfer behavior within and between individual layers. The optimal COF catalyst herein achieves an NADH regeneration capacity of 89% within 20 min, outperforming most of the reported nanomaterial photocatalysts. Based on this, an artificial photosynthesis system is constructed for the green synthesis of a high-value compound, L-glutamate, and its conversion efficiency significantly surpasses the enzymatic approach without the NADH photocatalytic cycle. This work offers new insights into the coenzyme regeneration by means of regulating the distal heterocyclic microenvironment of a COF skeleton, holding great potential for the green photosynthesis of important chemicals.

Viral shedding pattern of severe fever with thrombocytopenia syndrome virus in severely ill patients: A prospective, Multicenter cohort study.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is spreading rapidly in Asia. The pathway of SFTS virus shedding from patient and specific use of personal protective equipments (PPEs) against viral transmission have rarely been reported. The study was to determine SFTS virus (SFTSV) shedding pattern from the respiratory, digestive and urinary tract to outside in patients. Methods: Patients were divided into mild and severe groups in three sentinel hospitals for SFTS in Anhui province from April 2020 to October 2022. SFTSV level from blood, throat swabs, fecal/anal swabs, urine and bedside environment swabs of SFTS patients were detected by qRT-PCR. Specific PPEs were applied in healthcare workers contacting with the patients who had oropharyngeal virus shedding and hemorrhagic signs. Results: A total of 189 SFTSV-confirmed patients were included in the study, 54 patients died (case fatality rate, 28.57 %). Positive SFTSV in throat swabs (T-SFTSV), fecal/anal swabs (F-SFTSV) and urine (U-SFTSV) were detected in 121 (64.02 %), 91 (48.15 %) and 65 (34.4 %) severely ill patients, respectively. The levels of T-SFTSV, F-SFTSV and U-SFTSV were positively correlated with the load of SFTSV in blood. We firstly revealed that SFTSV positive rate of throat swabs were correlated with occurrence of pneumonia and case fatality rate of patients (P < 0.0001). Specific precaution measures were applied by healthcare workers in participating cardiopulmonary resuscitation and orotracheal intubation for severely ill patients with positive T-SFTSV, no event of SFTSV human-to-human transmission occurred after application of effective PPEs. Conclusions: Our research demonstrated SFTSV could shed out from blood, oropharynx, feces and urine in severely ill patients. The excretion of SFTSV from these parts was positively correlated with viral load in the blood. Effective prevention measures against SFTSV human-to-human transmission are needed.

A Synergetic Pore Compartmentalization and Hydrophobization Strategy for Synchronously Boosting the Stability and Activity of Enzyme.

Spatial immobilization of fragile enzymes using a nanocarrier is an efficient means to design heterogeneous biocatalysts, presenting superior stability and recyclability to pristine enzymes. An immobilized enzyme, however, usually compromises its catalytic activity because of inevasible mass transfer issues and the unfavorable conformation changes in a confined environment. Here, we describe a synergetic metal-organic framework pore-engineering strategy to trap lipase (an important hydrolase), which confers lipase-boosted stability and activity simultaneously. The hierarchically porous NU-1003, featuring interconnected mesopore and micropore channels, is precisely modified by chain-adjustable fatty acids on its mesopore channel, into which lipase is trapped. The interconnected pore structure ensures efficient communication between trapped lipase and exterior media, while the fatty acid-mediated hydrophobic pore can activate the opening conformation of lipase by interfacial interaction. Such dual pore compartmentalization and hydrophobization activation effects render the catalytic center of trapped lipase highly accessible, resulting in 1.57-fold and 2.46-fold activities as native lipase on ester hydrolysis and enantioselective catalysis. In addition, the feasibility of these heterogeneous biocatalysts for kinetic resolution of enantiomer is also validated, showing much higher efficiency than native lipase.

Enzyme-Immobilized Porous Crystals for Environmental Applications.

Developing efficient technologies to eliminate or degrade contaminants is paramount for environmental protection. Biocatalytic decontamination offers distinct advantages in terms of selectivity and efficiency; however, it still remains challenging when applied in complex environmental matrices. The main challenge originates from the instability and difficult-to-separate attributes of fragile enzymes, which also results in issues of compromised activity, poor reusability, low cost-effectiveness, etc. One viable solution to harness biocatalysis in complex environments is known as enzyme immobilization, where a flexible enzyme is tightly fixed in a solid carrier. In the case where a reticular crystal is utilized as the support, it is feasible to engineer next-generation biohybrid catalysts functional in complicated environmental media. This can be interpreted by three aspects: (1) the highly crystalline skeleton can shield the immobilized enzyme against external stressors. (2) The porous network ensures the high accessibility of the interior enzyme for catalytic decontamination. And (3) the adjustable and unambiguous structure of the reticular framework favors in-depth understanding of the interfacial interaction between the framework and enzyme, which can in turn guide us in designing highly active biocomposites. This Review aims to introduce this emerging biocatalysis technology for environmental decontamination involving pollutant degradation and greenhouse gas (carbon dioxide) conversion, with emphasis on the enzyme immobilization protocols and diverse catalysis principles including single enzyme catalysis, catalysis involving enzyme cascades, and photoenzyme-coupled catalysis. Additionally, the remaining challenges and forward-looking directions in this field are discussed. We believe that this Review may offer a useful biocatalytic technology to contribute to environmental decontamination in a green and sustainable manner and will inspire more researchers at the intersection of the environment science, biochemistry, and materials science communities to co-solve environmental problems.

Developing Covalent Organic Framework Biocatalysts through Enzyme Encapsulation.

Utilizing covalent organic frameworks (COFs) as porous supports to encapsulate enzyme represents an advanced strategy for constructing COFs biocatalysts, which has inspired numerous interests across various applications. As the structural advantages including ultrastable covalent-bonded linkage, tailorable pore structure, and metal-free biocompatibility, the resultant enzyme-COFs biocatalysts showcase functional enhancement in catalytic activity, chemical stability, long-term durability, and recyclability. This Concept describes the recent advances in the methodological strategies for engineering the COFs biocatalysts, with specific emphasis on the pore entrapment and in situ encapsulation strategies. The structural advantages of the COFs hybrid biocatalysts for organic synthesis, environment- and energy-associated applications are also canvassed. Additionally, the remaining challenges and the forward-looking directions in this field are also discussed. We believe that this Concept can offer useful methodological guidance for developing active and robust COFs biocatalysts.

The incidence and risk factors of marginal ulcers in the short and medium term in symptomatic post-pancreaticoduodenectomy patients ---- single-center experience.

BACKGROUND: Marginal ulcer (MU) is one of the postoperative complications of pancreaticoduodenectomy (PD), which needs particular attention in postoperative treatments. METHODS: The data of 190 patients who underwent PD and follow-up gastroscopic review due to upper GI symptoms within two years were retrospectively analyzed. The incidence of MU and risk factors were analyzed based on personal history, surgical procedure, past medical history, postoperative complications, and other relevant indicators. RESULTS: The proportion of MU in patients who underwent endoscopic follow-up for upper gastrointestinal symptoms in the postoperative period in this cohort was 10.5% (20/190). Advanced age (69y vs. 59y, P â€‹= â€‹0.012), alcohol consumption (20% vs. 8.2%, P â€‹= â€‹0.03), and cigarette smoking (35% vs. 14.7%, P â€‹= â€‹0.022) were associated with an increased incidence of MU. Longer surgery time (276.5min vs. 240min, P â€‹= â€‹0.049), postoperative bleeding (10% vs. 1.8%, P â€‹= â€‹0.030), and failure to take antacid regularly postoperatively (75% vs. 97.1%, P â€‹= â€‹0.000) would increase the risk of MU; taking antacid regularly was an independent protective factor for postoperative anastomotic ulceration (OR: 0.091, CI: 0.022-0.383, P â€‹= â€‹0.001). CONCLUSION: Advanced age, alcohol consumption, smoking, longer operation time, or postoperative extraluminal hemorrhage are associated with MU. Regular use of antacids is an independent protective factor against the development of MU.

A new classification of mandible defects and condyle changed after mandible reconstruction with FFF.

Objectives: To explore a new classification of mandibular defects and changes in the preserved condyle after mandibular reconstruction with free fibular flap(FFF). Study design: We reviewed patients who underwent mandibular reconstruction with FFF from 2015 to 2021 and classified the mandibular defects into five categories: classⅠ(unilateral-mandibular excluding condyle), classⅡ(unilateral-mandibular including condyle), classⅢ(bilateral-mandibular excluding condyle), classⅣ(bilateral-mandibular including one condyle), and classⅤ(bilateral-mandibular including both condyles). Cone Beam Computed Tomography (CBCT) data were collected preoperatively(T0), at 7-10 postoperative days(T1), 6 postoperative months(T2), and 1 postoperative year(T3). We calculated the condylar surface area, volume, and displacement. Results: 62 cases were collected. The condylar surface areas and volumes in T2 and T3 values were lower than those of T0 and T1(P < 0.01) The condylar displacement was the lowest in ClassI and the largest in ClassⅣ(P < 0.01), while no significant differences in classesⅠ-Ⅲ(P < 0.05). Displacement during T1-T0 was greater than that during T2-T0 and T3-T0(P < 0.05). Conclusion: Mandibular reconstruction with FFF results in displacement and alteration of the condyle within a time interval, and this alteration stabilizes after 6 months. Mandibular defects that do not reach the midline, surgical alteration to preserve the condyle are not required. However, when the defects cross the midline, the condyle should be preserved as much as possible.

Ionic Liquid-Mediated Dynamic Polymerization for Facile Aqueous-Phase Synthesis of Enzyme-Covalent Organic Framework Biocatalysts.

Utilizing covalent organic framework (COF) as a hypotoxic and porous scaffold to encapsulate enzyme (enzyme@COF) has inspired numerous interests at the intersection of chemistry, materials, and biological science. In this study, we report a convenient scheme for one-step, aqueous-phase synthesis of highly crystalline enzyme@COF biocatalysts. This facile approach relies on an ionic liquid (2 µL of imidazolium ionic liquid)-mediated dynamic polymerization mechanism, which can facilitate the in situ assembly of enzyme@COF under mild conditions. This green strategy is adaptive to synthesize different biocatalysts with highly crystalline COF "exoskeleton", as well evidenced by the low-dose cryo-EM and other characterizations. Attributing to the rigorous sieving effect of crystalline COF pore, the hosted lipase shows non-native selectivity for aliphatic acid hydrolysis. In addition, the highly crystalline linkage affords COF "exoskeleton" with higher photocatalytic activity for in situ production of H2 O2 , enabling us to construct a self-cascading photo-enzyme coupled reactor for pollutants degradation, with a 2.63-fold degradation rate as the poorly crystalline photo-enzyme reactor. This work showcases the great potentials of employing green and trace amounts of ionic liquid for one-step synthesis of crystalline enzyme@COF biocatalysts, and emphasizes the feasibility of diversifying enzyme functions by integrating the reticular chemistry of a COF.

An integrative model with HLA-DR, CD64, and PD-1 for the diagnostic and prognostic evaluation of sepsis.

BACKGROUND: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection and progressive immunosuppression with high mortality. HLA-DR, CD64, and PD-1 were assumed to be useful biomarkers for sepsis prediction. However, the ability of a combination of these biomarkers has not been clarified. METHODS: An observational case-control study was conducted that included 30 sepsis patients, 30 critically ill patients without sepsis admitted to the intensive care unit (ICU), and 32 healthy individuals. The levels of HLA-DR, CD64, and PD-1 expression in peripheral blood immune cells and subsets was assayed on Days 1, 3, and 5, and the clinical information of patients was collected. We compared these biomarkers between groups and evaluated the predictive validity of single and combined biomarkers on sepsis mortality. RESULTS: The results indicate that PD-1 expression on CD4- CD8- T (PD-1+ CD4- CD8- T) (19.19% ± 10.78% vs. 9.88% ± 1.79%, p = .004) cells and neutrophil CD64 index (nCD64 index) (9.15 ± 5.46 vs. 5.33 ± 2.34, p = .001) of sepsis patients were significantly increased, and HLA-DR expression on monocytes (mHLA-DR+ ) was significantly reduced (13.26% ± 8.06% vs. 30.17% ± 21.42%, p = 2.54 × 10-4 ) compared with nonsepsis critically ill patients on the first day. Importantly, the expression of PD-1+ CD4- CD8- T (OR = 0.622, 95% CI = 0.423-0.916, p = .016) and mHLA-DR+ (OR = 1.146, 95% CI = 1.014-1.295, p = .029) were significantly associated with sepsis mortality. For sepsis diagnosis, the mHLA-DR+ , PD-1+ CD4- CD8- T, and nCD64 index showed the moderate individual performance, and combinations of the three biomarkers achieved greater diagnostic value (AUC = 0.899, 95% CI = 0.792-0.962). When adding PCT into the combined model, the AUC increased to 0.936 (95% CI = 0.840-0.983). For sepsis mortality, combinations of PD-1+ CD4- CD8- T and mHLA-DR+ , have a good ability to predict the prognosis of sepsis patients, with an AUC = 0.921 (95% CI = 0.762-0.987). CONCLUSION: These findings indicate that the combinations of HLA-DR, CD64, and PD-1 outperformed each of the single indicator in diagnosis and predicting prognosis of sepsis.

Hydrogen-Bonded Supramolecular Nanotrap Enabling the Interfacial Activation of Hosted Enzymes.

Engineering nanotraps to immobilize fragile enzymes provides new insights into designing stable and sustainable biocatalysts. However, the trade-off between activity and stability remains a long-standing challenge due to the inevitable diffusion barrier set up by nanocarriers. Herein, we report a synergetic interfacial activation strategy by virtue of hydrogen-bonded supramolecular encapsulation. The pore wall of the nanotrap, in which the enzyme is encapsulated, is modified with methyl struts in an atomically precise position. This well-designed supramolecular pore results in a synergism of hydrogen-bonded and hydrophobic interactions with the hosted enzyme, and it can modulate the catalytic center of the enzyme into a favorable configuration with high substrate accessibility and binding capability, which shows up to a 4.4-fold reaction rate and 4.9-fold conversion enhancements compared to free enzymes. This work sheds new light on the interfacial activation of enzymes using supramolecular engineering and also showcases the feasibility of interfacial assembly to access hierarchical biocatalysts featuring high activity and stability simultaneously.

Sea-urchin-like covalent organic framework as solid-phase microextraction fiber coating for sensitive detection of trace pyrethroid insecticides in water.

Pyrethroid insecticides residues in water pose a critical threat to the environment from widespread production and overuse. Therefore, it is of major relevance to develop a sensitive and efficient method to detect pyrethroid insecticides in water. In this paper, a covalent organic framework (COF) with NHCO as the structural unit was synthesized using a simple condensation reaction of TTL (NH2) and TDBA (COOH). Various characterization results and density functional theory (DFT) calculations demonstrated that multiple interactions synergistically promoted the adsorption of pyrethroid insecticides on COFTDBA-TTL. Based on the excellent extraction capability of COFTDBA-TTL, efficient detection of 11 pyrethroid insecticides in water was achieved using COFTDBA-TTL-coated SPME fiber and gas chromatography-tandem mass spectrometry (GC-MS). The results showed that the extraction enhancement factors (EFs) of pyrethroid insecticides were as high as 2584-7199, and the extraction efficiencies were 3.28-446 times higher than that of commercial fiber, which reflected its high adsorption property. Meanwhile, the limits of detection (LODs) of the COFTDBA-TTL coated fiber were as low as 0.170-1.68 ng/L under the optimal conditions, and the recoveries of 11 pyrethroid insecticides in the actual water samples were 88.5-108 %. In conclusion, the SPME-GC-MS method based on COFTDBA-TTL coated fiber was simple, rapid, and efficient, and should have a promising application in trace detection of pyrethroid insecticides in the environment.

Pore-Engineered Hydrogen-Bonded Supramolecular Fluorosensor for Ultrasensitive Determination of Copper Ions.

The selective quantification of copper ions (Cu2+ ) in biosamples holds great importance for disease diagnosis, treatment, and prognosis since the Cu2+ level is closely associated with the physiological state of the human body. While it remains a long-term challenge due to the extremely low level of free Cu2+ and the potential interference by the complex matrices. Here, a pore-engineered hydrogen-bonded organic framework (HOF) fluorosensor is constructed enabling the ultrasensitive and highly selective detection of free Cu2+ . Attributing to atomically precise functionalization of active amino "arm" within the HOF pores and the periodic π-conjugated skeleton, this porous HOF fluorosensor affords high affinity toward Cu2+ through double copper-nitrogen (Cu─N) coordination interactions, resulting in specific fluorescence quenching of the HOF as compared with a series of substances ranging from other metal ions, metabolites, amino acids to proteins. Such superior fluorescence quenching effect endows the Cu2+ quantification by this new HOF sensor with a wide linearity of 50-20 000 nm, a low detection limit of 10 nm, and good recoveries (89.5%-115%) in human serum matrices, outperforming most of the reported approaches. This work highlights the practicability of hydrogen-bonded supramolecular engineering for designing facile and ultrasensitive biosensors for clinical free Cu2+ determination.

Structural and Functional Insights into the Biomineralized Zeolite Imidazole Frameworks.

Biomineralization is a natural process of mineral formation mediated by biomacromolecules, allowing access to hierarchical structures integrating biological, chemical, and material properties. In this contribution, we comprehensively investigate the biomineralization of zeolite imidazole frameworks (ZIFs) for one-step synthesis of an enzyme-MOF biocomposite, in terms of differential crystallization behaviors, fine microstructure of resultant ZIF biominerals, the enzyme's conformation evolution, and protective effect of ZIF mineral. We discover that the biomineralization ability is ZIF organic linker dependent and the biocatalytic function is highly related to the ZIF mineral species and their distinguishable topologies and defect structures. Importantly, a side-by-side analysis suggests that the protective effect of ZIF mineral toward the hosted enzyme is highly associated with the synergistic effect of size dimension and chemical microenvironment of the ZIF pores. This work provides important insight into the ZIF-dependent biomineralization behaviors and highlights the important role of the ZIF microstructure in its biocatalytic activity and durability, which has been underestimated previously.

Photonanozyme with Light Mediated Activity.

Since the discovery that Fe3 O4 nanoparticle has intrinsic natural peroxidase-like activity by Yan et al in 2007, mimicking native enzymes via nano-engineering (named as nanozyme) pays a new avenue to bypass the fragility and recyclability of natural enzymes and thus expedites the biocatalysis in multidisciplinary applications. In addition, the high programmability and structural stability attributes of nanozyme afford the ease of coupling with electromagnetic waves of different energies, providing great opportunities to construct photo-responsive nanozyme under user-defined electromagnetic waves, which is known as photo-nanozyme. In this concept, we aim to providing a summary of how electromagnetic waves with varying wavelengths can serve as external stimuli to induce or enhance the biocatalytic performance of photo-nanozymes, thereby offering fascinating functions that cannot be achieved by pristine nanozyme.

Protocol for mechanochemistry-guided assembly strategy for enzyme encapsulation using covalent organic frameworks.

Enzyme immobilization into porous frameworks is an emerging strategy for enhancing the stability of dynamic conformation and prolonging the lifespan of enzymes. Here, we present a protocol for a de novo mechanochemistry-guided assembly strategy for enzyme encapsulation using covalent organic frameworks. We describe steps for mechanochemical synthesis, enzyme loading measurements, and material characterizations. We then detail evaluations of biocatalytic activity and recyclability. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).1.

Green synthesis of stable hybrid biocatalyst using a hydrogen-bonded, π-π-stacking supramolecular assembly for electrochemical immunosensor.

Rational integration of native enzymes and nanoscaffold is an efficient means to access robust biocatalyst, yet remains on-going challenges due to the trade-off between fragile enzymes and harsh assembling conditions. Here, we report a supramolecular strategy enabling the in situ fusion of fragile enzymes into a robust porous crystal. A c2-symmetric pyrene tecton with four formic acid arms is utilized as the building block to engineer this hybrid biocatalyst. The decorated formic acid arms afford the pyrene tectons high dispersibility in minute amount of organic solvent, and permit the hydrogen-bonded linkage of discrete pyrene tectons to an extended supramolecular network around an enzyme in almost organic solvent-free aqueous solution. This hybrid biocatalyst is covered by long-range ordered pore channels, which can serve as the gating to sieve the catalytic substrate and thus enhance the biocatalytic selectivity. Given the structural integration, a supramolecular biocatalyst-based electrochemical immunosensor is developed, enabling the pg/mL detection of cancer biomarker.

Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworks.

Enzymes are outstanding natural catalysts with exquisite 3D structures, initiating countless life-sustaining biotransformations in living systems. The flexible structure of an enzyme, however, is highly susceptible to non-physiological environments, which greatly limits its large-scale industrial applications. Seeking suitable supports to immobilize fragile enzymes is one of the most efficient routes to ameliorate the stability problem. This protocol imparts a new bottom-up strategy for enzyme encapsulation using a hydrogen-bonded organic framework (HOF-101). In short, the surface residues of the enzyme can trigger the nucleation of HOF-101 around its surface through the hydrogen-bonded biointerface. As a result, a series of enzymes with different surface chemistries are able to be encapsulated within a highly crystalline HOF-101 scaffold, which has long-range ordered mesochannels. The details of experimental procedures are described in this protocol, which involve the encapsulating method, characterizations of materials and biocatalytic performance tests. Compared with other immobilization methods, this enzyme-triggering HOF-101 encapsulation is easy to operate and affords higher loading efficiency. The formed HOF-101 scaffold has an unambiguous structure and well-arranged mesochannels, favoring mass transfer and understanding of the biocatalytic process. It takes ~13.5 h for successful synthesis of enzyme-encapsulated HOF-101, 3-4 d for characterizations of materials and ~4 h for the biocatalytic performance tests. In addition, no specific expertise is necessary for the preparation of this biocomposite, although the high-resolution imaging requires a low-electron-dose microscope technology. This protocol can provide a useful methodology to efficiently encapsulate enzymes and design biocatalytic HOF materials.

Identification and validation of a novel ubiquitination-related gene UBE2T in Ewing's sarcoma.

Background: Ewing's sarcoma (ES) is one of the most prevalent malignant bone tumors worldwide. However, the molecular mechanisms of the genes and signaling pathways of ES are still not well sufficiently comprehended. To identify candidate genes involved in the development and progression of ES, the study screened for key genes and biological pathways related to ES using bioinformatics methods. Methods: The GSE45544 and GSE17618 microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified, and functional enrichment analysis was performed. A protein-protein interaction (PPI) network was built, and key module analysis was performed using STRING and Cytoscape. A core-gene was gained and was validated by the validation dataset GSE67886 and immunohistochemistry (IHC). The diagnostic value and prognosis evaluation of ES were executed using, respectively, the ROC approach and Cox Regression. Results: A total of 187 DEGs, consisting of 56 downregulated genes and 131 upregulated genes, were identified by comparing the tumor samples to normal samples. The enriched functions and pathways of the DEGs, including cell division, mitotic nuclear division, cell proliferation, cell cycle, oocyte meiosis, and progesterone-mediated oocyte maturation, were analyzed. There were 149 nodes and 1246 edges in the PPI network, and 15 hub genes were identified according to the degree levels. The core gene (UBE2T) showed high expression in ES, validated by using GSE67886 and IHC. The ROC analysis revealed UBE2T had outstanding diagnostic value in ES (AUC = 0.75 in the training set, AUC = 0.90 in the validation set). Kaplan-Meier (analysis of survival rate) and Cox Regression analyses indicated that UBE2T was a sign of adverse results for sufferers with ES. Conlusion: UBE2T was a significant value biomarker for diagnosis and treatment of ES, thereby presenting a novel potential therapeutic target for ES as well as a new perspective for assessing the effect of treatment and prognostic prediction.