GPNMB Modulates Autophagy to Enhance Functional Recovery After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) severely affects the quality of life and autonomy of patients, and effective treatments are currently lacking. Autophagy, an essential cellular metabolic process, plays a crucial role in neuroprotection and repair after SCI. Glycoprotein non-metastatic melanoma protein B (GPNMB) has been shown to promote neural regeneration and synapse reconstruction, potentially through the facilitation of autophagy. However, the specific role of GPNMB in autophagy after SCI is still unclear. In this study, we utilized the spinal cord transection method to establish SCI rats model and overexpressed GPNMB using adenoviral vectors. We assessed tissue damage using hematoxylin and eosin (H&E) and Nissl staining, and observed cell apoptosis using TUNEL staining. We evaluated the inflammatory response by measuring inflammatory factors using enzyme-linked immunosorbent assay (ELISA). In addition, we measured reactive oxygen species (ROS) levels using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and assessed oxidative stress levels by measuring malondialdehyde (MDA) and glutathione (GSH) using ELISA. To evaluate autophagy levels, we performed immunofluorescence staining for the autophagy marker Beclin-1 and conducted Western blot analysis for autophagy-related proteins. We also assessed limb recovery through functional evaluation. Meanwhile, we induced cell injury using lipopolysaccharide (LPS) and added an autophagy inhibitor to verify the impact of GPNMB on SCI through autophagy modulation. The results demonstrated that GPNMB alleviated the inflammatory response, reduced oxidative stress levels, inhibited cell apoptosis, and promoted autophagy following SCI. Inhibiting autophagy reversed the effects of GPNMB. These findings suggest that GPNMB promotes neural injury repair after SCI, potentially through attenuating the inflammatory response, reducing oxidative stress, and inhibiting cell apoptosis.

Cloning and Characterization of Chitin Deacetylase from Euphausia superba.

Chitin deacetylase (CDA) can catalyze the deacetylation of chitin to produce chitosan. In this study, we identified and characterized a chitin deacetylase gene from Euphausia superba (EsCDA-9k), and a soluble recombinant protein chitin deacetylase from Euphausia superba of molecular weight 45 kDa was cloned, expressed, and purified. The full-length cDNA sequence of EsCDA-9k was 1068 bp long and encoded 355 amino acid residues that contained the typical domain structure of carbohydrate esterase family 4. The predicted three-dimensional structure of EsCDA-9k showed a 67.32% homology with Penaeus monodon. Recombinant chitin deacetylase had the highest activity at 40 °C and pH 8.0 in Tris-HCl buffer. The enzyme activity was enhanced by metal ions Co2+, Fe3+, Ca2+, and Na+, while it was inhibited by Zn2+, Ba2+, Mg2+, and EDTA. Molecular simulation of EsCDA-9k was conducted based on sequence alignment and homology modeling. The EsCDA-9k F18G mutant showed a 1.6-fold higher activity than the wild-type enzyme. In summary, this is the first report of the cloning and heterologous expression of the chitin deacetylase gene in Euphausia superba. The characterization and function study of EsCDA-9k will serve as an important reference point for future application.

Construction of a myoglobin scaffold-based biocatalyst for the biodegradation of sulfadiazine and sulfathiazole.

Sulfonamide antibiotics, a family of broad-spectrum antibiotic drugs, are increasingly used in aquaculture and are frequently detected in aquatic environments. This poses a potential threat to organisms and may cause the evolution of antimicrobial resistance. Therefore, it is important to develop an environmentally friendly and efficient biocatalyst to degrade sulfonamides (SAs) such as sulfadiazine (SD) and sulfathiazole (ST). Here, we realized the direct and efficient degradation of SD and ST using a hydrogen peroxide-dependent artificial catalytic system based on myoglobin (Mb). The arrangements of amino acids at positions 29, 43, 64, and 68 were found to influence catalytic activity. An L29H/H64D/V68I myoglobin mutant showed the best catalytic efficiency (i.e., kcat/Km = 720.42 M-1 s-1) against SD. Next, mutant H64D/V68I showed the best degradation rate against SD (i.e., 91.45 ± 0.16%). Moreover, L29H/H64D/V68I Mb was found to efficiently catalyze ST oxidation (kcat/Km = 670.08 M-1 s-1), while H64D/V68I had the best degradation rate against ST (i.e., 99.45 ± 0.23%). Our results demonstrate that SAs can be efficiently degraded by artificial peroxygenases constructed using a myoglobin scaffold. This therefore provides a simple and economical method for the biodegradation of SD and ST.

The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

Temozolomide (TMZ) resistance presents a significant challenge in the treatment of gliomas. Although lysine demethylase 4A (KDM4A) has been implicated in various cancer-related processes, its role in TMZ resistance remains unclear. This study aims to elucidate the contribution of KDM4A to TMZ resistance in glioma cells and its potential implications for glioma prognosis. We assessed the expression of KDM4A in glioma cells (T98G and U251MG) using qRT-PCR and Western blot assays. To explore the role of KDM4A in TMZ resistance, we transfected siRNA targeting KDM4A into drug-resistant glioma cells. Cell viability was assessed using the CCK-8 assay and the TMZ IC50 value was determined. ChIP assays were conducted to investigate KDM4A, H3K9me3, and H3K36me3 enrichment on the promoters of ROCK2 and HUWE1. Co-immunoprecipitation confirmed the interaction between HUWE1 and ROCK2, and we examined the levels of ROCK2 ubiquitination following MG132 treatment. Notably, T98G cells exhibited greater resistance to TMZ than U251MG cells, and KDM4A displayed high expression in T98G cells. Inhibiting KDM4A resulted in decreased cell viability and a reduction in the TMZ IC50 value. Mechanistically, KDM4A promoted ROCK2 transcription by modulating H3K9me3 levels. Moreover, disruption of the interaction between HUWE1 and ROCK2 led to reduced ROCK2 ubiquitination. Inhibition of HUWE1 or overexpression of ROCK2 counteracted the sensitization effect of si-KDM4A on TMZ responsiveness in T98G cells. Our findings highlight KDM4A's role in enhancing TMZ resistance in glioma cells by modulating ROCK2 and HUWE1 transcription and expression through H3K9me3 and H3K36me3 removal.

Postoperative use of steroids for peri-electrode edema after deep brain stimulation surgery: A retrospective cohort study.

BACKGROUND: To review the incidence and extent of peri-electrode edema after DBS and to clarify the effect of postoperative use of steroids on the peri-electrode edema. METHODS: This retrospective cohort study included 250 patients who underwent bilateral subthalamic nucleus (STN) DBS surgery with intact MRI within 1 month after DBS surgery. Patients were divided into steroid and non-steroid groups, based on postoperative steroids use. The occurrence and extent of peri-electrode edema were compared between the two groups, and other associated factors were analyzed using univariate and multivariate methods. RESULTS: Peri-electrode edema >1 cm3 in at least one hemisphere was reported in 215 (86.00%) patients. The mean volume of peri-electrode edema observed in the steroid group was significantly smaller than in the non-steroid group (8.09 ± 8.47 cm3 vs 17.10 ± 16.90 cm3 , p < 0.001). In the steroid group, 104 (32.91%) of the 316 implanted electrodes present with edema less than 1 cm3 , whereas in the non-steroid group, only 27 (14.67%) of the 184 implanted electrodes present with edema less than 1 cm3 (p < 0.001). Multivariate analysis indicated that lesser peri-electrode edema was significantly associated with postoperative steroids use and general anesthesia. CONCLUSIONS: Peri-electrode edema is common after DBS surgery, and postoperative steroids use reduces the occurrence and extent of peri-electrode edema.

Hybrid Electrically Conductive Hydrogels with Local Nerve Growth Factor Release Facilitate Peripheral Nerve Regeneration.

It is challenging to treat peripheral nerve injury (PNI) clinically. As the gold standard for peripheral nerve repair, autologous nerve grafting remains a critical limitation, including tissue availability, donor-site morbidity, immune rejection, etc. Recently, conductive hydrogels (CHs) have shown potential applications in neural bioengineering due to their good conductivity, biocompatibility, and low immunogenicity. Herein, a hybrid electrically conductive hydrogel composed of acrylic acid derivatives, gelatin, and heparin with sustained nerve growth factor (NGF) release property was developed. The rat sciatic nerve injury (SNI) model (10 mm long segment defect) was used to investigate the efficacy of these hydrogel conduits in facilitating peripheral nerve repair. The results showed that the hydrogel conduits had excellent conductivity, mechanical properties, and biocompatibility. In addition, NGF immobilized in the hydrogel conduits had good sustained release characteristics. Finally, functional recovery and electrophysiological evaluations, together with histological analysis, indicated that the hydrogel conduits immobilizing NGF had superior effects on motor recovery, axon growth, and remyelination, thereby significantly accelerating the repairing of the sciatic nerve. This study demonstrated that hybrid electrically conductive hydrogels with local NGF release could be effectively used for PNI repair.

Exploiting and Engineering Neuroglobin for Catalyzing Carbene N-H Insertions and the Formation of Quinoxalinones.

It is desired to design and construct more efficient enzymes with better performance to catalyze carbene N-H insertions for the synthesis of bioactive molecules. To this end, we exploited and designed a series of human neuroglobin (Ngb) mutants. As shown in this study, a double mutant, A15C/H64G Ngb, with an additional disulfide bond and a modified heme active site, exhibited yields up to >99% and total turnover numbers up to 33000 in catalyzing the carbene N-H insertions for aromatic amine derivatives, including those with a large size such as 1-aminopyrene. Moreover, for o-phenylenediamine derivatives, they underwent two cycles of N-H insertions, followed by cyclization to form quinoxalinones, as confirmed by the X-ray crystal structures. This study suggests that Ngb can be designed into a functional carbene transferase for efficiently catalyzing carbene N-H insertion reactions with a range of substrates. It also represents the first example of the formation of quinoxalinones catalyzed by an engineered heme enzyme.

FOXM1 maintains fatty acid homoeostasis through the SET7-H3K4me1-FASN axis.

Reprogramming of metabolic genes and subsequent alterations in metabolic phenotypes occur widely in malignant tumours, including glioblastoma (GBM). FOXM1 is a potent transcription factor that plays an oncogenic role by regulating the expression of many genes. As a SET domain containing protein, SET7 is a protein lysine methyltransferase which monomethylates histone proteins and other proteins. The epigenetic modification of histones regulates gene expressions by epigenetically modifying promoters of DNAs and inter vening in tumor development. Activation of FASN increased de novo fatty acid (FA) synthesis, a hallmark of cancer cells. Here, we report that FOXM1 may directly promote the transcription of SET7 and activate SET7-H3K4me1-FASN axis, which results in the maintenance of de novo FA synthesis.

Operative treatment of cystic prolactinomas: a retrospective study.

BACKGROUND: The optimal therapeutic approach for cystic prolactinomas remains unclear. This study aimed to evaluate the remission rates of prolactinoma patients after surgical treatment and the risk factors affecting postoperative remission in cystic prolactinoma patients. METHODS: The clinical data were retrospectively compiled from 141 patients with prolactinomas (including 41 cases of cystic prolactinomas, 21 cases of solid microprolactinomas and 79 cases of solid macroprolactinomas) who underwent transsphenoidal surgery (TSS) between April 2013 and October 2021 at the First Affiliated Hospital of Sun Yat-sen University. RESULTS: Early postoperative remission was achieved in 65.83% (n = 27/41) of cystic prolactinomas, 80.95% (n = 17/21) of solid microprolactinomas and 40.51% (n = 32/79) of solid macroprolactinomas. The mean length of follow up in all patients was 43.95 ± 2.33 months (range: 6-105 months). The follow-up remission rates were 58.54%, 71.43% and 44.30% in cystic, solid micro- and solid macroprolactinomas, respectively. For cystic prolactinomas, the early postoperative remission rates in the patients with preoperative dopamine agonists (DA) treatment were significantly higher than those without preoperative DA treatment (p = 0.033), but the difference in the follow-up remission rates between these two groups was not significant (p = 0.209). Multivariate stepwise logistic regression analysis indicated that tumor size and preoperative prolactin (PRL) levels < 200 ng/ml were independent predictors for early postoperative remission in cystic prolactinomas. CONCLUSION: For cystic prolactinomas, tumor size and preoperative PRL levels were independent predictors of early postoperative remission. Preoperative DA therapy combined with TSS may be more beneficial to cystic prolactinoma patients.

The enormous repetitive Antarctic krill genome reveals environmental adaptations and population insights.

Antarctic krill (Euphausia superba) is Earth's most abundant wild animal, and its enormous biomass is vital to the Southern Ocean ecosystem. Here, we report a 48.01-Gb chromosome-level Antarctic krill genome, whose large genome size appears to have resulted from inter-genic transposable element expansions. Our assembly reveals the molecular architecture of the Antarctic krill circadian clock and uncovers expanded gene families associated with molting and energy metabolism, providing insights into adaptations to the cold and highly seasonal Antarctic environment. Population-level genome re-sequencing from four geographical sites around the Antarctic continent reveals no clear population structure but highlights natural selection associated with environmental variables. An apparent drastic reduction in krill population size 10 mya and a subsequent rebound 100 thousand years ago coincides with climate change events. Our findings uncover the genomic basis of Antarctic krill adaptations to the Southern Ocean and provide valuable resources for future Antarctic research.

Construction of Biocatalysts Using the P450 Scaffold for the Synthesis of Indigo from Indole.

With the increasing demand for blue dyes, it is of vital importance to develop a green and efficient biocatalyst to produce indigo. This study constructed a hydrogen peroxide-dependent catalytic system for the direct conversion of indole to indigo using P450BM3 with the assistance of dual-functional small molecules (DFSM). The arrangements of amino acids at 78, 87, and 268 positions influenced the catalytic activity. F87G/T268V mutant gave the highest catalytic activity with kcat of 1402 min-1 and with a yield of 73%. F87A/T268V mutant was found to produce the indigo product with chemoselectivity as high as 80%. Moreover, F87G/T268A mutant was found to efficiently catalyze indole oxidation with higher activity (kcat/Km = 1388 mM-1 min-1) than other enzymes, such as the NADPH-dependent P450BM3 (2.4-fold), the Ngb (32-fold) and the Mb (117-fold). Computer simulation results indicate that the arrangements of amino acid residues in the active site can significantly affect the catalytic activity of the protein. The DFSM-facilitated P450BM3 peroxygenase system provides an alternative, simple approach for a key step in the bioproduction of indigo.

Geometrical Bounds on Irreversibility in Squeezed Thermal Bath.

Irreversible entropy production (IEP) plays an important role in quantum thermodynamic processes. Here, we investigate the geometrical bounds of IEP in nonequilibrium thermodynamics by exemplifying a system coupled to a squeezed thermal bath subject to dissipation and dephasing, respectively. We find that the geometrical bounds of the IEP always shift in a contrary way under dissipation and dephasing, where the lower and upper bounds turning to be tighter occur in the situation of dephasing and dissipation, respectively. However, either under dissipation or under dephasing, we may reduce both the critical time of the IEP itself and the critical time of the bounds for reaching an equilibrium by harvesting the benefits of squeezing effects in which the values of the IEP, quantifying the degree of thermodynamic irreversibility, also become smaller. Therefore, due to the nonequilibrium nature of the squeezed thermal bath, the system-bath interaction energy has a prominent impact on the IEP, leading to tightness of its bounds. Our results are not contradictory with the second law of thermodynamics by involving squeezing of the bath as an available resource, which can improve the performance of quantum thermodynamic devices.

Phenoxazinone Synthase-like Activity of Rationally Designed Heme Enzymes Based on Myoglobin.

The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o-aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (pKa = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.

The Effect of Surgical Positioning on Pneumocephalus in Subthalamic Nucleus Deep Brain Stimulation Surgery for Parkinson Disease.

OBJECTIVES: This research analyzed the effect of surgical positioning on postoperative pneumocephalus and assessed additional potential risk factors of pneumocephalus in subthalamic nucleus (STN) deep brain stimulation (DBS) for Parkinson disease (PD). MATERIALS AND METHODS: In this study, 255 consecutive patients with PD who received bilateral STN DBS under general anesthesia were retrospectively included. Of these, 180 patients underwent surgery with their heads in an elevated position, and 75 patients underwent surgery in a supine position. The postoperative pneumocephalus volume was compared between the two groups. Other potential risk factors for pneumocephalus also were analyzed. RESULTS: The mean pneumocephalus volume for the group with elevated-head positioning (16.76 ± 15.23 cm3) was greater than for the supine group (3.25 ± 8.78 cm3) (p < 0.001). Multivariable analysis indicated that the pneumocephalus volume was related to surgical positioning, lateral trajectory angle, intraoperative mean arterial pressure (MAP), microelectrode recording (MER) passage number, brain atrophy degree, and the anterior trajectory angle. No correlation was found between pneumocephalus and age, sex, duration of PD, surgery length, or intracranial volume. In the subgroup analysis, the pneumocephalus volume exhibited a negative correlation with intraoperative MAP (r = -0.210, p = 0.005) and positive correlations with degree of brain atrophy (r = 0.242, p = 0.001) and MER passage number (r = 0.184, p = 0.014) in the elevated-head group. Specifically, an MER passage number > 3 was a significant risk factor for pneumocephalus in the elevated-head group. A positive correlation was observed between the pneumocephalus volume and the lateral trajectory angle in both groups (elevated-head positioning, r = 0.153, p = 0.041; supine positioning, r = 0.546, p < 0.001). CONCLUSIONS: In patients with PD who were anesthetized and receiving STN DBS, supine positioning reduced pneumocephalus volume compared with patients with PD receiving STN DBS with their heads elevated. The pneumocephalus volume was negatively correlated with intraoperative MAP and positively correlated with the degree of brain atrophy, the lateral trajectory angle, and the MER passage number.

Efficient Degradation of Tetracycline Antibiotics by Engineered Myoglobin with High Peroxidase Activity.

Tetracyclines are one class of widely used antibiotics. Meanwhile, due to abuse and improper disposal, they are often detected in wastewater, which causes a series of environmental problems and poses a threat to human health and safety. As an efficient and environmentally friendly method, enzymatic catalysis has attracted much attention. In previous studies, we have designed an efficient peroxidase (F43Y/P88W/F138W Mb, termed YWW Mb) based on the protein scaffold of myoglobin (Mb), an O2 carrier, by modifying the heme active center and introducing two Trp residues. In this study, we further applied it to degrade the tetracycline antibiotics. Both UV-Vis and HPLC studies showed that the triple mutant YWW Mb was able to catalyze the degradation of tetracycline, oxytetracycline, doxycycline, and chlortetracycline effectively, with a degradation rate of ~100%, ~98%, ~94%, and ~90%, respectively, within 5 min by using H2O2 as an oxidant. These activities are much higher than those of wild-type Mb and other heme enzymes such as manganese peroxidase. As further analyzed by UPLC-ESI-MS, we identified multiple degradation products and thus proposed possible degradation mechanisms. In addition, the toxicity of the products was analyzed by using in vitro antibacterial experiments of E. coli. Therefore, this study indicates that the engineered heme enzyme has potential applications for environmental remediation by degradation of tetracycline antibiotics.

Withdrawn: MiR-101-3p inhibits MTX-mediated HMGB1 release and reduces recruitment and M1-like polarization of macrophages through the UBE2D1/KAT2B pathway.

The above article, published online on 5 December 2022, on Wiley Online Library (https://onlinelibrary.wiley.com/doi/abs/10.1002/htj.22448), has been withdrawn by agreement between the journal Editor in Chief, Hari Bhat, and Wiley Periodicals, LLC. The withdrawal has been agreed due to a technical error at the publisher that caused the article to be mistakenly published online although publication had been canceled because the authors did not approve their proof.

Nomogram for Prediction of Postoperative Delirium after Deep Brain Stimulation of Subthalamic Nucleus in Parkinson's Disease under General Anesthesia.

Introduction: Postoperative delirium can increase cognitive impairment and mortality in patients with Parkinson's disease. The purpose of this study was to develop and internally validate a clinical prediction model of delirium after deep brain stimulation of the subthalamic nucleus in Parkinson's disease under general anesthesia. Methods: We conducted a retrospective observational cohort study on the data of 240 patients with Parkinson's disease who underwent deep brain stimulation of the subthalamic nucleus under general anesthesia. Demographic characteristics, clinical evaluation, imaging data, laboratory data, and surgical anesthesia information were collected. Multivariate logistic regression was used to develop the prediction model for postoperative delirium. Results: A total of 159 patients were included in the cohort, of which 38 (23.90%) had postoperative delirium. Smoking (OR 4.51, 95% CI 1.56-13.02, p < 0.01) was the most important risk factor; other independent predictors were orthostatic hypotension (OR 3.42, 95% CI 0.90-13.06, p=0.07), inhibitors of type-B monoamine oxidase (OR 3.07, 95% CI 1.17-8.04, p=0.02), preoperative MRI with silent brain ischemia or infarction (OR 2.36, 95% CI 0.90-6.14, p=0.08), Hamilton anxiety scale score (OR 2.12, 95% CI 1.28-3.50, p < 0.01), and apolipoprotein E level in plasma (OR 1.48, 95% CI 0.95-2.29, p=0.08). The area under the receiver operating characteristic curve (AUC) was 0.76 (95% CI 0.66-0.86). A nomogram was established and showed good calibration and clinical predictive capacity. After bootstrap for internal verification, the AUC was 0.74 (95% CI 0.66-0.83). Conclusion: This study provides evidence for the independent inducing factors of delirium after deep brain stimulation of the subthalamic nucleus in Parkinson's disease under general anesthesia. By predicting the development of delirium, our model may identify high-risk groups that can benefit from early or preventive intervention.

Risk Factors for Hiccups after Deep Brain Stimulation of Subthalamic Nucleus for Parkinson's Disease.

Background: After deep brain stimulation (DBS), hiccups as a complication may lead to extreme fatigue, sleep deprivation, or affected prognosis. Currently, the causes and risk factors of postoperative hiccups are unclear. In this study, we investigated the risk factors for hiccups after DBS of the subthalamic nucleus (STN) for Parkinson's disease (PD) under general anesthesia. Methods: We retrospectively included patients who underwent STN DBS in the study, and collected data of demographic characteristics, clinical evaluations, and medications. According to the occurrence of hiccups within seven days after operation, the patients were divided into a hiccups group and non-hiccups group. The potentially involved risk factors for postoperative hiccups were statistically analyzed by logistic regression analysis. Results: A total of 191 patients were included in the study, of which 34 (17.80%) had postoperative transient persistent hiccups. Binary univariate logistic regression analysis showed that male, higher body mass index (BMI), smoker, Hoehn and Yahr stage (off), preoperative use of amantadine, hypnotic, Hamilton anxiety scale and Hamilton depression scale scores, and postoperative limited noninfectious peri-electrode edema in deep white matter were suspected risk factors for postoperative hiccups (p < 0.1). In binary multivariate logistic regression analysis, male (compared to female, OR 14.00; 95% CI, 1.74−112.43), postoperative limited noninfectious peri-electrode edema in deep white matter (OR, 7.63; 95% CI, 1.37−42.37), preoperative use of amantadine (OR, 3.64; 95% CI, 1.08−12.28), and higher BMI (OR, 3.50; 95% CI, 1.46−8.36) were independent risk factors for postoperative hiccups. Conclusions: This study is the first report about the risk factors of hiccups after STN DBS under general anesthesia for PD patients. The study suggests that male, higher BMI, preoperative use of amantadine, and postoperative limited noninfectious peri-electrode edema in deep white matter are independent risk factors for postoperative hiccups of STN-DBS for PD patients. Most hiccups after STN-DBS for PD patients were transient and self-limiting.

Cloning and Characterization of Fructose-1,6-Bisphosphate Aldolase from Euphausia superba.

Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli (E. coli). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei. Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA's enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA.

Engineering globins for efficient biodegradation of malachite green: two case studies of myoglobin and neuroglobin.

Malachite green (MG)-contaminated wastewater resulting from industrialization causes a global problem because of its toxicity and widespread usage. Compared with traditional physical and chemical approaches, biodegradation provides a new route for the degradation of MG. As promising candidates for native enzymes, artificial enzymes have received tremendous attention for potential applications due to unlimited possibilities based on precise design. In this study, we rationally engineered artificial enzymes based on myoglobin (Mb) and neuroglobin (Ngb). We introduced an aspartic acid (H64D mutation) in the heme pocket of Mb. A distal histidine (F43H mutation) was further introduced into H64D Mb to obtain a double mutant of F43H/H64D Mb. Moreover, we used A15C/H64D Ngb as designed recently for comparison studies. The H64D Mb, F43H/H64D Mb, and A15C/H64D Ngb were found to catalyze MG degradation efficiently, with activities much higher than those of native enzymes, such as dye-decolorizing peroxidase and laccase (83-205-fold). The crystal structure of H64D Mb was solved and the interactions of MG and H64D Mb and A15C/H64D Ngb were investigated by using both experimental and molecular docking studies. The biodegradation products of MG were also revealed by ESI-MS analysis. Therefore, these artificial enzymes have potential applications in the biodegradation of MG in textile industries and fisheries.