Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin.

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.

Advance in the pharmacological and comorbidities management of heart failure with preserved ejection fraction: evidence from clinical trials.

The prevalence of heart failure with preserved ejection fraction (HFpEF) accounts for approximately 50% of the total heart failure population, and with the aging of the population and the increasing prevalence of hypertension, obesity, and type 2 diabetes (T2DM), the incidence of HFpEF continues to rise and has become the most common subtype of heart failure. Compared with heart failure with reduced ejection fraction, HFpEF has a more complex pathophysiology and is more often associated with hypertension, T2DM, obesity, atrial fibrillation, renal insufficiency, pulmonary hypertension, obstructive sleep apnea, and other comorbidities. HFpEF has generally been considered a syndrome with high phenotypic heterogeneity, and no effective treatments have been shown to reduce mortality to date. Diuretics and comorbidity management are traditional treatments for HFpEF; however, they are mostly empirical due to a lack of clinical evidence in the setting of HFpEF. With the EMPEROR-Preserved and DELIVER results, sodium-glucose cotransporter 2 inhibitors become the first evidence-based therapies to reduce rehospitalization for heart failure. Subgroup analyses of the PARAGON-HF, TOPCAT, and CHARM-Preserved trials suggest that angiotensin receptor-neprilysin inhibitors, spironolactone, and angiotensin II receptor blockers may be beneficial in patients at the lower end of the ejection fraction spectrum. Other potential pharmacotherapies represented by non-steroidal mineralocorticoid receptor antagonists finerenone and antifibrotic agent pirfenidone also hold promise for the treatment of HFpEF. This article intends to review the clinical evidence on current pharmacotherapies of HFpEF, as well as the comorbidities management of atrial fibrillation, hypertension, T2DM, obesity, pulmonary hypertension, renal insufficiency, obstructive sleep apnea, and iron deficiency, to optimize the clinical management of HFpEF.

Phosphine-Catalyzed Ring-Opening Regioselective Addition of Cyclopropenones with Amides.

A series of amides, including α-bromo hydroxamates, N-alkoxyamides, and N-aryloxyamides, were subjected to phosphine-catalyzed ring-opening O-selective addition with cyclopropenones, producing various special α,ß-unsaturated esters containing oxime ether motif, in moderate to excellent yields, with high regioselectivity, and exclusive O-selectivity. The methodology is highly atom-economical, with simple operation procedures, and compatible with a wide substrate scope (more than 44 examples).

Rational Design of an Artificial Metalloenzyme by Constructing a Metal-Binding Site Close to the Heme Cofactor in Myoglobin.

In this study, we constructed a metal-binding site close to the heme cofactor in myoglobin (Mb) by covalently attaching a nonnative metal-binding ligand of bipyridine to Cys46 through the F46C mutation in the heme distal site. The X-ray structure of the designed enzyme, termed F46C-mBpy Mb, was solved in the Cu(II)-bound form, which revealed the formation of a heterodinuclear center of Cu-His-H2O-heme. Cu(II)-F46C-mBpy Mb exhibits not only nitrite reductase reactivity but also cascade reaction activity involving both hydrolysis and oxidation. Furthermore, F46C-mBpy Mb displays Mn-peroxidase activity by the oxidation of Mn2+ to Mn3+ using H2O2 as an oxidant. This study shows that the construction of a nonnative metal-binding site close to the heme cofactor is a convenient approach to creating an artificial metalloenzyme with a heterodinuclear center that confers multiple functions.

Peroxidase activity of a Cu-Fe bimetallic hydrogel and applications for colorimetric detection of ascorbic acid.

A Cu-Fe bimetallic hydrogel (2-QF-CuFe-G) was constructed through a simple method. The 2-QF-CuFe-G metallohydrogel possesses excellent peroxidase-like activity to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. The catalytic mechanism was confirmed by the addition of •OH radical scavenger isopropyl alcohol (IPA), tert-butyl alcohol (TBA) and ˙OH trapping agent terephthalic acid (TA). Remarkably, the resultant blue ox-TMB system can be used to selectively and sensitively detect ascorbic acid (AA) with an LOD of 0.93 µM in the range of 4-36 µM through the colorimetric method. Moreover, the assay based on the 2-QF-CuFe-G metallohydrogel can be successfully applied to detect AA in fresh fruits.

Exogenous titanium dioxide nanoparticles alleviate cadmium toxicity by enhancing the antioxidative capacity of Tetrastigma hemsleyanum.

Nanotechnology is one of the most recent approaches employed to defend plants against both biotic and abiotic stress including heavy metals such as Cadmium (Cd). In this study, we evaluated the effects of titanium dioxide (TiO2) nanoparticles (TiO2 NPs) in alleviating Cd stress in Tetrastigma hemsleyanum Diels et Gilg. Compared with Cd treatment, TiO2 NPs decreased leaf Cd concentration, restored Cd exposure-related reduction in the biomass to about 69% of control and decreased activities of antioxidative enzymes. Integrative analysis of transcriptome and metabolome revealed 325 differentially expressed genes associated with TiO2 NP treatment, most of which were enriched in biosynthesis of secondary metabolites. Among them, the flavonoid and phenylpropanoid biosynthetic pathways were significantly regulated to improve the growth of T. hemsleyanum when treated with Cd. In the KEGG Markup Language (KGML) network analysis, we found some commonly regulated pathways between Cd and Cd+TiO2 NP treatment, including phenylpropanoid biosynthesis, ABC transporters, and isoflavonoid biosynthesis, indicating their potential core network positions in controlling T. hemsleyanum response to Cd stress. Overall, our findings revealed a complex response system for tolerating Cd, encompassing the transportation, reactive oxygen species scavenging, regulation of gene expression, and metabolite accumulation in T. hemsleyanum. Our results indicate that TiO2 NP can be used to reduce Cd toxicity in T. hemsleyanum.

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.

Effect of Adjuvant Steroid Therapy in Type 3 Biliary Atresia: A Single-Center, Open-Label, Randomized Controlled Trial.

OBJECTIVE: To evaluate the efficacy and side effects of additional postoperative steroid therapy for type 3 BA versus the current routine care. SUMMARY BACKGROUND DATA: Whether steroid therapy post-Kasai portoen-terostomy improves the outcomes of BA remains controversial. Clinical evidence from 2 randomized trials in the UK and USA do not support the routine use of steroid in the treatment of BA. METHODS: In this open-label randomized controlled trial, patients with type 3 BA were randomized to routine postoperative treatment with or without 10 to 12 weeks of adjuvant steroid treatment. The primary outcome was the postoperative jaundice clearance rate with native liver at 6 months. The secondary outcomes included postoperative jaundice clearance rate at 3, 12, and 24 months, survival with native liver at 12 and 24 months, and SAEs within 3 months. RESULTS: Overall, 200 participants were randomized and allocated into either steroid or control group (n = 100/group). The proportion of participants that are jaundice free without liver transplantation was significantly higher in the steroid group than in the control group at 6 months (54.1% vs 31.0%, P = 0.0015). The native liver survival rate was higher postoperatively in the steroid group than in the control group at 12 (66.3% vs 50.0%, P = 0.02) and 24 (57.1% vs 40.0%, P = 0.02) months. The survival time with native liver was significantly longer in the steroid group than in the control group (median survival, steroid vs control: not reached vs 1.21 years, P = 0.02). There were no significant differences between the 2 groups in the mean occurrence of SAEs within 3 months (steroid vs control: 0.63 vs 0.45, P = 0.20). CONCLUSIONS: Postoperative adjuvant steroid intervention improved bile drainage and survival with native liver in type 3 BA patients, without increasing early-stage SAEs.

A fluorescent probe based on a phenylalanine derivative is capable of sequential detection of Zn2+ and Cys/His.

A facile and dual fluorescent chemosensor (named 7-IDF) based on a phenylalanine derivative with an indole group was designed and synthesized. 7-IDF can selectively and sensitively detect Zn2+ via obvious fluorescence enhancement in an aqueous solution. Remarkably, the 7-IDF-Zn complex with blue luminescence has higher selectivity toward cysteine (Cys) and histidine (His) than for other amino acids. Intriguingly, 7-IDF can also be used as an excellent probe to detect Zn2+ in real water samples. Moreover, 7-IDF and 7-IDF-Zn possess excellent biocompatibility and cell permeability, and 7-IDF can consecutively detect Zn2+ and Cys/His in Hela cells through fluorescence imaging experiments. This study suggests that the phenylalanine-based chemosensor possesses great potential applications for the sequential detection of Zn2+ and Cys/His in biosystems.

Synthesis of CF3-Containing 2-Imidazolines and Imidazoles via a Iodide-Promoted [3 + 2] Cyclization Reaction.

We report herein a general and effective system achieving cyclization of ß-trifluoromethyl enones with amidines in the presence of 1,3-diiodo-5,5-dimethylhydantoin (DIH), which affords a range of trifluoromethylated 2-imidazolines in synthetically useful yields with good diastereoselectivities (up to 95% yield, up to 98:2 dr) and good functional group tolerance. Furthermore, the one-pot synthesis of trifluoromethylated imidazoles via sequential cyclization and oxidation is demonstrated. More significantly, the reaction mechanism was verified by ESI-MS studies of possible intermediates, and a reasonable reaction mechanism was proposed.

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.

Application of engineered myoglobins for biosynthesis of clofazimine by integration with chemical synthesis.

Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2-N-(4-chlorophenyl) benzene-1,2-diamine (N-4-CPBDA) in the presence of H2O2, with 97% yields. The overall catalytic efficiency (kcat/Km) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.

Azo-Functionalized Thermoplastic Polyurethane for Light-Driven Shape Memory Materials.

Shape memory polymers have great potential in the fields of soft robotics, injectable medical devices, and as essential materials for advanced electronic devices. Herein, light-triggered shape-memory thermoplastic polyurethane (TPU) is reported using azido TPU grafted by the photoswitchable azo compound. The trans-cis transitions of the azobenzene on the side chain of the TPU induce the recoiling of the main chain, leading to shaping memory behavior. Under UV irradiation, cis-azo allows the oriented main chain to recoil to release residual stress and realize light-triggered shape memory behavior. The facile method proposed here for the preparation of azo-functionalized TPU can provide viable opportunities for soft robotics and smart TPU applications.

White-light-emitting Ln-hydrogels with multi-stimuli responsiveness and applications in environmental sensing and anti-counterfeiting.

The construction of smart materials, especially white light emitting (WLE) hydrogels with multi-stimuli responsive properties, has received widespread attention from researchers. In this study, a WLE hydrogel was obtained by the in situ doping of Eu3+ and Tb3+ into a blue emission low molecular weight gelator (MPF). Remarkably, the prepared WLE hydrogel possessed excellent stimuli responsiveness to pH, temperature and chemicals, and could be used as a soft thermometer and a selective sensor for Cu2+. The correlated color temperature of the WLE hydrogel was calculated to be 5063 K, suggesting a potential application in cool white light. Moreover, a series of metallohydrogels with different colors were obtained by modulating the ratio of MPF, Eu3+ and Tb3+ or changing the excitation wavelength, which was an excellent candidate to construct soft materials of a full-color system. Additionally, the WLE hydrogel could be used for constructing anti-counterfeiting materials. Therefore, this study provides a new approach for preparing smart WLE hydrogels with multiple functions.

An Analysis of the Clinical and Radiological Prognostic Factors Affecting the Outcomes of Lumbar Intradiscal Biacuplasty.

Purpose: Intradiscal biacuplasty (IDB) has been proven to be effective for treating lumbar degenerative disc disease (DDD). However, there has not been a reported prognostic factor for IDB. The present study meticulously evaluates the general and radiographic features that may serve as markers for predicting the therapeutic outcome of IDB. Methods: A prospective case series study was conducted, following time-series analysis moving averages models, with forty-one patients suffering from chronic discogenic lower back pain for more than six months. These patients subsequently received lumbar cool radiofrequency IDB and were enrolled in the study. Thirty-seven patients completed follow-up questionnaires at 1, 3, 6, and 12 months. The surgical outcomes were reported using visual analogue scale (VAS), Oswestry disability index (ODI), and the consumption of nonsteroidal anti-inflammatory drugs (NSAID). Furthermore, a univariate analysis was performed to identify prognostic factors associated with pain relief from age, gender, body mass index (BMI), and pre-operative lumbar magnetic resonance imaging reading. Results: Significant reductions were found in estimated VAS and ODI at the post-operative period at 1, 3, 6, and 12 months (P < 0.001). The NSAID dosage was significantly decreased at 3-month and 1-year follow-up (P < 0.05). No procedure-related complications were detected. The prognosis of IDB was not related to disc height, Pfirrmann grading or Modic endplate change. However, disc extrusions were associated with promising outcomes (VAS improvement ≥ 50%) on pain relief (P < 0.05). Conclusion: IDB is a good alternative choice for treating lumbar DDD. Patients with a painful extrusion lumbar disc may gain some benefits after receiving IDB following a period of failed conservative treatment. These findings may also add some references for physicians in the decision making when treating lumbar DDD.

A five-parameter score for predicting saphenous vein graft degenerative and/or occlusive disease in recurring ischemic symptoms after one year post coronary artery bypass grafting.

BACKGROUND: The recurrence rate of ischemic symptoms after coronary artery bypass grafting (CABG) is increasing in recent years. How to prevent and treat saphenous vein graft disease (SVGD [symptomatic ⩾50% stenosis in at least one Saphenous vein graft]) has been a clinical challenge to date. Different pathogenesis may exist in SVGD of different periods. There are currently few available scores for estimating the risk of SVGD after one year post CABG. OBJECTIVE: We sought to develop and validate a simple predictive clinical risk score for SVGD with recurring ischemia after one year post CABG. METHODS AND RESULTS: This was a cross-sectional study and the results were validated using bootstrap resampling on a separate cohort. A nomogram and risk scoring system were developed based on retrospective data from a training cohort of 606 consecutive patients with recurring ischemia >1 year after CABG. Logistic regression model was used to find the predictive factors and to build a nomogram. To assess the generalization, models were validated using bootstrap resampling and an external cross-sectional study of 187 consecutive patients in four other hospitals. In multivariable analysis of the primary cohort, native lesion vessel number, SVG age, recurring ischemia type, very low-density lipoprotein level, and left ventricular end-diastolic diameter were independent predictors. A summary risk score was derived from nomogram, with a cut-off value of 15. In internal and external validation, the C-index was 0.86 and 0.82, indicating good discrimination. The calibration curve for probability of SVGD showed optimal agreement between actual observations and risk score prediction. CONCLUSION: A simple-to-use risk scoring system based on five easily variables was developed and validated to predict the risk of SVGD among patients who recurring ischemia after one year post CABG. This score may be useful for providing patients with individualized estimates of SVGD risk.

The referral pattern and treatment modality for peri-implant disease between periodontists and non-periodontist dentists.

OBJECTIVES: This study is to investigate the referral pattern and treatment modality of dentists in the management of peri-implant diseases between periodontists and non-periodontist dentists (NPDs). MATERIALS AND METHODS: A total of 167 validated questionnaires were obtained from periodontists and NPDs, who had experience of placing implants for at least one year. Question I to IV asked how the dentist would respond if a patient came for treatment of their peri-implant diseases with four different scenarios according to resource of patient and disease severity. For each Scenario, dentists also replied which treatment procedures they would use if they decide to treat the patient. RESULTS: Periodontal training, resource of patient, and disease severity were shown to significantly influence the referral pattern and treatment modality in the management of peri-implant disease (p < 0.05). Periodontists were more likely to use variable treatment procedures, including occlusal adjustment (OR = 2.283, p < 0.01), oral hygiene instruction (OR = 3.751, p < 0.001), topical antiseptic agent (OR = 2.491, p < 0.005), non-surgical mechanical therapy (OR = 2.689, p < 0.001), surgical therapy (OR = 2.009, p < 0.01), and remove implant (OR = 3.486, p < 0.001) to treat peri-implant diseases, compared to NPDs. CONCLUSION: The periodontal specialty training, resource of patient, and disease severity significantly influenced the referral pattern and treatment modality of dentist treating an implant diagnosed with peri-implant disease. This study also highlighted the importance of educating basic periodontal and peri-implant disease-related knowledge to all dentists regularly performing dental implant treatments. CLINICAL RELEVANCE: Peri-implant diseases are highly prevalent among patients with dental implants. Periodontal specialty training could enhance using variable treatment procedures to treat peri-implant diseases for dentists.

The X-ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand-binding sites.

Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.

A novel insight into the molecular mechanism of human soluble guanylyl cyclase focused on catalytic domain in living cells.

Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α1C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from "open to "closed" upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity.

Improving the cell-membrane-penetrating activity of globins by introducing positive charges on protein surface: A case study of sperm whale myoglobin.

Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.