Synthetic Substrates Research Articles

Detection Strategies for Sialic Acid and Sialoglycoconjugates.

Glycoconjugates are a vast class of biomolecules implicated in biological processes important for human health and disease. The structural complexity of glycoconjugates remains a challenge to deciphering their precise biological roles and for their development as biomarkers and therapeutics. Human glycoconjugates on the outside of the cell are modified with sialic (neuraminic) acid residues at their termini. The enzymes that install sialic acids are sialyltransferases (SiaTs), a family of 20 different isoenzymes. The removal and degradation of sialic acids is mediated by neuraminidase (NEU; sialidase) enzymes, of which there are four isoenzymes. In this review, we discuss chemical and biochemical approaches for the detection and analysis of sialoglycoconjugate (SGC) structures and their enzymatic products. The most common methods include affinity probes and synthetic substrates. Fluorogenic and radiolabelled substrates are also important tools for many applications, including screening for enzyme inhibitors. Strategies that give insight into the native substrate-specificity of enzymes that regulate SGCs (SiaT & NEU) are necessary to improve our understanding of the role of sialic acid metabolism in health and disease.

Mechanotransductive N-cadherin binding induces differentiation in human neural stem cells

The neural stem cell niche is a complex microenvironment that includes cellular factors, secreted factors, and physical factors that impact stem cell behavior and development. Cellular interactions through cadherins, cell–cell binding proteins, have implications in embryonic development and mesenchymal stem cell differentiation. However, little is known about the influence of cadherins within the neural stem cell microenvironment and their effect on human stem cell maintenance and differentiation. Therefore, the purpose of this study was to develop synthetic substrates to examine the effect of cadherin mechanotransduction on human neural stem cells. Glass substrates were fabricated using silane, protein A, and recombinant N-cadherin; we used these substrates to examine the effect of N-cadherin binding on neural stem cell proliferation, cytoskeletal structure and morphology, Yes-associated protein-1 (YAP) translocation, and differentiation. Bound exogenous N-cadherin induced concentration-dependent increases in adherens junction formation, YAP translocation, and early expression of neurogenic differentiation markers. Strong F-actin ring structures were initiated by homophilic N-cadherin binding, eliciting neuronal differentiation of cells within 96 ​h without added soluble differentiation factors. Our findings show that active N-cadherin binding plays an important role for differentiation of human iPS-derived neural stem cells towards neurons, providing a new tool to differentiate cells in vitro.

An Integrated Module Performs Selective 'Online' Epoxidation in the Biosynthesis of the Antibiotic Mupirocin.

The delineation of the complex biosynthesis of the potent antibiotic mupirocin, which consists of a mixture of pseudomonic acids (PAs) isolated from Pseudomonas fluorescens NCIMB 10586, presents significant challenges, and the timing and mechanisms of several key transformations remain elusive. Particularly intriguing are the steps that process the linear backbone from the initial polyketide assembly phase to generate the first cyclic intermediate PA-B. These include epoxidation as well as incorporation of the tetrahydropyran (THP) ring and fatty acid side chain required for biological activity. Herein, we show that the mini-module MmpE performs a rare online (ACP-substrate) epoxidation and is integrated ('in-cis') into the polyketide synthase via a docking domain. A linear polyketide fragment with six asymmetric centres was synthesised using a convergent approach and used to demonstrate substrate flux via an atypical KS0 and a previously unannotated ACP (MmpE_ACP). MmpE_ACP-bound synthetic substrates were critical in demonstrating successful epoxidation in vitro by the purified MmpE oxidoreductase domain. Alongside feeding studies, these results confirm the timing as well as chain length dependence of this selective epoxidation. These mechanistic studies pinpoint the location and nature of the polyketide substrate prior to the key formation of the THP ring and esterification that generate PA-B.

Identification of inhibitors of human ChaC1, a cytoplasmic glutathione degrading enzyme through high throughput screens in yeast.

The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. Synthetic substrate analogs were also not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.

Distinct Adjacent Substrate Binding Pocket Regulates the Activity of a Decameric Feruloyl Esterase from Bacteroides thetaiotaomicron.

Understanding how the human gut microbiota contribute to the metabolism of dietary carbohydrates is of great interest, particularly those with ferulic acid (FA) decorations that have manifold health benefits. Here, we report the crystal structure of a decameric feruloyl esterase (BtFae) from Bacteroides thetaiotaomicron in complex with methyl ferulate (MFA), revealing that MFA is situated in a noncatalytic substrate binding pocket adjacent to the catalytic pocket. Molecular docking and mutagenesis studies further demonstrated that the adjacent pocket affects substrate binding in the active site and negatively regulates the BtFae activity on both synthetic and natural xylan substrates. Additionally, quantum mechanics (QM) calculations were employed to investigate the catalytic process of BtFae from substrate binding to product release, and identified TS_2 in the acylation step is rate-limiting. Collectively, this study unmasks a novel regulatory mechanism of FAE activity, which may contribute to further investigation of FA-conjugated polysaccharides metabolism in the human gut.

Inhibition of starch hydrolysis during in vitro co-digestion of pasta with phenolic compound-rich vegetable foods

The ability of phenolic compounds to inhibit amylolytic enzymes activities has been investigated, suggesting their possible role in type-2 diabetes management. However, these studies have been carried out with purified enzymes and synthetic substrates and are distant from simulating a real physiological situation. The objective of the present research was to evaluate the ability of phenolic-rich vegetable foods to inhibit starch hydrolysis during in vitro co-digestion with pasta resulting in a potential anti-diabetic effect and mimicking as closely as possible a real scenario. Some tested vegetable foods, such as capers, red-skinned onion, red radish, and olives, determined a decrease in starch hydrolysis by 21.5%–31.7% during in vitro co-digestion with pasta. The quali-quantitative phenolic profiles of in vitro co-digested samples were elucidated and selected standard compounds were tested for their ability to inhibit porcine pancreatic α-amylase and mammalian α-glucosidase. The inhibitory potential of these compounds, especially against α-glucosidase, explained the effect observed during co-digestion experiments. The most active phenolic compounds against α-glucosidase were quercetin-4’-O-glucoside, quercetin-3-O-rutinoside, luteolin-7-O-glucoside and quercetin-3-O-glucoside-4’-O-glucoside with IC50 values of 20.67, 52.23, 68.84 and 87.58 μmol/L, respectively. This is the first report suggesting that these compounds are potent inhibitors of mammalian α-glucosidase. This study indicates that consuming starchy foods (i.e., pasta) with phenolic-rich vegetable foods may result in an inhibition of starch digestion possibly reducing the post-prandial glucose levels.

Synthetic Hydrogel Substrate for Human Induced Pluripotent Stem Cell Definitive Endoderm Differentiation

Human induced pluripotent stem cells (hiPSCs) can give rise to multiple lineages derived from three germ layers, endoderm, mesoderm and ectoderm. Definitive endoderm (DE) cell types and tissues have great potential for regenerative medicine applications. Current hiPSC differentiation protocols focus on the addition of soluble factors; however, extracellular matrix properties are known to also play a role in dictating cell fate. Matrigel™ is the gold standard for DE differentiation, but this xenogeneic, poorly defined basement membrane extract limits the clinical translatability of DE-derived tissues. Here we present a fully defined PEG-based hydrogel substrate to support hiPSC-derived DE differentiation. We screened hydrogel formulations presenting different adhesive peptides and matrix stiffness. Our results demonstrate that presenting a short peptide, cyclic RGD, on the engineered PEG hydrogel supports the transition from undifferentiated hiPSCs to DE using a serum-free, commercially available kit. We show that increasing substrate stiffness (G’=1.0-4.0 kPa) results in an increased linear response in DE differentiation efficiency. We also include a temporal analysis of the expression of integrin and syndecan receptors as the hiPSCs undergo specification towards DE lineage. Finally, we show that focal adhesion kinase activity regulates hiPSC growth and DE differentiation efficiency. Overall, we present a fully defined matrix as a synthetic alternative for Matrigel™ supporting DE differentiation.

A green, cheap and robust method for selective hydrogenation of nitroarenes

A commercially available nickel was utilized as an effective catalyst for the exclusive hydrogenation of nitroarenes to primary anilines in gram-scale without additional column chromatographic purification. This novel synthetic hydrogenation strategy features wide substrate scope, mild conditions and operational simplicity in aqueous media at room temperature. Further transformations resulted in the delivery of important amine-containing drugs and pharmaceutical intermediates.

Heterologous expression, characterisation and 3D-structural insights of GH18 chitinases derived from sago palm (Metroxylon sagu)

Although plants don't have chitins, they produce chitinases to protect themselves from biotic and abiotic stressors. There are two forms of chitinases found in organisms: glycosyl hydrolase 18 (GH18) and 19 (GH19) families. Plant GH19 chitinases are well known for their role in protecting against pathogens, but the roles of GH18 chitinases have not been fully elucidated. This study aimed to produce and characterise two recombinant GH18 chitinases from Metroxylon sagu. Two GH18 chitinase genes, MsChi1 and MsChi2, were identified, with nucleotide sequences of 1009 and 1308 bp, respectively. The proteins encoded by MsChi1 and MsChi2 genes were single polypeptide chains of 310 and 300 amino acids with predicted molecular masses of 31.21 and 30.15 kDa, respectively. Both cDNAs were cloned and expressed in the GS115 strain of Pichia pastoris. Recombinant MsChi1 and MsChi2 exhibited optimal activity at 60 °C with acidic pH 4.0 and 5.0, respectively. Both recombinant enzymes could hydrolyze synthetic and natural substrates (colloidal chitin). rMsChi1 preferred 4-nitrophenol N,N′-diacetyl-β-D chitobioside, while rMsChi2 preferred 4-nitrophenol N,N′,N″-triacetyl-β-D chitotriose, suggesting they might function as exochitinase and endochitinase, respectively. They also demonstrated antifungal activities against tested fungi. Homology modeling indicated ASP and GLU as essential residues for proton donation and acceptance.

Synthesis of a natural core substrate with lignin-xylan cross-linkage for unveiling the productive kinetic parameters of glucuronoyl esterase

Lignin-carbohydrate complexes (LCCs) present a considerable hurdle to the economic utilization of lignocellulosic biomass. Glucuronoyl esterase (GE) is an LCC-degrading enzyme that catalyzes the cleavage of the cross-linkages between lignin and xylan in LCCs. Benzyl-d-glucuronate (Bn-GlcA), a commercially available substrate, is widely used to evaluate GE activity assays. However, since Bn-GlcA lacks the structural backbone of naturally occurring LCCs, the mechanisms underlying the activity of GEs and their diversity in the structure-activity relationship are not fully understood. Herein, we provided a synthesis scheme for designing 1,23-α-d-(6-benzyl-4-O-methyl-glucuronyl)-1,4-β-d-xylotriose (Bn-MeGlcA3Xyl3) as a natural core substrate bearing cross-linkage between lignin and glucuronoxylan. A well-defined and yet more realistic synthetic substrate was successfully synthesized via a key step of the benzyl esterification of 4-O-methyl-glucuronyl-1,4-β-d-xylotriose (MeGlcA3Xyl3), a minimized fragment of glucuronoxylan enzymatically digested by β-1,4-xylanase. To the best of our knowledge, this is the first report of the productive GE kinetic analysis using this substrate. Kinetic parameters of the GE from the fungal Pestalotiopsis sp. AN-7 (PesGE), i.e., the Km, Vmax, and kcat of Bn-MeGlcA3Xyl3, were 0.43 mM, 55.5 μmol min−1·mg−1, and 35.8 s−1, respectively. On the other hand, as reported to date, the productive kinetic parameters for Bn-GlcA were not obtained because of its excessively high Km value (>16 mM). The substantial variance in the enzymatic activity of PesGE regarding substrate-binding affinity between Bn-MeGlcA3Xyl3 and Bn-GlcA was also demonstrated using in silico docking simulation. These results suggested that the extended xylan fragment is a key structural determinant affecting PesGE's substrate recognition. Furthermore, the presence of a natural xylan backbone allows for evaluating the enzyme activity of xylan-degrading enzymes. Accordingly, the synthesized substrate with the natural core structure of LCC allowed us to unveil the productive kinetic parameters of GEs, serving as a versatile substrate for further elucidating the cascade reaction of GE and xylan-degrading enzymes.

Production of milk-coagulating protease by fungus Pleurotus djamor through solid state fermentation using wheat bran as the low-cost substrate

Proteases are enzymes that hydrolyze peptide bonds present in proteins and peptides. They are widely used for various industrial applications, such as in the detergent, food, and dairy industries. Cheese is one of the most important products of the dairy industry, and the coagulation stage is crucial during the cheese-making process. Enzymatic coagulation is the most common technique utilized for this purpose. Microbial enzymes are frequently used for coagulation due to their advantages in terms of availability, sustainability, quality control, product variety, and compliance with dietary and cultural/religious requirements. In the present study, we identified and subsequently characterized milk coagulant activity from the fungus Pleurotus djamor PLO13, obtained during a solid-state fermentation process, using the agro-industrial residue, wheat bran, as the fermentation medium. Maximum enzyme production and caseinolytic activity occurred 120 h after cultivation. When the enzyme activity against various protease-specific synthetic substrates and inhibitors was analyzed, the enzyme was found to be a serine protease, similar to elastase 2. This elastase-2-like serine protease was able to coagulate pasteurized whole and reconstituted skim milk highly efficiently in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time, and calcium concentration. We demonstrate here, for the first time, an elastase-2-like enzyme in a microorganism and its potential application in the food industry for cheese production.

Effects on Iron Metabolism and System Xc- /GPX4 Pathway from Hydroquinone Suggest Ferroptosis of Jurkat Cells.

Prolonged exposure to hydroquinone (HQ), a metabolite of benzene, can cause severe haematologic disorders in humans. However, the mechanism is still unclear. In the present study, we investigated whether HQ can induce haematological diseases through ferroptosis, which is another form of cell death apart from apoptosis. The results showed that HQ inhibited the viability of Jurkat cells in a dose-dependent and time-dependent manner. The half inhibitory concentrations (IC50s) of HQ-treated Jurkat cells for 12 h, 24 h and 48 h were 107.16 μmol/L, 33.29 μmol/L, and 14.78 μmol/L. The exposure of Jurkat cells to HQ increased intracellular Fe2+, malondialdehyde (MDA) and lipid reactive oxygen species (ROS) levels and down-regulated glutathione (GSH) levels. We used erastin-treated cells as a positive control and cells treated with HQ combined with deferoxamine mesylate (DFO) and ferrostain-1 (Fer-1)-treated cells as the negative controls. DFO and Fer-1 partially restored the degradation of cell viability and GSH content and the accumulation of Fe2+, MDA and lipid ROS caused by HQ. In addition, we found that cellular mitochondria in the HQ-treated group showed a decrease in volume, an increase in the density of the bilayer membrane and a decrease or disappearance of mitochondrial cristae. Changes in the erastin-treated group were similar to those in the HQ-treated group. We inferred that HQ induces ferroptosis in Jurkat cells. Subsequently, we found that HQ up-regulated the levels of transferrin receptor 1 (TFRC) mRNA and protein expression and down-regulated FTH1, SLC7A11 and synthetic substrate of antioxidant enzyme 4 (GPX4) mRNA levels and protein expression levels. However, the exposure of Jurkat cells to HQ with DFO and Fer-1 alleviated these changes. Notably, the activation of TFRC and the inhibition of FTH1 and System Xc- (cystine-glutamate reverse transporter protein) /GPX4 were associated with HQ-induced ferroptosis. These results provide novel insights into how HQ exacerbates haematopoietic cytotoxicity and provide potential targets for the prevention of HQ-induced diseases.

Synthetic Amine Linkers for Efficient Sortagging.

Enzymatic site-specific bioconjugation techniques, in particular sortase-mediated ligation, are increasingly used to generate conjugated proteins for a wide array of applications. Extension of the utility and practicality of sortagging for diverse purposes is critically dependent on further improvement of the efficiency of sortagging reactions with a wider structural variety of substrates. We present a comprehensive comparative mass spectrometry screening study of synthetic nonpeptidic incoming amine nucleophile substrates of Staphylococcus aureus Sortase A enzyme. We have identified the optimal structural motifs among the chemically diverse set of 452 model primary and secondary amine-containing sortagging substrates, and we demonstrate the utility of representative amine linkers for efficient C-terminal biotinylation of nanobodies.

Exploring stevioside binding affinity with various proteins and receptors actively involved in the signaling pathway and a future candidate for diabetic patients.

Introduction and Background: Diabetes is a chronic metabolic disease characterized by elevated blood glucose levels and is one of the main global health concerns. Synthetic sugar substrate has many side effects such as leukemia, bladder cancer, hepatotoxicity, breast cancer, headache, and brain toxicity. The WHO and FDA has recently banned some of the synthetic sugar alternatives due to their carcinogenic effects. Objective and Methodology: Therefore, the main objective of the current study was to investigate the safety and binding affinity of Stevioside with Glucose Transpoter-4 (GLUT-4), Akt, Insulin Receptor (IR) and Insulin Receptor Substrate-1 (IRS-1) to confirmed that Stevioside is one the potent natural sweetener/drug for diabetes. This study delves into the molecular interaction between Stevioside and key diabetic proteins: GLUT-4, Akt, IR and IRS-1. A precise molecular docking approach was used to simulate the binding affinity of Stevioside to these proteins. The pharmacokinetic properties of the molecule should be taken into consideration as important variables throughout the virtual screening process. Results: The result of active site analysis of GLUT-4, Akt, IR and IRS-1 showed a zone of 2158.359 Ǻ2, 579.259 Ǻ2, 762.651 Ǻ2, and 152.167 Ǻ2 and a volume of 2765.094 Ǻ³, 355.567 Ǻ³, 686.806 Ǻ³, and 116.874 Ǻ³, respectively. Docking analysis of the Stevioside compound showed the highest docking energy with scores of -9.9 with GLUT-4, -6.7 with Akt, -8.0 with IR and -8.8 with IRS-1. Studies indicated that it remains undigested by stomach acids and enzymes and is not absorbed in the upper small intestine. Further, tests revealed no hepatotoxicity, AMES toxicity, or skin sensitivity, making it a promising candidate for safe consumption as drug metabolism. Conclusion and Recommendations: Instead of other sugar alternatives, Stevioside will help diabetic patients with a lower chance of infections, lowered blood pressure/blood sugar, and increased glucose uptake in diabetic muscles. Stevioside is a natural sweetener, and the current study recommends its usage in various dietary products for diabetic patients.

An Integrated Module Performs Selective ‘On‐Line’ Epoxidation in the Biosynthesis of the Antibiotic Mupirocin.

The delineation of the complex biosynthesis of the potent antibiotic mupirocin, which consists of a mixture of pseudomonic acids (PAs) isolated from Pseudomonas fluorescens NCIMB 10586, presents significant challenges and the timing and mechanisms of several key transformations remain elusive. Particularly intriguing are the steps that process the linear backbone from the initial polyketide assembly phase to generate the first cyclic intermediate PA‐B. These include epoxidation as well as incorporation of the tetrahydropyran (THP) ring and fatty acid sidechain required for biological activity. Here, we show that the mini‐module MmpE performs a rare online (ACP‐substrate) epoxidation and is integrated (‘in‐cis’) into the polyketide synthase via a docking domain. A linear polyketide fragment with 6 asymmetric centres was synthesised using a convergent approach and used to demonstrate substrate flux via an atypical KS0 and a previously unannotated ACP (MmpE_ACP). MmpE_ACP‐bound synthetic substrates were critical in demonstrating successful epoxidation in vitro by the purified MmpE oxidoreductase domain. Alongside feeding studies, these results confirm the timing as well as chain length dependence of this selective epoxidation. These mechanistic studies pinpoint the location and nature of the polyketide substrate prior to the key formation of the THP ring and esterification that generate PA‐B.

Fe3O4@silica-thermolysin: A robust, advantageous, and reusable microbial nanobiocatalyst for proteolysis and milk-clotting

Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

Tailoring the specificity of ficin versus large hemoglobin and small casein by co-immobilizing inert proteins on the immobilized enzyme layer and further modification with aldehyde dextran

Ficin has been immobilized at full loading on glyoxyl agarose beads. Then, ficin was blocked with 2,2′-dipyridyldisulfide. To be effective, the modification must be performed in the presence of 0.5 M urea, as the enzyme was not inhibited under standard conditions, very likely because the catalytic Cys was not fully exposed to the medium. Activity could be fully recovered by incubation with 1 M mercaptoethanol. This biocatalyst could hydrolyze hemoglobin and casein. The objective of this paper was to increase the enzyme specificity versus small proteins by generating steric hindrances to the access of large proteins. The step by step blocking via ionic exchange of the biocatalyst with aminated bovine serum albumin (BSA), aldehyde dextran and a second layer of aminated BSA produced a biocatalyst that maintained its activity versus small synthetic substrates, increased the biocatalyst stability, while reduced its activity to over 50 % versus casein. Interestingly, this treatment almost fully annulled the activity versus hemoglobin, more effectively at 37 °C than at 55 °C. The biocatalyst could be reused 5 times without changes in activity. The changes could be caused by steric hindrances, but it cannot be discarded some changes in enzyme sequence specificity caused by the modifications.

TiO2 nanofilms for surface-enhanced Raman scattering analysis of urea

In this study, titanium dioxide (TiO2) nanofilms with nanoparticle structure were grown in situ on metallic aluminum (Al) sheets using a simple sol-hydrothermal method. Al sheets were chosen because they can form Schottky junctions with TiO2 during the calcination process, thus achieving a tight bonding between the nanoparticles and the solid substrate, which cannot be achieved with conventional glass substrates. The substrates synthesized with different contents of titanium butoxide [Ti(OBu)4] were investigated using 4-mercaptobenzoic acid as a probe molecule, and the results showed that the substrate with 9 % of the total volume of Ti(OBu)4 had the highest surface-enhanced Raman scattering (SERS) performance. As a low-cost SERS substrate that is simple to synthesize, it has excellent signal reproducibility, with a relative standard deviation of 4.51 % for the same substrate and 6.43 % for different batches of synthesized substrates. Meanwhile, the same batch of substrate can be stored at room temperature for at least 20 weeks and still maintain stable SERS signals. In addition, the synthetic substrate was used to quantitatively detect urea with a detection limit of 4.23 × 10−3 mol/L, which is comparable to the application of noble metal substrates. The feasibility of this method was verified in human urine, and the results were consistent with the clinical results, indicating that this method has great potential for clinical application.

Recent Progress in Synthesis of Alkyl Fluorinated Compounds with Multiple Contiguous Stereogenic Centers.

Organic fluorides are widely used in pharmaceuticals, agrochemicals, material sciences, and other fields due to the special physical and chemical properties of fluorine atoms. The synthesis of alkyl fluorinated compounds bearing multiple contiguous stereogenic centers is the most challenging research area in synthetic chemistry and has received extensive attention from chemists. This review summarized the important research progress in the field over the past decade, including asymmetric electrophilic fluorination and the asymmetric elaboration of fluorinated substrates (such as allylic alkylation reactions, hydrofunctionalization reactions, Mannich addition reactions, Michael addition reactions, aldol addition reactions, and miscellaneous reactions), with an emphasis on synthetic methodologies, substrate scopes, and reaction mechanisms.

Innovative Substrate Design with Basement Membrane Components for Enhanced Endothelial Cell Function and Endothelization.

Enhancing endothelial cell growth on small-diameter vascular grafts produced from decellularized tissues or synthetic substrates is pivotal for preventing thrombosis. While optimized decellularization protocols can preserve the structure and many components of the extracellular matrix (ECM), the process can still lead to the loss of crucial basement membrane proteins, such as laminin, collagen IV, and perlecan, which are pivotal for endothelial cell adherence and functional growth. This loss can result in poor endothelialization and endothelial cell activation causing thrombosis and intimal hyperplasia. To address this, the basement membrane's ECM is emulated on fiber substrates, providing a more physiological environment for endothelial cells. Thus, fibroblasts are cultured on fiber substrates to produce an ECM membrane substrate (EMMS) with basement membrane proteins. The EMMS then underwent antigen removal (AR) treatment to eliminate antigens from the membrane while preserving essential proteins and producing an AR-treated membrane substrate (AMS). Subsequently, human endothelial cells cultured on the AMS exhibited superior proliferation, nitric oxide production, and increased expression of endothelial markers of quiescence/homeostasis, along with autophagy and antithrombotic factors, compared to those on the decellularized aortic tissue. This strategy showed the potential of pre-endowing fiber substrates with a basement membrane to enable better endothelization.