Boron-Nitrogen-Embedded Polycyclic Aromatic Hydrocarbon-Based Controllable Hierarchical Self-Assemblies through Synergistic Cation-π and C-H···π Interactions for Bifunctional Photo- and Electro-Catalysis.

Boron-Nitrogen-embedded polycyclic aromatic hydrocarbons (BN-PAHs) as novel π-conjugated systems have attracted immense attention owing to their superior optoelectronic properties. However, constructing long-range ordered supramolecular assemblies based on BN-PAHs remains conspicuously scarce, primarily attributed to the constraints arising from coordinating multiple noncovalent interactions and the intrinsic characteristics of BN-PAHs, which hinder precise control over delicate self-assembly processes. Herein, we achieve the successful formation of BN-PAH-based controllable hierarchical assemblies through synergistically leveraged cation-π and C-H···π interactions. By carefully adjusting the solvent conditions in two progressive assembly hierarchies, the one-dimensional (1D) supramolecular assemblies with "rigid yet flexible" assembled units are first formed by cation-π interactions, and then they can be gradually fused into two-dimensional (2D) structures under specific C-H···π interactions, thus realizing the precise control of the transformation process from BN-PAH-based 1D primary structures to 2D higher-order assemblies. The resulting 2D-BNSA, characterized by enhanced electrical conductivity and ordered 2D layered structure, provides anchoring and dispersion sites for loading two appropriate nanocatalysts, thus facilitating the efficient photocatalytic CO2 reduction (with a remarkable CH4 evolution rate of 938.7 µmol g-1 h-1) and electrocatalytic acetylene semihydrogenation (reaching a Faradaic efficiency for ethylene up to 98.5%).

Evolving ω-amine transaminase AtATA guided by substrate-enzyme binding free energy for enhancing activity and stability against non-natural substrates.

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from Aspergillus terreus (AtATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate (R)-(+)-1-(1-naphthyl)ethylamine [(R)-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 (AtATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to AtATA structure, catalytic activity, and stability in silico analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing (R)-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the AtATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from Aspergillus terreus toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The robust combinational variant acquired displayed significant application value for pushing the asymmetric synthesis of aromatic chiral amines to a higher level.

Intramolecular Cation-π Interactions Organize Bowl-Shaped, Luminescent Molecular Containers.

Molecules with nonplanar architectures are highly desirable due to their unique topological structures and functions. We report here the synthesis of two molecular containers (1 ⋅ 3Br- and 1 ⋅ 3Cl-), which utilize intramolecular cation-π interactions to enforce macrocylic arrangements and exhibit high binding affinity and luminescent properties. Remarkably, the geometry of the cation-π interaction can be flexibly tailored to achieve a precise ring arrangement, irrespective of the angle of the noncovalent bonds. Additionally, the C-H⋅⋅⋅Br- hydrogen bonds within the container are also conducive to stabilizing the bowl-shaped conformation. These bowl-shaped conformations were confirmed both in solution through NMR spectroscopy and in the solid state by X-ray studies. 1 ⋅ 3Br- shows high binding affinity and selectivity: F->Cl-, through C-H⋅⋅⋅X- (X=F, Cl) hydrogen bonds. Additionally, these containers exhibited blue fluorescence in solution and yellow room-temperature phosphorescence (RTP) in the solid state. Our findings illustrate the utility of cation-π interactions in designing functional molecules.

Atomistic insight into the binding mode and self-regulation mechanism of IsPETase towards PET substrates with different polymerization degrees.

Poly(ethylene terephthalate) (PET) is one of the most widely used synthetic polyesters, however, its extensive use creates a long-term environmental burden. Unlike traditional recycling methods, biodegradation is a sustainable strategy. The emergence of PETase from Ideonella sakaiensis 201-F6 (IsPETase) has brought great potential for the industrialization of degradable PET. In this work, models of enzyme-substrate complexes with different degrees of polymerization were established to study the binding mode using molecular dynamics simulation. We found that the whole binding site can be further subdivided into three parts, including head, middle and tail binding regions. Most importantly, the presence of the middle region formed by both ends of Ser93 and Ser236 provides a potential possibility for the binding of substrates with different chain lengths, and exerts the self-regulation ability of enzymes to accommodate substrates. Meanwhile, the 'pocket bottom' Arg280 in the tail region echoes the 'pocket mouth' Trp185 in the head region, defining the substrate binding region. This work reveals the self-regulation of IsPETase, as well as the key residues for the substrate binding. The solution to these problems enables us to better understand the function of enzymes and design high-performance degradation enzymes, which is of great significance for industrial application research.

A Bioinspired Free-Standing 2D Crown-Ether-Based Polyimine Membrane for Selective Proton Transport.

Biological proton channels play important roles in the delicate metabolism process, and have led to great interest in mimicking selective proton transport. Herein, we designed a bioinspired proton transport membrane by incorporating flexible 14-crown-4 (14C4) units into rigid frameworks of polyimine films by an interfacial Schiff base reaction. The Young's modulus of the membrane reaches about 8.2 GPa. The 14C4 units could grab water, thereby forming hydrogen bond-water networks and acting as jumping sites to lower the energy barrier of proton transport. The molecular chains present a vertical orientation to the membrane, and the ions travel between the quasi-planar molecular sheets. Furthermore, the 14C4 moieties could bond alkali ions through host-guest interactions. Thus, the ion conductance follows H+ ≫K+ >Na+ >Li+ , and an ultrahigh selectivity of H+ /Li+ (ca. 215) is obtained. This study provides an effective avenue for developing ion-selective membranes by embedding macrocycle motifs with inherent cavities.

Caveolin-1 increases glycolysis in pancreatic cancer cells and triggers cachectic states.

In pancreatic cancer, autocrine insulin-like growth factor-1 (IGF-1) and paracrine insulin stimulate both IGF-1 receptor (IGF1R) and insulin receptor (IR) to increase tumor growth and glycolysis. In pancreatic cancer patients, cancer-induced glycolysis increases hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis and, thereby, causes cancer cachexia. As a protein coexisting with IGF1R and IR, caveolin-1 (cav-1) may be involved in pancreatic cancer-induced cachexia. We undertook the present study to test this hypothesis. Out of wild-type MiaPaCa2 and AsPC1 human pancreatic cancer cell lines, we created their stable sub-lines whose cav-1 expression was diminished with RNA interference or increased with transgene expression. When these cells were studied in vitro, we found that cav-1 regulated IGF1R/IR expression and activation and also regulated cellular glycolysis. We transplanted the different types of MiaPaCa2 cells in growing athymic mice for 8 weeks, using intact athymic mice as tumor-free controls. We found that cav-1 levels in tumor grafts were correlated with expression levels of the enzymes that regulated hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis in the respective tissues. When the tumors had original or increased cav-1, their carriers' body weight gain was less than the tumor-free reference. When cav-1 was diminished in tumors, the tumor carriers' body weight gain was not changed significantly, compared to the tumor-free reference. In conclusion, cav-1 in pancreatic cancer cells stimulated IGF1R/IR and glycolysis in the cancer cells and triggered cachectic states in the tumor carrier.

Pathogenesis and Therapeutic Strategies Related to Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), one of the most common types of chronic liver disease, is strongly correlated with obesity, insulin resistance, metabolic syndrome, and genetic components. The pathological progression of NAFLD, consisting of non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and liver cirrhosis, is characterized by a broad spectrum of clinical phenotypes. Although patients with mild NAFL are considered to show no obvious clinical symptoms, patients with long-term NAFL may culminate in NASH and further liver fibrosis. Even though various drugs are able to improve NAFLD, there are no FDA-approved medications that directly treat NAFLD. In this paper, the pathogenesis of NAFLD, the potential therapeutic targets, and their underlying mechanisms of action were reviewed.

Self-Assembly of Adaptive Chiral [1]Rotaxane for Thermo-Rulable Circularly Polarized Luminescence.

It remains a formidable challenge to realize thermo-rulable circularly polarized luminescence (CPL). Herein, we propose a new strategy by covalently "potting" the emitter into a flexible chiral macrocycle as an adaptive chiral [1]rotaxane to address this issue. Different from its included analogue, the potted emitter [DS] showed adaptive conformers with tunable CPL in varied solvents. Significantly, upon controlled self-assembly, [DS] formed a channel-type nanoarchitecture ([DS]A ), which exhibited an extraordinary thermo-rulable CPL between 25-100 °C. Impressively, the glum was large as 0.024 at 25 °C, which was amplified to 0.040 at 90 °C with a decrease of QY (from 92 % to 80 %). By comparison, the assemblies of cage-type [DS]B and the unpotted emitter showed irreversible or unanswered thermal behaviors. The work demonstrated the thermo-rulable and durable CPL for the first time and provided an insightful understanding of thermal effect on high-temperature and switchable CPL materials.

Semirational engineering of an aldo-keto reductase KmAKR for overcoming trade-offs between catalytic activity and thermostability.

Enzyme engineering usually generates trade-offs between activity, stability, and selectivity. Herein, we report semirational engineering of an aldo-keto reductase (AKR) KmAKR for simultaneously enhancing its thermostability and catalytic activity. Previously, we constructed KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C), which showed outstanding activity towards t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-CDHH), and t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, the key chiral building blocks of rosuvastatin and atorvastatin. Under the guidance of computer-aided design including consensus residues analysis and molecular dynamics (MD) simulations, K164, S182, S232, and Q266 were dug out for their thermostability conferring roles, generating the "best" mutant KmAKRM13 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The Tm and T5015 values of KmAKRM13 were 10.4 and 6.1°C higher than that of KmAKRM9 , respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over KmAKRM9 . Structural analysis indicated that stabilization of the α-helixes mainly contributed to thermostability enhancement. Under the optimized conditions, KmAKRM13 completely asymmetrically reduced 400 g/l t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) in 8.0 h at a high substrate to catalyst ratio (S/C) of 106.7 g/g, giving diastereomerically pure (3R,5S)-CDHH (>99.5% d.e.P ) with a space-time yield (STY) of 449.2 g/l·d.

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase.

Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (kcat /KM = 47.3 s-1 ·mM-1 ), improved stereoselectivity (from moderate selectivity (deP = 66.7%) to strict (S)-selectivity (deP > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.

The introduction of L-phenylalanine into antimicrobial peptide protonectin enhances the selective antibacterial activity of its derivative phe-Prt against Gram-positive bacteria.

Protonectin was a typical amphiphilic antimicrobial peptide with potent antimicrobial activity against Gram-positive and Gram-negative bacteria. In the present study, when its eleventh amino acid in the sequence was substituted by phenylalanine, the analog named phe-Prt showed potent antimicrobial activity against Gram-positive bacteria, but no antimicrobial activity against Gram-negative bacteria, indicating a significant selectivity between Gram-positive bacteria and Gram-negative bacteria. However, when Gram-negative bacteria were incubated with EDTA, the bacteria were susceptible to phe-Prt. Next, the binding effect of phe-Prt with LPS was determined. Our result showed that LPS could hamper the bactericidal activity of phe-Prt against Gram-positive bacteria. The result of zeta potential assay further confirmed the binding effect of phe-Prt with LPS for it could neutralize the surface charge of E. coli and LPS. Then, the effect of phe-Prt on the integrity of outer membrane of Gram-negative bacteria was determined. Our results showed that phe-Prt had a much weaker disturbance to the outer membrane of Gram-negative bacteria than the parent peptide protonectin. In summary, the introduction of L-phenylalanine into the sequence of antimicrobial peptide protonectin made phe-Prt show significant selectivity against Gram-positive bacteria, which could partly be attributed to the delay effect of LPS for phe-Prt to access to cell membrane. Although further study is still needed to clarify the exact mechanism of selectivity, the present study provided a strategy to develop antimicrobial peptides with selectivity toward Gram-positive and Gram-negative bacteria.

Thiol-Rich Multifunctional Macromolecular Crosslinker for Gelatin-Norbornene-Based Bioprinting.

Extrusion-based bioprinting is an emerging and most frequently used technique for the fabrication of cell-laden constructs. A suitable hydrogel-based bioink for cell encapsulation and protection is critical for printability, structural stability, and post-printing cell viability. The thiol-ene chemistry-based gelatin-norbornene (GelNB) hydrogels have drawn much attention as a promising substitution of gelatin methacryloyl (GelMA), owing to the fast and controllable step-growth polymerization mechanism, as well as a significant reduction in reactive oxygen species (ROS) accumulation. Herein, thiolated heparin (HepSH) was synthesized and used as a macromolecular crosslinker for GelNB-based bioprinting, so that GelNB gelation became less sensitive to the thiol/ene ratio. The mechanical stability and moduli of GelNB/HepSH hydrogels were easily manipulated by the concentration and/or degree of thiol substitution. The GelNB/HepSH hydrogel allowed little intracellular ROS for encapsulated cells but provided vascular endothelial growth factor binding affinity for potential facilitation of neovascularization. Finally, the GelNB/HepSH bioink enabled a convenient printing process for both complex-structured bioscaffolds and cell-laden constructs, and resulted in good printability and high post-crosslinking cell viability. The crosslinker HepSH may serve as a multifunctional macromolecule that enables GelNB-based bioprinting in broad applications in regenerative medicine.

Tailoring an aldo-keto reductase KmAKR for robust thermostability and catalytic efficiency by stepwise evolution and structure-guided consensus engineering.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an advanced chiral diol intermediate of the cholesterol-lowering drug atorvastatin. KmAKRM5 (W297H/Y296W/K29H/Y28A/T63M) constructed in our previous work, displayed good biocatalytic performance on (3R,5R)-2. In the present work, stepwise evolution was applied to further enhance the thermostability and activity of KmAKRM5. For thermostability enhancement, N109 and S196 located far from the active site were picked out by structure-guided consensus engineering, and mutated by site-directed mutagenesis (SDM). For catalytic efficiency improvement, the residues A30 and T302 adjacent to the substrate-binding pocket were subjected to site-saturation mutagenesis (SSM). As a result, the "best" mutant KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C) was developed, of which T5015 and Tm were 5.0 °C and 8.2 °C higher than those of KmAKRM5. Moreover, compared to KmAKRM5, KmAKRM9 displayed a 1.9-fold (846 vs 2436 min) and 6.7-fold (126 vs 972 min) longer half-lives at 40 and 50 °C, respectively. Structural analysis suggested that beneficial mutations introduced additional hydrophobic interactions and hydrogen bonds, contributing rigidification of the flexible loops and the increase of internal forces, hence increasing the thermostability and activity. 5 g DCW (dry cell weight) L-1KmAKRM9 completely reduced 350 g L-1t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate ((5R)-1), within 3.7 h at 40 °C, yielding optically pure (3R,5R)-2 (d.e.p > 99.5%) with a space-time yield (STY) of 1.82 kg L-1 d-1. Hence, KmAKRM9 is a robust biocatalyst for the synthesis of (3R,5R)-2.

Female-specific activation of pregnane X receptor mediates sex difference in fetal hepatotoxicity by prenatal monocrotaline exposure.

Pyrrolizidine alkaloids (PAs) are a group of hepatic toxicant widely present in plants. Cytochrome P450 (CYP) 3A plays a key role in metabolic activation of PAs to generate electrophilic metabolites, which is the main cause of hepatotoxicity. We have previously demonstrated the sex difference in developmental toxicity and hepatotoxicity in fetal rats exposed to monocrotaline (MCT), a representative toxic PA. The aim of this study was to explore the underlying mechanism. 20 mg·kg-1·d-1 MCT was intragastrically given to pregnant Wistar rats from gestation day 9 to 20. CYP3As expression and pregnane X receptor (PXR) activation were specifically enhanced in female fetal liver. After MCT treatment, we also observed a significant increase of CYP3As expression in LO2 cells (high PXR level) or hPXR-transfected HepG2 cells (low PXR level). Employing hPXR and CYP3A4 dual-luciferase reporter gene assay, we confirmed the agonism effect of MCT on PXR-dependent transcriptional activity of CYP3A4. Agonism and antagonism of the androgen receptor (AR) either induced or blocked MCT-induced PXR activation, respectively. This study was the first report identifying that MCT served as PXR agonist to induce CYP3A expression. CYP3A induction may increase self-metabolic activation of MCT and subsequently lead to more severe hepatotoxicity in female fetus. While in male, during the intrauterine period, activated AR by testosterone secretion from developing testes represses MCT-induced PXR activation and CYP3A induction, which may partially protect male fetus from MCT-induced hepatotoxicity.

Weak-field polaron dynamics in organic ferromagnets.

With a nonadiabatic dynamical method the polaron dynamics in organic ferromagnets with spin radicals is investigated under weak electric fields. The results reveal two novel phenomena different from those in normal polymers due to the existence of spin radicals. One is that the velocity of the polaron is asymmetric upon the reversal of the applied electric field, which is explained from the asymmetric polarity of the polaron charge density in different directions of the field, and hence its effect on the lattice distortion. The other is the 'intermittent rebound' of the polaron, where the polaron intermittently moves against the electric field force during a short interval behaving like a negative current. The details of lattice distortion and charge distribution of the polaron during the process have been revealed. We further found that there exist different critical fields for the above two phenomena. With an increase of the electric field, the 'intermittent rebound' of the polaron vanishes first and subsequently the asymmetric polaron velocity. This work demonstrates the unique properties of polaron transport in organic ferromagnets, and will be helpful in the future design of organic ferromagnetic devices.

Co-evolution of activity and thermostability of an aldo-keto reductase KmAKR for asymmetric synthesis of statin precursor dichiral diols.

Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the "best" variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose Km, kcat/Km towards (5R)-1 was 0.66 mM and 210.77 s-1 mM-1, respectively, with improved thermostability (half-life of 14.13 h at 40 °C). Combined with 1.5 g dry cell weight (DCW) L-1Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW L-1Variant VI completely reduced (5R)-1 of up to 450 g L-1 within 7.0 h at 40 °C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-3, >99.5% d.e.p) with a space-time yield (STY) of 1.24 kg L-1 day-1, and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW L-1Variant VI completely reduced 400 g L-1 (5S)-2, within 5.0 h at 40 °C, yielding optically pure tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-4, >99.5% d.e.p) with a STY of 1.34 kg L-1 day-1. In summary, Variant VI displayed industrial application potential in statins biomanufacturing.

Increased dietary fatty acids determine the fatty-acid profiles of human pancreatic cancer cells and their carrier's plasma, pancreas and liver.

Primary contents of dietary fat are three or four types of fatty acids, namely saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), n6-polyunsaturated fatty acid (n6PUFA) and, to less extent, n3-polyunsaturated fatty acid (n3PUFA). Previous studies suggest that increased SFA, MUFA, and n6PUFA in high fat diets (HFDs) stimulate the origination, growth, and liver metastasis of pancreatic cancer cells, whereas increased n3PUFA has the opposite effects. It is unclear whether the fatty acid-induced effects are based on changed fatty-acid composition of involved cells. Here, we investigated whether increased SFA, MUFA, n6PUFA, and n3PUFA in different HFDs determine the FA profiles of pancreatic cancer cells and their carrier's plasma, pancreas, and liver. We transplanted MiaPaCa2 human pancreatic cancer cells in athymic mice and fed them normal diet or four HFDs enriched with SFA, MUFA, n6PUFA, and n3PUFA, respectively. After 7 weeks, fatty acids were profiled in tumor, plasma, pancreas, and liver, using gas chromatography. When tumor carriers were fed four HFDs, the fatty acids that were increased dietarily were also increased in the plasma. When tumor carriers were fed MUFA-, n6PUFA-, and n3PUFA-enriched HFDs, the dietarily increased fatty acids were also increased in tumor, pancreas, and liver. When tumor-carriers were fed the SFA-enriched HFD featuring lauric and myristic acids (C12:0 and C14:0), tumor, pancreas, and liver showed an increase not in the same SFAs but palmitic acid (C16:0) and/or stearic acid (C18:0). In conclusion, predominant fatty acids in HFDs determine the fatty-acid profiles of pancreatic cancer cells and their murine carriers.

Cu2+ reduces hemolytic activity of the antimicrobial peptide HMPI and enhances its trypsin resistance.

Nowadays, drug-resistant microbes are becoming a serious clinical problem threatening people's health and life. Antimicrobial peptides (AMPs) are believed to be potential alternatives of conventional antibiotics to combat the threat of drug-resistant microbes. However, the susceptibility of AMPs toward proteases is one of the major problems limiting their clinical use. In the present study, we reported the effect of Cu2+ on the bioactivity of AMP HMPI. We found that the addition of Cu2+ could improve the protease resistance of AMP HMPI without affecting its bioactivity. Notably, after the binding of Cu2+ with HMPI, the hemolytic activity of HMPI was greatly decreased. In addition, our results also demonstrated that the addition of Cu2+ increased the production of reactive oxygen species in the fungal cells, which may be a supplement for the antifungal activity of HMPI. In conclusion, the introduction of Cu2+ may provide an inorganic strategy to improve the stability and decrease the hemolytic activity of AMP HMPI, while maintaining its antifungal activity.

Pancreatobiliary Diversion in the Mouse.

The gut hormone cholecystokinin (CCK) is primarily secreted from I-cells in the duodenum and proximal jejunum. CCK secretion is stimulated by food digests and inhibited by proteases from pancreatic juice. CCK regulates digestion and appetite, stimulates pancreatic growth, and participates in pancreatic carcinogenesis. The molecular mechanisms of CCK-induced effects are not fully understood. When the mechanisms are studied in animals, the surgical model of pancreatobiliary diversion (PBD) is frequently used. After animals have had PBD, their CCK secretion is no longer inhibited by pancreas-derived proteases, so circulating CCK is increased. PBD is established in rats and hamsters, but not in mice. In this study, we modified PBD procedures and established the model in the mouse. In an experiment, we performed PBD and sham operation (SO) in two groups of mice (20 mice per group). Twenty days after operation, 75% of the PBD mice and all SO mice survived. When plasma CCK was determined by radioimmunoassay, the PBD group had higher levels than the SO group (p < 0.001). To assess pancreatic growth, we determined pancreatic weight and pancreatic contents of protein and DNA. We also stained pancreatic sections by immunohistochemistry to show the proliferating cells that either expressed the proliferating cell nuclear antigen or were labeled with 5-bromo-2'-deoxyuridine. As a result, the pancreases of the PBD mice were heavier (p < 0.001) and had more protein (p < 0.001), DNA (p < 0.01), and proliferating cells (p < 0.01) than those of the SO counterparts. Thus, pancreatic growth was increased as a result of PBD-induced hypercholecystokininemia. The plasma and pancreatic data demonstrated that the PBD model was a success. This model may be used in CCK-related research. For instance, pancreatic cancer is frequently studied in transgenic mice. PBD may be combined with the cancer model to study the role of CCK in the molecular biology of pancreatic cancer.

Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The Km values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate vmax was 55.56 µmol/min/mg. Compared with M1, the catalytic efficiency kcat/Km of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e.P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.