Discovery and Mechanistic Understanding of a Lipase from Rhizorhabdus dicambivorans for Efficient Ester Aminolysis in Aromatic Amines.

The formation of amide bonds via aminolysis of esters by lipases generates a diverse range of amide frameworks in biosynthetic chemistry. Few lipases have satisfactory activity towards bulky aromatic amines despite numerous attempts to improve the efficiency of this transformation. Here, we report the discovery of a new intracellular lipase (Ndbn) with a broad substrate scope. Ndbn turns over a range of esters and aromatic amines in the presence of water (2 %; v/v), producing a high yield of multiple valuable amides. Remarkably, a higher conversion rate was observed for the synthesis of amides from substrates with aromatic amine rather than aliphatic amines. Molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies showcase the mechanism for the preference for aromatic amines, including a more suitable orientation, shorter catalytic distances in the active site pocket and a lower reaction barrier for aromatic than for aliphatic amines. This unique lipase is thus a promising biocatalyst for the efficient synthesis of aromatic amides.

Case Report: Novel LIM domain-binding protein 3 (LDB3) mutations associated with hypertrophic cardiomyopathy family.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant cardiomyopathy, which is one of the most common reasons for cardiac arrest in children or adolescents. It is characterized by ventricular hypertrophy (usually left ventricle), small ventricular cavity, and reduced ventricular diastolic compliance found by echocardiography in the absence of abnormal load (such as hypertension or aortic stenosis). HCM is usually caused by mutations in genes encoding sarcomere or sarcomere-related genes. Whole exome sequencing (WES) is performed to identify probable causative genes. Through WES, we identified LIM domain-binding protein 3 (LDB3) mutations (R547Q and P323S) respectively in an 11-year-old HCM girl and a 6-year-old HCM boy. Neural network analyses showed that the LDB3 (R547Q and P323S) mutation decreased its protein stability, with confidence scores of -0.9211 and -0.8967. The STRUM server also confirmed that the mutation decreased its protein stability. Thus, LDB3 mutation may be associated with heritable HCM. To our knowledge, this is the first time to report LDB3 heterozygous variants (R547Q and P323S) responsible for heritable HCM.

Characterization of the complete chloroplast genome of Clematis potaninii (Ranunculaceae), a medicinal and ornamental plant.

Clematis potaninii Maxim. is an important medicinal and ornamental plant. The length of C. potaninii chloroplast genome was 159,691 bp, with a large single-copy region of 79,503 bp, a small single-copy region of 18,106 bp, and two inverted repeat regions of 31,041 bp each. The chloroplast genome contains 138 genes including 94 protein-coding, eight rRNA, and 36 tRNA genes. Phylogenetic analysis showed that C. potaninii is closely related to C. alternata.

The adaptation mechanisms of Acidithiobacillus caldus CCTCC M 2018054 to extreme acid stress: Bioleaching performance, physiology, and transcriptomics.

To understand the acid-resistant mechanism of bioleaching microorganism Acidithiobacillus caldus CCTCC M 2018054, its physiology and metabolic changes at the transcriptional level under extreme acid stress were systemically studied. Scanning electron microscopy (SEM), Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) showed that with an increase in acidity, the absorption peak of sulfur oxidation-related functional groups such as S-O decreased significantly, and a dense sulfur passivation film appeared on the surface of the ore. Confocal laser scanning microscopy (CLSM) revealed that coverage scale of extracellular polymeric substance (EPS) and biofilm fluctuated accordingly along with the increasing acid stress (pH-stat 1.5, 1.2 0.9 and 0.6) during the bioleaching process. In response to acid stress, the increased levels of intracellular glutamic acid, alanine, cysteine, and proline contributed to the maintenance of intracellular pH homeostasis via decarboxylation and alkaline neutralization. Higher unsaturated fatty acid content was closely related to membrane fluidity. Up to 490 and 447 differentially expressed genes (DEGs) were identified at pH 1.5 vs pH 1.2 and pH 1.2 vs pH 0.9, respectively, and 177 common DEGs were associated with two-component system (TCS) regulation, transporter regulation, energy metabolism, and stress response. The upregulation of kdpB helped cells defend against proton invasion, whereas the downregulation of cysB and cbl implied stronger oxidation of sulfur compounds. The transcriptional level of sqr, sor, and soxA was significantly increased and consolidated the energy supply needed for resisting acid stress. Furthermore, eight of the identified DEGs (sor, cbl, ompA, atpF, nuoH, nuoC, sqr, grxB) were verified as being related to the acid stress response process. This study contributes toward expanding the application of these acidophiles in industrial bioleaching.

Identification of Antitumor Active Constituents in Polygonatum sibiricum Flower by UPLC-Q-TOF-MSE and Network Pharmacology.

We aimed to investigate the material basis and mechanisms underlying the antitumor activity of Polygonatum sibiricum flower by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MSE). A compound-protein interaction network for cancer was constructed to identify potential drug targets, and then the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted to elucidate the pathways involved in the antitumor activity of P. sibiricum flower. Subsequently, molecular docking was performed to determine whether the identified proteins are a target of the compounds of P. sibiricum flower. Sixty-four compounds were identified in P. sibiricum flower. Among these, 35 active constituents and 72 corresponding targets were found to be closely associated with the antitumor activity of P. sibiricum flower. By constructing and analyzing the compound-target-pathway network, five key compounds and 10 key targets were obtained. The five key compounds were wogonin, rhamnetin, dauriporphine, chrysosplenetin B, and 5-hydroxyl-7,8-panicolin. The 10 key targets were PIK3CG, AKT1, PTGS1, PTGS2, MAPK14, CCND1, TP53, GSK3B, NOS2, and SCN5A. In addition, 34 antitumor-related pathways were identified using the KEGG pathway analysis. To further verify the results of network pharmacology screening, molecular docking was performed with the five key compounds and the top three targets based on degree ranking, namely, PIK3CG, AKT1, and PTGS2; the results of molecular docking were consistent with those of network pharmacology. P. sibiricum flower can exert its antitumor activity via multicomponent, multitarget, and multichannel mechanisms of action. In this study, we identified the antitumor active constituents of P. sibiricum flower and their potential mechanisms of action.

Specific mechanism of Acidithiobacillus caldus extracellular polymeric substances in the bioleaching of copper-bearing sulfide ore.

This study aimed to reveal the specific mechanism of extracellular polymeric substances (EPS) in the bioleaching of copper-bearing sulfide ore by moderately thermophilic bacterium Acidithiobacillus caldus. The bioleaching performance of blank control (BC), planktonic cell deficient (PD), attached cell deficient (AD), and EPS deficient (ED) systems were compared, to investigate the specific functions of "non-contact" and "contact" (including direct contact and, EPS-mediated contact) mechanisms. The detailed mechanics of bioleaching were studied using µx of cell growth, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The µx of cell growth demonstrated that EPS favors planktonic and attached cell growth. SEM observation revealed that intensive micro-pores on slag benefitted from the "EPS-mediated contact" mechanism. XRD identification indicated that additional chemical derivatives were produced via "EPS-mediated contact" mechanism, because of the active iron/sulfur metabolism. FTIR analysis revealed that the absorption peaks of C-O-S, sulfate, and S = O, which are closely associated with sulfur metabolism, have significant influences of EPS secretion. Taken together, the "EPS-mediated contact" mechanism contributed to almost half of the "contact" mechanism efficiency and a quarter of the total bioleaching efficiency. The proposed specific mechanism of EPS can deepen our understanding of similar bioleaching processes.

Enhanced "contact mechanism" for interaction of extracellular polymeric substances with low-grade copper-bearing sulfide ore in bioleaching by moderately thermophilic Acidithiobacillus caldus.

In order to enhance the "contact mechanism" governing the interaction of extracellular polymeric substances (EPS) with low-grade copper-bearing sulfide ore for the bioleaching of copper, moderately thermophilic Acidithiobacillus caldus was subjected to exogenous intervention with iron and sulfur. The enhancement of the contact mechanism was systematically investigated by evaluating the attached cells/EPS dynamics, intracellular adenosine triphosphate (ATP), cell functional groups, gene transcriptional level, and ore characteristics. Confocal laser scanning microscopy (CLSM) revealed that exogenous intervention with iron and sulfur led to the production of a denser EPS layer and faster adsorption of the attached cells to the ore based on differential fluorescence staining, which indicated enhancement of the "contact mechanism". The increased intracellular ATP content of the attached cells in the exogenous substrate system provided the required energy for the adsorption processes associated with the "contact mechanism". Fourier-transform infrared spectroscopic (FTIR) analysis of the attached cells and the ore showed a dramatic shift of the NH and COS peaks (associated with EPS formation), whereas the FTIR peaks of SO and SO42- associated with sulfur metabolism were also significantly influenced. Moreover, reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the expression of genes related to cellular energy metabolism (nuoB, nuoC, atpE, atpF), sulfur metabolism (sor, sqr, sdo, soxA), biofilm formation (pgaA, pgaB), and cell colonization (acfA, acfB, acfC, acfD) was up-regulated after exogenous intervention, verifying enhancement of the "contact mechanism" at the transcriptional level. In addition, scanning electron microscopy (SEM) indicated more obvious adsorption traces on the ore surface. X-ray diffraction (XRD) indicated the presence of more complex derivatives, such as Fe3(SO4)4, FeSO4, Fe2(SO4)3, and Cu2S, which is suggestive of more active iron/sulfur metabolism with addition of the exogenous iron and sulfur. Overall, a model for bioleaching of low-grade copper-bearing sulfide ore by moderately thermophilic A. caldus was constructed. The results of this investigation should provide a guide for similar industrial bioleaching processes.

Endothelium-independent vasodilator effect of isocorynoxeine in vitro isolated from the hook of Uncaria rhynchophylla (Miquel).

This study was designed to investigate the vasorelaxant effects and underlying mechanism of isocorynoxeine (ICN), one of the indole alkaloids from Uncaria hooks, on isolated mesenteric arteries in vitro. The myograph system was applied for isometric tension recording in the vascular rings. ICN relaxed both endothelium-intact and endothelium-denuded rat vascular rings precontracted with phenylephrine or KCl in a dose-dependent manner. Propranolol, tetraethylammonium, BaCl2, and glibenclamide had no influence on the vasodilator effect of ICN on phenylephrine-primed vascular rings, while 4-aminopyridine decreased the maximum relaxation. Furthermore, ICN produced a significant drop in maximum response in the PE log concentration-response curve without shifting to the right. In the Ca2+-free Kreb's-Henseleit buffer, ICN inhibited the contraction in vascular rings evoked by PE, but not by KCl. The phasic contractions of segments in the Ca2+-free Kreb's-Henseleit buffer induced by CaCl2 were restrained by ICN, while contractions elicited by caffeine displayed no differences. Furthermore, the phasic vasodilation of ICN was significantly lower than controls when pretreated with nifedipine and heparin. Both BAYK8644- and PE-evoked responses were significantly inhibited in the presence of 100 µM of ICN in human vascular smooth muscle cells loaded with the fluorescent Ca2+ indicator Fluo-4-AM. All these results suggest that ICN act in an endothelium-independent manner on the mesenteric artery. Its mechanisms of vasorelaxant action were produced by the inhibition of L-type calcium channel-mediated external Ca2+ influx and α1A-adrenoceptor-mediated intracellular Ca2+ release in vascular smooth muscle cells, and the participation of the Kv channel.

Four Main Active Ingredients Derived from a Traditional Chinese Medicine Guanxin Shutong Capsule Cause Cardioprotection during Myocardial Ischemia Injury Calcium Overload Suppression.

Guanxin Shutong capsule is a traditional Chinese medicine for the treatment of myocardial ischemia (MI). Previous studies have shown that the formula has four main active ingredients (FMAI), protocatechuic acid, cryptotanshinone, borneol, and eugenol. However, the mechanisms of action of these FMAI against MI injury are still not well known. The aim of the present study was to evaluate the protective effects of the FMAI on MI in vitro and in vivo. In vitro, rat neonatal cardiomyocytes were isolated, the cell viability and apoptosis rate were, respectively, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and fluorescence activating cell sorter, and the intracellular calcium concentration ([Ca2+ ]i ) and CaM and CaMKII δ mRNA as well as protein levels were determined. Meanwhile, their downstream targets of RyR2 and PLB were also measured by western blot. In vivo, a rat model of coronary artery ligation was used to evaluate the cardioprotective effects. Infarct sizes of heart tissues and levels of serum biochemical indicators, including creatine kinase, lactate dehydrogenase, superoxide dismutase, and glutamate oxaloacetic transaminase, were measured. The in vitro results showed that the FMAI inhibited cell apoptosis, reduced [Ca2+ ]i , decreased the expression of CaM and CaMKII δ, and increased the expression of RyR2 and PLB. In vivo, the FMAI diminished infract size, reduced creatine kinase, lactate dehydrogenase, and aspartate aminotransferase levels, and enhanced superoxide dismutase activity. In conclusion, our data suggest that the FMAI suppressed calcium overload and exerted its protective effect via its antioxidant, antiinflammatory, and anti-apoptosis activities. Copyright © 2017 John Wiley & Sons, Ltd.

Protective effects of Guanxin Shutong capsule drug-containing serum on tumor necrosis factor-α-induced endothelial dysfunction through nicotinamide adenine dinucleotide phosphate oxidase and the nitric oxide pathway.

The Chinese medicinal formula Guanxin Shutong capsule (GXSTC) has been used for almost 10 years as a clinical treatment for chest pain, depression, palpitation and cardiovascular diseases. The aim of this study was to investigate the effects of GXSTC drug-containing serum on tumor necrosis factor-α (TNF-α)-stimulated endothelial cells. Cell viability was measured by MTT assay, and nitric oxide (NO) levels and NO synthase (NOS) activity were measured as standards of endothelial dysfunction. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were evaluated using commercial kits. In addition, the protein expression of endothelial NOS (eNOS), AKT and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits was examined to evaluate the effect of GXSTC drug-containing serum on ECV304 cells. GXSTC significantly reversed the decrease in NO production induced by TNF-α (5 ng/ml) in ECV304 cells. The expression of NADPH oxidase subunits was increased by TNF-α treatment, but markedly inhibited by treatment with GXSTC in TNF-α-stimulated cells. In summary, GXSTC increased the production of NO in ECV304 cells and exerted a protective effect on ECV304 cells stimulated with TNF-α by upregulating the mRNA and protein expression of eNOS. This was accompanied by increased SOD activity and reduced MDA levels. These results suggested that GXSTC protects the endothelium via the NO pathway and exhibits antioxidant effects.

Chinese medicinal formula Guanxin Shutong capsule protects the heart against oxidative stress and apoptosis induced by ischemic myocardial injury in rats.

Guanxin Shutong capsule (GXSTC) is a Chinese medicinal formula that has been used clinically for the treatment of chest pain, depression, palpitation and cardiovascular diseases in China for almost 10 years. The aim of the present study was to investigate the protective mechanisms against oxidative stress and apoptosis that GXSTC exhibits in the hearts of rats with myocardial ischemia (MI). Infarct size and the levels of marker enzymes, including serum creatine kinase-isoenzyme (CK-MB), lactate dehydrogenase (LDH) and glutamate oxaloacetic transaminase (GOT), as well as the levels of nitric oxide (NO) and NO synthase (NOS) in the heart were measured by biochemical analysis assays. Levels of the antioxidants superoxide dismutase (SOD), catalase (CATA), and glutathione (GSH), and the oxidative stress marker malondialdehyde (MDA), were also determined. Following a 6-week period of ischemia, myocardial apoptosis, as well as the protein and mRNA expression of NADPH oxidase, was evaluated. Myocardial NADPH oxidase activity was measured by protein expression of p47phox and gp91phox using western blot analysis and mRNA expression of p22phox, p47phox, p67phox and gp91phox using reverse transcription polymerase chain reaction. The results showed that daily oral treatment of the rats with GXSTC reduced infarct size, myocardial apoptosis, the levels of serum MDA, LDH, CK-MB and GOT and heart GOT, and increased the activities of total SOD, CATA, NOS and the levels of NO and GSH compared with those in vehicle-treated MI model rats. Administration of GXSTC for 6 weeks also reduced the mRNA expression of the NADPH oxidase subunits p47phox and gp91phox protein, as well as the expression of Bax and caspase-3 proteins. By contrast, Bcl-2 protein expression increased. In conclusion, the results demonstrate that GXSTC attenuates myocardial injury via antioxidative and antiapoptotic effects.