Production of capsaicinoid nonivamide from plant oil and vanillylamine via whole-cell biotransformation.

Capsaicinoids are mostly derived from chili peppers and have widespread applications in food, feed, and pharmacology. Compared with plant extraction, the use of microbial cell factories for capsaicinoids production is considered as a more efficient approach. Here, the biotransformation of renewable plant oil and vanillylamine into capsaicinoid nonivamide was investigated. Nonivamide biosynthesis using nonanoic acid and vanillylamine as substrates was achieved in Escherichia coli by heterologous expression of genes encoding amide-forming N-acyltransferase and CoA-ligase. Through increasing nonanoic acid tolerance of chassis cell, screening key enzymes involved in nonivamide biosynthesis and optimizing biotransformation conditions, the nonivamide titer reached 0.5 g/L. By further integrating a route for conversion of oleic acid to nonanoic acid, nonivamide biosynthesis was finally achieved using olive oil and vanillylamine as substrates, yielding a titer of approximately 10.7 mg/L. Results from this study provide valuable information for constructing highly efficient cell factories for the production of capsaicinoid compounds.

Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.

Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.

Progress in diagnosis and treatment of ovarian metastases in rectal cancer / 中华胃肠外科杂志

Rectal cancer is the most common tumor of digestive tract. For female patients, ovarian metastasis ranks the second place in intraperitoneal organ metastasis. Its symptoms are occult, easily missed and insensitive to systemic treatment, so the prognosis is poor. Surgery is the treatment of choice for patients with rectal ovarian metastases, whether R0 resection is possible or not, and reducing tumor load is associated with better prognosis. With the continuous development of hyperthermic intraperitoneal chemotherapy (HIPEC), tumor reduction can reach the cellular level, which can significantly improve survival. Prophylactic ovariectomy remains a controversial issue in patients at high risk of ovarian metastasis. In this review, we summarize the diagnosis, treatment and prevention strategies of rectal cancer ovarian metastases, hoping to provide some reference for clinical practice.

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism.

BACKGROUND: The methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production. RESULTS: In this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain). CONCLUSIONS: The GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

[Active components and action mechanism of Shenmai Injection in treatment of atrial fibrillation based on network pharmacology and molecular docking].

This study aims to explore the active components and molecular mechanism of Shenmai Injection in the treatment of atrial fibrillation(AF) based on the application of network pharmacology and molecular docking technology. The chemical components of single herbs of Shenmai Injection were collected from TCMSP and TCMID, with the standard chemical name and PubChem CID(referred to as CID) obtained from PubChem database. The active components were screened using SwissADME, and their targets were predicted using SwissTargetPrediction. Targets related to AF treatment were identified using GeneCards, OMIM, and other databases. Venn diagram was constructed using Venny 2.1 to obtain the intersection targets. The single herb-active component-potential target network was constructed using Cytoscape, and the clusterProfiler R function package was used to perform the gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment. The protein-protein interaction(PPI) network of intersection targets was generated based on the STRING database. The hub target protein was identified by visualization using Cytoscape, and then docked to its reverse-selected active components. The analysis showed that there were 65 active components with 681 corresponding targets in Shenmai Injection, 2 798 targets related to AF treatment, and 235 intersection targets involving 2 549 GO functions and 153 KEGG pathways. Finally, hub target proteins, including RAC-alpha serine/threonine-protein kinase(AKT1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3 CA), and estrogen receptor 1(ESR1), were screened out by PPI network visualization. The molecular docking was performed for 39 active components screened out in reverse, among which 30 active components de-monstrated high affinity. Among them, homoisoflavanoids CID 10871974, CID 5319742, and CID 10361149 had stronger affinity docking with AKT1. This study preliminarily indicates that Shenmai Injection treats AF through multiple components, multiple targets, and multiple pathways. Homoisoflavonoids of Ophiopogon japonicus are its important active components, which target AKT1 to regulate metabolism, inflammation, and apoptosis in AF treatment.

Expression and characterization of a pectin methylesterase from Aspergillus niger ZJ5 and its application in fruit processing.

A pectin methylesterase gene, pme-zj5a, from Aspergillus niger ZJ5 was cloned and high-level expressed in Pichia pastoris. The highest PME activity was 71.11 U/ml after induction with methanol for 20 h at 30 °C. The molecular mass of purified PME-ZJ5A was estimated to be 37 kDa by SDS-PAGE, and its Km, Vmax and kcat values of PME-ZJ5A were determined to be 3.27 mg/ml, 5.36 µmol/min/mg, and 22.33 s-1 with pectin. Purified recombinant PME-ZJ5A exhibited optimal activity at pH 3.8 and 45 °C. It retained more than 60% of its maximum activity at 10 °C. Moreover, recombinant PME-ZJ5A can increase the transmittance of pineapple juice by 60.8%, and increase the firmness of pineapple cubes nearly double when combined with CaCl2, which showed good potential in fruit processing.

Myocardial protective effect of extracellular superoxide dismutase gene modified bone marrow mesenchymal stromal cells on infarcted mice hearts.

AIM: Extracellular superoxide dismutase (ecSOD) is a unique scavenger of superoxide anions and a promising target of gene therapy for ischemia/reperfusion injury (I/R). However, conventional gene therapies have limitation in effectiveness and efficiency. This study aimed to investigate the protective effects of ecSOD gene modified bone marrow mesenchymal stromal cells (BMSCs) on cardiac function improvement in mice infarcted heart. METHODS & RESULTS: BMSCs were isolated from Fluc(+) transgenic mice (Tg FVB[Fluc(+)]) and transfected by adenovirus combined with human ecSOD gene. ELISA was performed to determine ecSOD protein level. Female syngeneic FVB mice were randomized into 5 groups: (1) Sham group (sham); (2) MI group (MI); (3) MI+BMSCs group (BMSC); (4) MI+BMSCs-vector group (BMSC-vector); (5) MI+ BMSCs-ecSOD group (BMSC-ecSOD). MI was accomplished by ligation of the left anterior descending artery. BMSCs (2 x 10(6)) were injected into the border zone of infarction. In vivo bioluminescence imaging (BLI) was performed to monitor transplanted BMSCs viability. Echocardiography and histological staining revealed that BMSCs-ecSOD significantly reduced myocardial infarction size and improved cardiac function. Lucigenin chemiluminescence, DHE and TUNEL staining demonstrated that BMSCs-ecSOD delivery reduced ROS level and cell apoptosis both in vivo and in vitro. Western blot assay revealed that ecSOD supplementation increased FoxO3a phosphorylation in cardiomyocytes. Moreover, quantitative real-time PCR showed that pro-apoptotic factors (bim and bax) were decreased while the anti-apoptotic factor mir-21 expression was increased after ecSOD intervention. CONCLUSION: Intra-myocardial transplantation of adenovirus-ecSOD transfected BMSCs could exert potential cardiac protection against MI, which may be partly through reduction of oxidative stress and improvement of BMSCs survival.

Rosuvastatin enhances the therapeutic efficacy of adipose-derived mesenchymal stem cells for myocardial infarction via PI3K/Akt and MEK/ERK pathways.

The poor viability of transplanted stem cells hampers their therapeutic efficacy for treatment of myocardial infarction. The aim of this study was to investigate whether rosuvastatin improved survival of adipose-derived mesenchymal stem cells (AD-MSCs) after transplantation into infarcted hearts. AD-MSCs isolated from Tg(Fluc-egfp) mice which constitutively express both firefly luciferase (Fluc) and enhanced green fluorescent protein were transplanted into infarcted hearts with or without rosuvastatin administration. Longitudinal in vivo bioluminescence imaging and histological staining revealed that rosuvastatin enhanced the survival of engrafted AD-MSCs. Furthermore, combined therapy of AD-MSC and rosuvastatin reduced fibrosis, decreased cardiomyocyte apoptosis, and preserved heart function. AD-MSCs were then subjected to hypoxia and serum deprivation injury in vitro to mimic the ischemic environment. Rosuvastatin (10(-6) mmol/L) enhanced the viability and paracrine effect of AD-MSCs, and decreased their apoptotic rate. Western blotting revealed that rosuvastatin supplementation increased Akt and ERK phosphorylation, which resulted in FoxO3a phosphorylation and nuclear export. In addition, rosuvastatin administration decreased the pro-apoptotic proteins Bim and Bax, and increased the anti-apoptotic proteins Bcl-xL and Bcl-2. Furthermore, these effects were abolished by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126. This study demonstrates that rosuvastatin may improve the survival of engrafted AD-MSCs at least in part through the PI3K/Akt and MEK/ERK1/2 signaling pathways. Combination therapy with rosuvastatin and AD-MSCs has a synergetic effect on improving myocardial function after infarction.

Effect of Pinus massoniana Lamb. bark extract on lytic cycle of Epstein-Barr virus.

Pinus massoniana bark extract (PMBE) at a concentration of 60 microg/mL or more inhibits the expression of Epstein-Barr virus (EBV) lytic proteins, such as Rta, Zta, and EA-D. EBV lytic cycle was blocked by inhibiting the transcription of immediate-early genes. The results suggest that the PMBE has anti-EBV activity. Thus, the extract is potentially useful in preventing the lytic development of EBV in vitro.

Efficacy evaluation of acupoints massage on asthenopia of video display terminal under different exposure dose / 中国针灸

<p><b>OBJECTIVE</b>To observe the efficacy differences of acupoints massage for asthenopia of video display terminal (VDT) under different exposure dose.</p><p><b>METHODS</b>One hundred and two cases (204 eyes) were divided into a low exposure group and a high exposure group, fifty-one cases in each group. The same intervention of acupoints massage on Cuanzhu (BL 2), Jingming (BL 1), Sizhukong (TE 23), Sibai (ST 2) and Taiyang(EX-HN 5) were given to the two groups, one acupoint for 5 min and once everyday, one month of which made a course. The symptom score, tear film break-up time (BUT) and Schirmer I test(SIT) were observed before and after treatment.</p><p><b>RESULTS</b>(1) The correlation coefficient of cubic curve model of the exposure dose was the biggest with symptom improvement index (P = 0.000), which indicated that the lower VDT exposure index was, the more obvious the symptom improved. The symptom improvement indices of low exposure group and high exposure group, which were (52.31 +/- 16.65)% and (28.93 +/- 13.35)% respectively, were statistical significant difference (P = 0.000). (2) Compared to before treatment, the levels of BUT and SIT in the two groups were both significantly higher (P < 0.05). Compared with the high exposure group, the levels of BUT and SIT in the low exposure group were increased by 0.826 s (P = 0.022) and 1.029 mm (P = 0.033), respectively, after the impact of BUT and SIT was corrected before the research.</p><p><b>CONCLUSION</b>The acupoints massage can improve the symptoms and ocular physiology for patients with VDT asthenpia, and it is more effective for the low exposure cases.</p>

Effect of electrochemical treatment on high-flow vascular malformations in the maxillofacial region.

We explored the effect of electrochemical therapy for the treatment of high-flow venous malformations in the maxillofacial region in 32 patients. We used a method of anaesthesia that was suitable to the site and size of the lesion, and then inserted platinum needles into the lesion in a sterile environment. We protected the normal skin by inserting the cannulas into plastic sheaths, and connected the needles to an electrochemical machine. The common voltage, electric current, and amount of electricity were 6-8 V, 80-100 mA, and 10-20 C/cm(2), respectively. During a follow up period of two months to three years, 18 (56%) patients had a complete response, and 14 a partial response (44%). Electrochemical treatment is a simple, relatively atraumatic method of dealing with high-flow venous malformations that leaves no scars.

[Clinical study on Shen Shuai Fang in treating chronic renal failure].

OBJECTIVE: To explore the effect of Shen Shuai Fang in treating Chronic Renal Failure (CRF) of deficiency of spleen and kidney with turbid damp and blood stagnation symptom. METHODS: 125 patients with CRF of deficiency of spleen and kidney with turbid damp and blood stagnation symptom were randomly divided into two groups, 75 patients in treatment group were treated with Shen Shuai Fang adding western medicine and the other patients in control group were treated only with western medicine. We observed the effect and indexes change including renal function, Hb, proteinuria, lipid before and after treatment. RESULTS: After six months' treatment, the general effective rate in treatment group was 77.33%, which was higher than that in control group obviously (44.00%, P < 0.01). So treatment group was obviously better than control group on decreasing proteinuria, improving renal function, increasing Hb, ameliorating lipid metabolism (P < 0.05 or P < 0.01). CONCLUSION: Shen Shuai Fang is effective to treat CRF of deficiency of spleen and kidney with turbid damp and blood stagnation symptom.

[A clinical research of Naoxinqing tablet's effects on blood fat and viscosity].

OBJECTIVE: To investigate the effects on blood fat and viscosity of Naoxinqing tablet. METHODS: 66 patients of primary hyperlipoidemia were randomly divided into three groups. The first group was treated by Naoxinqing tablet, the second group was treated by Naoxinqing and combined with half dose Simvastatin, the third group was treated by full dose Simvastatin. Then the clinical effect, fasting plasma lipid, the index of blood viscosity and side effect of the three group' patients were compared after 8 weeks. RESULTS: The index of blood fat improved obviously after treatment of the three groups ( P < 0.05, P < 0. 01), group 2 and 3 were significantly superior to group 1 (P < 0.05), and no significance between group 2 and 3 (P > 0.05). CONCLUSIONS: The Naoxinqing tablet can improve the metabolism of blood fat, decrease blood viscosity obviously and treat hyperlipoidemia effectively with little side effect.

[Clinical study on Dan Shao Tang in treating diabetic nephropathy of deficiency of Yin with damp-heat symptom].

OBJECTIVE: To explore the effect of Dan Shao Tang (DST) in treating diabetic nephropathy (DN) of deficiency of Yin with damp-heat symptom. METHODS: The 72 patients with DN of deficieny of Yin with damp-heat symptom were randomly divided into two groups. Among them, the 42 patients in treatment group were treated with DST and western medicine and the 30 patients in control group were treated only with western medicine. Before and after treatment, the effect and indexes change including blood glucose, renal function, proteinuria, microproteins, lipid metabolism and hyperviscosity were observed. RESULTS: After three months treatment, the general effective rate in treatment group was 76.19%, which was markedly higher than that in control group (50.00%, P < 0.01). Moreover, treatment group was obviously better than control group on decreasing blood glucose proteinuria and microproteins, improving renal function, ameliorating lipuid metabolism and hyperviscosity (P < 0.01 or P < 0.05). CONCLUSION: DST is effective ton treatment on DN of deficiency of Yin with damp-heat symptom.