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The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82 kDa monomeric enzyme by SDS-PAGE and gel filtration chromatography analysis. LOX was identified as a single-dioxygenating arachidonate (ARA) 9S-LOX by analyzing ARA-derived bioconversion products using high-performance liquid chromatography with reverse-, normal-, and chiral-phase columns and evaluating kinetic parameters for C20- and C22-polyunsaturated fatty acids (PUFAs). The catalytic efficiency (k

Peroxidation and reduction of 11S- and 13S-positions on C20 and C22 polyunsaturated fatty acids (PUFAs) by Escherichia coli expressing highly active arachidonate (ARA) 11S-lipoxygenase (11S-LOX) from Enhygromyxa salina with the reducing agent cysteine.The specific activity and catalytic efficiency of ARA 11S-LOX from E. salina were 4.1- and 91-fold higher than those of only reported ARA 11S-LOX from Myxococcus xanthus, respectively. The hydroxy fatty acids (HFAs) obtained by the biotransformation of ARA, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexanoic acid (DHA) by Escherichia coli expressing 11S-LOX from E. salina in the presence of cysteine were identified as 11S-hydroxyeicosatetraenoic acid (11S-HETE), 11S-hydroxyeicosapentaenoic acid (11S-HEPE), 13S-hydroxydocosapentaenoic acid (13S-HDPA), and 13S-hydroxydocosahexaenoic acid (13S-HDHA), respectively. The recombinant cells converted 3 mM of ARA, EPA, DPA, and DHA into 2.9 mM of 11S-HETE, 2.4 mM 11S-HEPE, 1. 9 mM 13S-HDPA, and 2.2 mM 13S-HDHA in 60, 80, 120, and 120 min, corresponding to productivities of 72.5, 40.4, 18.5, and 22.4 μM minWe report the highest concentrations, conversion yields, and productivities of 11S- and 13S-hydroxy fatty acids from C20- and C22-PUFAs achieved via E. coli expressing highly active E. salina 11S-LOX.

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder linked to numerous rare, inherited and arisingde novogenetic variants. ASD often co-occurs with attention-deficit hyperactivity disorder and epilepsy, which are associated with hyperexcitability of neurons. However, the physiological and molecular mechanisms underlying hyperexcitability in ASD remain poorly understood. Transient receptor potential canonical-6 (TRPC6) is a Ca2+-permeable cation channel that regulates store-operated calcium entry (SOCE) and is a candidate risk gene for ASD. Using human pluripotent stem cell (hPSC)-derived cortical neurons, single cell calcium imaging, and electrophysiological recording, we show that TRPC6 knockout (KO) reduces SOCE signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data provide evidence that reduction of SOCE by TRPC6 KO results in neuronal hyperexcitability, which we hypothesize is an important contributor to the cellular pathophysiology underlying hyperactivity in some ASD.

Genome-wide chromosomal microarray is extensively used to detect copy number variations (CNVs), which can diagnose microdeletion and microduplication syndromes. These small unbalanced chromosomal structural rearrangements ranging from 1 kb to 10 Mb comprise up to 15% of human mutations leading to monogenic or contiguous genomic disorders. Albeit rare, CNVs at 1p13.3 cause a variety of neurodevelopmental disorders (NDDs) including development delay (DD), intellectual disability (ID), autism, epilepsy, and craniofacial anomalies (CFA). Most of the 1p13.3 CNV cases reported in the pre-microarray era encompassed a large number of genes and lacked the demarcating genomic coordinates, hampering the discovery of positional candidate genes within the boundaries. In this study, we present four subjects with 1p13.3 microdeletions displaying DD, ID, autism, epilepsy, and CFA. In silico comparative genomic mapping with three previously reported subjects with CNVs and 22 unreported DECIPHER CNV cases has resulted in the identification of four different sub-genomic loci harboring five positional candidate genes for DD, ID, and CFA at 1p13.3. Most of these genes have pathogenic variants reported, and their interacting genes are involved in NDDs. RT-qPCR in various human tissues revealed a high expression pattern in the brain and fetal brain, supporting their functional roles in NDDs. Interrogation of variant databases and interacting protein partners led to the identification of another set of 11 potential candidate genes, which might have been dysregulated by the position effect of these CNVs at 1p13.3. Our studies define 1p13.3 as a genomic region harboring 16 NDD candidate genes and underscore the critical roles of small CNVs in in silico comparative genomic mapping for disease gene discovery. Our candidate genes will help accelerate the isolation of pathogenic heterozygous variants from exome/genome sequencing (ES/GS) databases.

Cholesterol is essential for neuronal activity and function. Cholesterol depletion in the plasma membrane impairs synaptic transmission. However, the molecular mechanisms by which cholesterol deficiency leads to defects in vesicle fusion remain poorly understood. Here we show that cholesterol is required for Ca2+-dependent native vesicle fusion using the in-vitro reconstitution of fusion and amperometry to monitor exocytosis in chromaffin cells. Purified native vesicles were crucial for the reconstitution of physiological Ca2+-dependent fusion, whereas vesicle-mimicking liposomes failed to reproduce the cholesterol effect. Intriguingly, cholesterol had no effect on membrane binding of synaptotagmin-1, a Ca2+ sensor for ultrafast fusion. Cholesterol stabilizes local membrane bending induced by synaptotagmin-1, thereby lowering the energy barrier for Ca2+-dependent fusion to occur. Our data provide evidence that cholesterol depletion abolishes Ca2+-dependent vesicle fusion by disrupting synaptotagmin-1-induced membrane bending, and suggests that cholesterol is an important lipid regulator for Ca2+-dependent fusion.

Platycodon grandiflorum (balloon flower) root (Platycodi radix, PR) is used as a health supplement owing to its beneficial bioactive properties. In the present study, the anti-inflammatory, antioxidant, and whitening effects of deglycosylated platycosides (saponins) from PR biotransformed by pectinase from Aspergillus aculeatus were investigated. The bioactivities of the platycosides improved when the number of sugar moieties attached to the aglycone platycosides was decreased. The deglycosylated saponins exhibited higher lipoxygenase inhibitory activities (anti-inflammatory activities) than the precursor platycosides and the anti-inflammatory compound baicalein. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the pectinase-treated PR extract was higher than that of the non-treated PR extract. The trolox-equivalent antioxidant capacity (TEAC) assay showed improved values as the saponins were hydrolyzed. The tyrosinase inhibitory activities (whitening effects) of deglycosylated platycosides were higher than those of the precursor platycosides. Furthermore, 3-O-β-D-glucopyranosyl platycosides showed higher anti-inflammatory, antioxidant, and whitening activities than their precursor glycosylated platycosides. Therefore, 3-O-β-D-glucopyranosyl platycosides may improve the beneficial effects of nutritional supplements and cosmetic products.

Genome-wide chromosomal microarray is extensively used to detect copy number variations (CNVs), which can diagnose microdeletion and microduplication syndromes. These small unbalanced chromosomal structural rearrangements ranging from 1 kb to 10 Mb comprise up to 15% of human mutations leading to monogenic or contiguous genomic disorders. Albeit rare, CNVs at 1p13.3 cause a variety of neurodevelopmental disorders (NDDs) including development delay (DD), intellectual disability (ID), autism, epilepsy, and craniofacial anomalies (CFA). Most of the 1p13.3 CNV cases reported in the pre-microarray era encompassed a large number of genes and lacked the demarcating genomic coordinates, hampering the discovery of positional candidate genes within the boundaries. In this study, we present four subjects with 1p13.3 microdeletions displaying DD, ID, autism, epilepsy, and CFA.

Recombinant Escherichia coli cells expressing 8,11-linoleate diol synthase (LDS) from Penicillium chrysogenum convert oleic and palmitoleic acids to 8-hydroperoxy-9(Z)-octadecenoic acid (HPOME) and 8-hydroperoxy-9(Z)-hexadecenoic acid (HPHME) only, respectively. However, recombinant E. coli cells expressing Q889A variant 6,8-LDS from Penicillium oxalicum as an 8,11-LDS converted oleic and palmitoleic acids to 8,11-dihydroxy-9(Z)-octadecenoic acid (DiHOME) and 8,11-dihydroxy-9(Z)-hexadecenoic acid (DiHHME), respectively, which were identified using liquid chromatography-tandem mass spectrometry analysis. To select suitable variants for producing these compounds, position 889 of 6,8-LDS from P. oxalicum was substituted with other amino acids, and recombinant E. coli cells expressing Q889L and Q889A variants were chosen as the best biocatalysts for producing 8,11-DiHOME and 8,11-DiHHME, respectively. The optimal conditions for producing 8,11-DiHOME or 8,11-DiHHME using cells expressing Q889L or Q889A variant 6,8-LDS were pH 6.5 and 30 °C with 5% (v/v) dimethyl sulfoxide, 60 g L

Xylan and pectin are major structural components of plant cell walls. There are two independent catabolic pathways for xylan and pectin. UxaE bridges these two pathways by reversibly epimerizing D-fructuronate and D-tagaturonate. The crystal structure of UxaE from Cohnella laeviribosi (ClUxaE) shows a core scaffold of TIM-barrel with a position-changing divalent metal cofactor. ClUxaE has the flexible metal-coordination loop to allow the metal shift and the extra domains to bind a phosphate ion in the active site, which are important for catalysis and substrate specificity. Elucidation of the structure and mechanism of ClUxaE will assist in understanding the catalytic mechanism of UxaE family members, which are useful for processing both xylan and pectin-derived carbohydrates for practical and industrial purposes, including the transformation of agricultural wastes into numerous valuable products.

Compound K (C-K) is one of the most pharmaceutically effective ginsenosides, but it is absent in natural ginseng. However, C-K can be obtained through the hydrolysis of protopanaxadiol-type ginsenosides (PPDGs) in natural ginseng. The aim of this study was to obtain the high concentration of food-available C-K using PPDGs in Korean ginseng extract by an extracellular enzyme from Aspergillus niger KACC 46495. A. niger was cultivated in the culture medium containing the inducer carboxymethyl cellulose (CMC) for 6 days. The extracellular enzyme extracted from A. niger was prepared from the culture broth by filtration, ammonium sulfate, and dialysis. The extracellular enzyme was used for C-K production using PPDGs. The glycoside-hydrolyzing pathways for converting PPDGs into C-K by the extracellular enzyme were Rb1 → Rd → F2 → C-K, Rb2 → Rd or compound O → F2 or compound Y → C-K, and Rc → Rd or compound Mc1 → F2 or compound Mc → C-K. The extracellular enzyme from A. niger at 8.0 mg/ml, which was obtained by the induction of CMC during the cultivation, converted 6.0 mg/ml (5.6 mM) PPDGs in Korean ginseng extract into 2.8 mg/ml (4.5 mM) food-available C-K in 9 h, with a productivity of 313 mg/l/h and a molar conversion of 80%. To the best of our knowledge, the productivity and concentration of C-K of the extracellular enzyme are the highest among those by crude enzymes from wild-type microorganisms.

d-Tagatose, a low-calorie functional sweetener, is biotransformed from lactose via galactose. The discovery of an enzyme with 4-epimerization activity toward d-fructose to produce d-tagatose has be...

Platycodon grandiflorum root (Platycodi radix) saponins, platycosides, have been used as health supplements and food items for the treatment of respiratory disorders and pulmonary diseases. Deglycosylated saponins have been known to exert stronger biological effects than their glycosylated forms. In the present study, glycosylated platycosides in Platycodi radix extract were biotransformed into deglycosylated 3-O-β-D-glucopyranosyl platycosides, including 3-O-β-Dglucopyranosyl platycodigenin, 3-O-β-D-glucopyranosyl polygalacic acid, and 3-O-β-Dglucopyranosyl platyconic acid, by pectinase from Aspergillus aculeatus. This is the first report on the quantitative enzymatic production of 3-O-β-D-glucopyranosyl platycosides. The chemical structures of 3-O-β-D-glucopyranosyl platycosides were identified with LC/MS. Moreover, the biotransformation pathways of the three types of platycosides in Platycodi radix into 3-O-β-Dglucopyranosyl platycosides were established.

Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. In this study, using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. As a neuronal EV marker, neural cell adhesion molecule (NCAM) is highly enriched in EVs released from induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Our results suggest that plasma NCAM-positive neuronal EVs can be used to discover biomarkers for neurological disorders.

Platycosides, Platycodi radix (

Platycosides, saponins contained in balloon flower, which have been used as food health supplements for respiratory diseases, have diverse pharmacological effects. Platycosides exhibit better pharmacological activity by hydrolyzing their own sugars. However, to date, there have been no studies on the production of deglucosylated platycodin D suitable for food applications. In this study, Pluszyme 2000P, which was derived from Aspergillus niger, a food-grade microorganism, was used to completely convert platycoside E into deglucosylated platycodin D. For an efficient and economical production of deglucosylated platycodin D, the productivity was improved approximately 2.4 times by application of high hydrostatic pressure and the discarded balloon flower leaf was used as a substrate. As a result, deglucosylated platycodin D was produced with the highest concentration (3.49 mg/mL) and productivity (581.7 mg/L/h) reported so far. Our results contribute to functional saponin production and the related food industries., Platycoside; Pluszyme 2000P; Food additive; Aspergillus niger; Balloon flower; Medicinal herb.

Background: Rapid lung function decliners have been considered a unique subgroup of patients with chronic obstructive pulmonary disease (COPD). A rapid decline manifests early and is related with a poor prognosis. Therefore, pre-emptive identification of risk factors for rapid decliner is necessary. We aimed to determine those risk factors in Korean patients.Methods: A longitudinal, observational study was conducted on the KOCOSS cohort (NCT02800499), consisting of patients assessed from January 2012 to December 2019 at 54 medical centers in South Korea. Eligible patients were adults followed up for 3 years with serial spirometric tests. We calculated the annualized percentage change in lung function from baseline. Rapid decliners were defined as the quartile of patients with the highest annualized percentage decline in lung function. Results: Among the 518 included patients, 130 were rapid decliners. Rapid decliners lost 6.2%/year and 100 ml/year of forced expiratory volume in 1 second from baseline. Rapid decliners had a higher rate of severe exacerbations than non-rapid decliners (0.2/year vs. 0.1/year, P=0.032). Upon multivariable logistic regression, male sex, being a current smoker, a blood eosinophil count decline compared to mono-bronchodilator therapy.Conclusions: Identification of risk and aggravating factors for rapid lung function decline may assist physicians in providing earlier intervention for high-risk patients with COPD.

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

Sepsis is a disease responsible for the death of almost all critical patients. Once infected by virus or bacteria, patients can die due to systemic inflammation within a short period of time. Cytokine storm plays an essential role in causing organ dysfunction and septic shock. Thus, inhibition of cytokine secretion is considered very important in sepsis therapy. In this study, we found that TFP, an antipsychotic drug mainly used to treat schizophrenia by suppressing dopamine secretion, inhibited cytokine release from activated immune cells both in vitro and in vivo. Trifluoperazine (TFP) decreased the levels of pro-inflammatory cytokines without altering their transcription level. In LPS-induced endotoxemia and cecal content injection (CCI) models, TFP intraperitoneal administration improved survival rate. Thus, TFP was considered to inhibit the secretion of proteins through a mechanism similar to that of W7, a calmodulin inhibitor. Finally, we confirmed that TFP treatment relieved organ damage by estimating the concentrations of aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) in the serum. Our findings were regarded as a new discovery of the function of TFP in treating sepsis patients. Key messages • TFP inhibits LPS-induced activation of DCs by suppressing pro-inflammatory cytokine. • Treatment of TFP increases survival of LPS-induced endotoxemia and CCI sepsis models. • TFP exerted a protective effect against tissue or organ damage in animal models. Electronic supplementary material The online version of this article (10.1007/s00109-019-01762-4) contains supplementary material, which is available to authorized users.

To quantitatively hydroxylate 8S- and 10S-positions on polyunsaturated fatty acids by recombinant Escherichia coli cells expressing mouse arachidonate 8S-lipoxygenase (8S-LOX). Hydroxylated products gained from the conversion of arachidonic acid (20:4Δ5Z,8Z,11Z,14Z, AA), eicosapentanoic acid (20:5Δ5Z,8Z,11Z,14Z,17Z, EPA), and (22:6Δ4Z,7Z,10Z,13Z,16Z,19Z, DHA) by recombinant E. coli cells containing 8S-LOX from mouse were identified as 8S-hydroxy-5,9,11,14(Z,E,Z,Z)-eicosatetranoic acid (8S-HETE), 8S-hydroxy-5,9,11,14,17(Z,E,Z,Z,Z)-eicosapentanoic acid (8S-HEPE), and 10S-hydroxy-4,8,12,14,16,19(Z,E,Z,Z,Z,Z)-docosahexaenoic acid (10S-HDoHE), respectively. Under the optimal hydroxylation conditions of pH 7.5, 30 °C, 5% (v/v) ethanol, 15 g cells l−1, and 5 mM substrate, AA, EPA, and DHA were hydroxylated into 4.37 mM 8S-HETE, 3.77 mM 8S-HEPE, and 3.13 mM 10S-HDoHE for 60, 90, and 60 min, with 87, 75, and 63% molar conversions, respectively. To the best of our knowledge, this is the first quantitatively biotechnological production of 8S-HETE, 8S-HEPE, and 10S-HDoHE.

This study was conducted to characterize recombinant α-l-rhamnosidase from Chloroflexus aurantiacus and apply the enzyme in the production of isoquercitrin from rutin. The α-l-rhamnosidase from C. aurantiacus was cloned and expressed in Escherichia coli BL21 and purified as a soluble enzyme. α-l-rhamnosidase purified from C. aurantiacus has a molecular mass of approximately 105 kDa and is predicted to exist as a homodimer with a native enzyme of 200 kDa. The purified enzyme exhibited the highest specific activity for rutin among the reported isoquercitrin producing α-l-rhamnosidases and was applied in the production of isoquercitrin from rutin. Under the optimised conditions of pH 6.0, 50 °C, 0.6 U mL−1 α-l-rhamnosidase, and 30 mM rutin, α-l-rhamnosidase from C. aurantiacus produced 30 mM isoquercitrin after 2 h with a 100% conversion yield and productivity of 15 mM h−1. We achieved a high productivity of isoquercitrin from rutin. Moreover, these results suggest that α-l-rhamnosidase from C. aurantiacus is an effective isoquercitrin producer.

The Platycodon grandiflorum root, Platycodi radix, a common vegetable, and its extract with glycosylated saponins, platycosides, have been used as food items and food health supplements for pulmonary diseases and respiratory disorders. Enzymes convert glycosylated saponins into deglycosylated saponins, which exhibit higher biological activity than glycosylated saponins. In this study, β-glucosidase from the hyperthermophilic bacterium Dictyoglomus turgidum converted platycosides in the Platycodi radix extract into deglucosylated platycosides. In addition, the enzyme completely converted platycoside E (PE), platycodin D3 (PD3), and platycodin D (PD) in Platycodi radix extract into deglucosylated platycodin D (deglu PD), which was first identified by nuclear magnetic resonance. The anti-inflammatory activities of deglu PD and deglucosylated Platycodi radix extract were higher than those of PE, PD3, PD, Platycodi radix extract, and baicalein, an anti-inflammatory agent. Therefore, deglucosylated Platycodi radix extract is expected to be used as improved functional food supplements.