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Abstract Compound K (C-K), one of the most bioactive ginsenoside, is produced by hydrolyzing the glycoside moieties of protopanaxadiol (PPD)-type glycosylated ginsenosides in the ginseng extract. To enhance the biotransformation of PPD-type ginsenosides in American ginseng extract (AGE) into C-K, the optimization of the feed type, concentration, and period for the carbon source sucrose and the reactant AGE was performed in fed-batch fermentation of Aspergillus tubingensis using a fermenter. The concentration (3.94 g/L) and productivity (27.4 mg/L/h) of C-K after feed optimization in fed-batch fermentation increased 3.1-fold compared to those (1.29 g/L and 8.96 mg/L/h) in batch fermentation, and a molar conversion of 100% was achieved. To the best of our knowledge, this is the first trial of fed-batch fermentation to convert ginseng extract into deglycosylated ginsenoside and the highest reported C-K concentration and productivity using ginseng extract via fermentation. After ethanol and resin treatments, C-K solids with purities of 59% and 96% were obtained from the fermentation broth as food- and pharmaceutical-grade products, respectively. Graphical Abstract

An isolated bacterium from soil that highly hydrolyzes cellulose was identified as Paenibacillus peoriae and named P. peoriae MK1. The cellulase from P. peoriae MK1 was cloned and expressed in Escherichia coli. The purified recombinant cellulase, a soluble protein with 13.2-fold purification and 19% final yield, displayed a specific activity of 77 U/mg for CM-cellulose and existed as a metal-independent monomer of 65 kDa. The enzyme exhibited maximum activity at pH 5.0 and 40 °C with a half-life of 9.5 h in the presence of Ca2+ ion. The highest activity was observed toward CM-cellulose as an amorphous substrate, followed by swollen cellulose, and sigmacell cellulose and α-cellulose as crystalline substrates. The enzyme and substrate concentrations for the hydrolysis of CM-cellulose were optimized to 133 U/mL and 20 g/L CM-cellulose, respectively. Under these conditions, CM-cellulose was hydrolyzed to reducing sugars composed mostly of oligosaccharides by cellulase from P. peoriae MK1 as an endo-type cellulase with a productivity of 11.1 g/L/h for 10 min. Our findings will contribute to the industrial usability of cellulase and the research for securing cellulase sources.

Platycosides, saponins from balloon flower root (Platycodi radix), have diverse health benefits, such as antioxidant, anti-inflammatory, anti-tussive, anti-cancer, anti-obesity, anti-diabetes, and whitening activities. Deglycosylated platycosides, which show greater biological effects than glycosylated platycosides, are produced by the hydrolysis of glycoside moieties in glycosylated platycosides. In this review, platycosides are classified according to the chemical structures of the aglycone sapogenins and also divided into natural platycosides, including major, minor, and rare platycosides, depending on the content in Platycodi radix extract and biotransformed platycosides. The biological activities of platycosides are summarized and methods for deglycosylation of saponins, including physical, chemical, and biological methods, are introduced. The biotransformation of glycosylated platycosides into deglycosylated platycosides was described based on the hydrolytic pathways of glycosides, substrate specificity of glycosidases, and specific productivities of deglycosylated platycosides. Methods for producing diverse and/or new deglycosylated platycosides are also proposed.

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.

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.

β-Carotene 15,15′-oxygenase (BCO1) and β-carotene 9′,10′-oxygenase (BCO2) are potential producers of vitamin A derivatives, since they can catalyze the oxidative cleavage of dietary provitamin A carotenoids to retinoids and derivative such as apocarotenal. Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it, and are essential nutrients for humans and highly valuable in the food and cosmetics industries. β-carotene oxygenases (BCOs) from various organisms have been overexpressed in heterogeneous bacteria, such as Escherichia coli, and their biochemical properties have been studied. For the industrial production of retinal, there is a need for increased production of a retinal producer and biosynthesis of retinal using biocatalyst systems improved by enzyme engineering. The current review aims to discuss BCOs from animal, plants, and bacteria, and to elaborate on the recent progress in our understanding of their functions, biochemical properties, substrate specificity, and enzyme activities with respect to the production of retinoids in whole-cell conditions. Moreover, we specifically propose ways to integrate BCOs into retinal biosynthetic bacterial systems to improve the performance of retinal production.

d-Tagatose, a functional sweetener, is converted from d-galactose by l-arabinose isomerase, which catalyzes the conversion of l-arabinose to l-ribulose. In this study, the araA gene encoding l-arabinose isomerase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli, and the expressed enzyme was purified and characterized. The purified l-arabinose isomerase, a soluble protein with 11.6-fold purification and a 22% final yield, displayed a specific activity of 1.8 U/mg for d-galactose and existed as a homohexamer of 336 kDa. The enzyme exhibited maximum activity at pH 8.0 and 40 °C in the presence of Mn2+ and relative activity for pentoses and hexoses in the order l-arabinose > d-galactose > l-ribulose > d-xylulose > d-xylose > d-tagatose > d-glucose. The thermal stability of recombinant E. coli cells expressing l-arabinose isomerase from K. pneumoniae was higher than that of the enzyme. Thus, the reaction conditions of the recombinant cells were optimized to pH 8.0, 50 °C, and 4 g/L cell concentration using 100 g/L d-galactose with 1 mM Mn2+. Under these conditions, 33.5 g/L d-tagatose was produced from d-galactose with 33.5% molar yield and 67 g/L/h productivity. Our findings will help produce d-tagatose using whole-cell reactions, extending its industrial application.

Isoflavone glycosides are commonly biotransformed into isoflavone aglycones due to the superior biological activities of the latter. Wild soybeans contain a higher isoflavone content than domesticated soybeans due to their high level of genetic diversity. In this study, we cloned and characterized a thermostable β-galactosidase from the extreme thermophile Thermoproteus uzoniensis for potential application in isoflavone conversion in Korean wild soybeans. The purified recombinant enzyme exhibited a maximum specific activity of 1103 μmol/min/mg at pH 5.0 and 90 °C with a half-life of 46 h and exists as a homodimer of 113 kDa. The enzyme exhibited the highest activity for p-nitrophenyl (pNP)-β-D-galactopyranoside among aryl glycosides and it hydrolyzed isoflavone glycosides in the order genistin > daidzin > ononin > glycitin. The enzyme completely hydrolyzed 2.77 mM daidzin and 3.85 mM genistin in the seed extract of wild soybean after 80 and 70 min with productivities of 1.86 and 3.30 mM/h, respectively, and 9.89 mM daidzin and 1.67 mM genistin in the root extract after 180 and 30 min, with the highest productivities of 3.30 and 3.36 mM/h, respectively, compared to other glycosidases. Our results will contribute to the industrial production of isoflavone aglycone using wild soybean and this is the first report on the enzymatic production of isoflavone aglycones from isoflavone glycosides in wild soybeans.

Abstract Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as anti-inflammatory and anti-stress activities. However, the complete conversion of all typical glycosylated protopanaxatriol ginsenosides to APPT has not been achieved to date. β-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues at C-6 and the inner glucose at C-20 in protopanaxatriol (PPT), but not the outer rhamnose residues at C-6. In contrast, β-glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer rhamnose residue at C-6 but not the inner glucose residues at C-6 and C-20 in PPT. Thus, the combined use of DT-bgl and PF-bgl resulted in the complete the conversion of all typical glycosylated PPT ginsenosides, including R1, R2, Re, Rg1, Rg2, Rh1, Rf, F1, F3, and F5, to APPT. DT-bgl combined with PF-bgl completely hydrolyzed 1.0 mg ml−1 R1 and 1.0 mg ml−1 total PPT-type ginsenosides in Panax notoginseng root extract to 0.5 and 0.63 mg ml−1 APPT for 4 and 3 h, with molar conversions of 100% and productivities of 125 and 210 mg l−1 h−1, respectively. To the best of our knowledge, this is the first report of the complete conversion of all typical glycosylated PPT ginsenosides to APPT and the highest productivity of APPT obtained from ginseng extract achieved to date.

Platycosides are the functional saponins present in balloon flowers that exert diverse biological effects, and which can be further improved by their deglycosylation. Deapiose-xylosylated platycodin D, which is absent in balloon flowers, can be generated only by cytolase PCL5 by acting on platycoside E. To improve cytolase PCL5-catalyzed production of deapiose-xylosylated platycodin D from platycoside E, we explored the use of high hydrostatic pressure (HHP). At an HHP of 150 MPa, the optimal temperature of cytolase PCL5 activity for converting platycoside E into deapiose-xylosylated platycodin D shifted from 50 to 55 °C, and increased the activity and stability of the enzyme by 5- and 4.9-fold, respectively. Under HHP, the enzyme completely converted 1 mM platycoside E into deapiose-xylosylated platycodin D within 4 h, with a 3.75-fold higher productivity than that under atmospheric pressure. Our results suggest that the application of HHP is a potential method for the economical production of platycosides and enzyme-catalyzed biotransformation of functional saponins.

Abstract 20(S)-Protopanaxadiol (APPD) has potential uses in the pharmaceutical, cosmetic, and food industries because of its anti-stress, anti-fatigue, anti-cancer, anti-inflammatory, and anti-wrinkle properties. However, APPD production is difficult because β-glycosidases that convert the protopanaxadiol (PPD)-type ginsenoside compound K to APPD are rare. β-Glycosidase from Dictyoglomus turgidum (DT-bgl) has the highest specific activity for converting compound K to APPD, but exhibits no activity towards the α-l-arabinopyranoside moiety in compound Y. Therefore, β-glycosidase from Caldicellulosiruptor bescii (CB-bgl), which has a strong α-l-arabinopyranosidase activity, was used along with DT-bgl. The volumetric and specific productivities of the two-enzyme system for APPD using ginseng root extract were 38.4- and 38.7-fold higher, respectively, than those of β-glycosidase from Pyrococcus furiosus, which had the highest volumetric productivity previously reported, at the same enzyme and substrate concentrations. Thus, DT-bgl combined with CB-bgl completely converted PPD-type ginsenosides to APPD with the highest volumetric and specific productivities reported thus far.

Abstract Ginsenoside compound K has been used as a key nutritional and cosmetic component because of its anti-fatigue and skin anti-aging effects. β-Glycosidase from Sulfolobus solfataricus (SS-BGL) is known as the most efficient enzyme for compound K production. The hydrolytic pathway from ginsenoside Rb1 to compound K via Rd and F2 is the most important because Rb1 is the most abundant component in ginseng extract. However, the enzymatic conversion of ginsenoside Rd to F2 is a limiting step in the hydrolytic pathway because of the relatively low activity for Rd. A V209 residue obtained from error-prone PCR was related to Rd-hydrolyzing activity, and a docking pose showing an interaction with Val209 was selected from numerous docking poses. W361F was obtained by rational design using the docking pose that exhibited 4.2-fold higher activity, 3.7-fold higher catalytic efficiency, and 3.1-fold lower binding energy for Rd than the wild-type enzyme, indicating that W361F compensated for the limiting step. W361F completely converted Rb1 to compound K with a productivity of 843 mg l−1 h−1 in 80 min, and showed also 7.4-fold higher activity for the flavanone, hesperidin, than the wild-type enzyme. Therefore, the W361F variant SS-BGL can be useful for hydrolysis of other glycosides as well as compound K production from Rb1, and semi-rational design is a useful tool for enhancing hydrolytic activity of β-glycosidase.

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus.

Petasites japonicus have been used since a long time in folk medicine to treat diseases including plague, pestilential fever, allergy, and inflammation in East Asia and European countries. Bioactive compounds that may prevent and treat infectious diseases are identified based on their ability to inhibit bacterial neuraminidase (NA). We aimed to isolate and identify bioactive compounds from leaves and stems of P. japonicas (PJA) and elucidate their mechanisms of NA inhibition. Key bioactive compounds of PJA responsible for NA inhibition were isolated using column chromatography, their chemical structures revealed using 1 H NMR, 13 C NMR, DEPT, and HMBC, and identified to be bakkenolide B (1), bakkenolide D (2), 1,5-di-O-caffeoylquinic acid (3), and 5-O-caffeoylquinic acid (4). Of these, 3 exhibited the most potent NA inhibitory activity (IC50 = 2.3 ± 0.4 μM). Enzyme kinetic studies revealed that 3 and 4 were competitive inhibitors, whereas 2 exhibited non-competitive inhibition. Furthermore, a molecular docking simulation revealed the binding affinity of these compounds to NA and their mechanism of inhibition. Negative-binding energies indicated high proximity of these compounds to the active site and allosteric sites of NA. Therefore, PJA has the potential to be further developed as an antibacterial agent for use against diseases associated with NA.

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in α-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α-l-arabinofuranosidase activity. A L213A variant β-glycosidase with increased α-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α-l-arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

A d-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65°C, and 1 mM Co2+ with a half-life of 40 min at 65°C, Km of 162 mM, and kcat of 25280 1/s. For increased d-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v) toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L d-allulose from 750 g/L d-fructose after 40 min, with a conversion rate of 31% (w/w) and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively.

Ginsenoside compound K (C-K) is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protopanaxadiol (PPD)-type ginsenosides in red-ginseng extract (RGE), the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf) was used along with the β-D-glucopyranoside/α-L-arabinopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bgly) because SS-bgly showed very low hydrolytic activity on the α-L-arabinofuranoside linkage in ginsenosides. The optimal reaction conditions for C-K production were as follows: pH 6.0, 80°C, 2 U/mL SS-bgly, 3 U/mL CS-abf, and 7.5 g/L PPD-type ginsenosides in RGE. Under these optimized conditions, SS-bgly supplemented with CS-abf produced 4.2 g/L C-K from 7.5 g/L PPD-type ginsenosides in 12 h without other ginsenosides, with a molar yield of 100% and a productivity of 348 mg/L/h. To the best of our knowledge, this is the highest concentration and productivity of C-K from ginseng extract ever published in literature.

We investigated whether a homozygous recessive genetic variant of NSF attachment protein beta (NAPB) gene inherited by monozygotic triplets contributed to their phenotype of early-onset epilepsy and autism. Induced pluripotent stem cell (iPSC) lines were generated from all three probands and both parents. The NAPB genetic variation was corrected in iPSC lines from two probands by CRISPR/Cas9 gene editing. Cortical neurons were produced by directed, in vitro differentiation from all iPSC lines. These cell line-derived neurons enabled us to determine that the genetic variation in the probands causes exon skipping and complete absence of NAPB protein. Electrophysiological and transcriptomic comparisons of cortical neurons derived from parents and probands cell lines indicate that loss of NAPB function contributes to alterations in neuronal functions and likely contributed to the impaired neurodevelopment of the triplets. [ABSTRACT FROM AUTHOR]

D-Allose is a rare natural sugar, which is attractive as a sweetener alternative to sugar because of its biological functions and low-calorie content. However, there is a lack of basic information regarding the use of D-allose as a food ingredient. In this study D-allose toxicity to ICR mice was investigated. The mice were treated with a single dose of D-allose (1,250, 2,500 or 5,000 mg/kg body weight). No organ injury was observed, nor any changes observed in the clinical sign, body weight and the blood serum chemistry. The approximate lethal dose was estimated to exceed 5,000 mg/kg in mice. These results suggest that D-allose is practically non-toxic to mice. [ABSTRACT FROM AUTHOR]

Abstract The brain contains the highest concentration of cholesterol in the human body, which emphasizes the importance of cholesterol in brain physiology. Cholesterol is involved in neurogenesis and synaptogenesis, and age-related reductions in cholesterol levels can lead to synaptic loss and impaired synaptic plasticity, which potentially contribute to neurodegeneration. The maintenance of cholesterol homeostasis in the neuronal plasma membrane is essential for normal brain function, and imbalances in cholesterol distribution are associated with various neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. This review aims to explore the molecular and pathological mechanisms by which cholesterol imbalance can lead to neurotransmission defects and neurodegeneration, focusing on four key mechanisms: (1) synaptic dysfunction, (2) alterations in membrane structure and protein clustering, (3) oligomers of amyloid beta (Aβ) protein, and (4) α-synuclein aggregation.

Abstract Rhabdomyosarcoma 2-associated transcript (RMST) long non-coding RNA has previously been shown to cause Kallmann syndrome (KS), a rare genetic disorder characterized by congenital hypogonadotropic hypogonadism (CHH) and olfactory dysfunction. In the present study, we generated large deletions of approximately 41.55 kb in the RMST gene in human pluripotent stem cells using CRISPR/Cas9 gene editing. To evaluate the impact of RMST deletion, these cells were differentiated into hypothalamic neurons that include 10–15% neurons that express gonadotrophin-releasing hormone (GnRH). We found that deletion in RMST did not impair the neurogenesis of GnRH neurons, however, the hypothalamic neurons were electro-physiologically hyperactive and had increased calcium influx activity compared to control. Transcriptomic and epigenetic analyses showed that RMST deletion caused altered expression of key genes involved in neuronal development, ion channels, synaptic signaling and cell adhesion. The in vitro generation of these RMST-deleted GnRH neurons provides an excellent cell-based model to dissect the molecular mechanism of RMST function in Kallmann syndrome and its role in hypothalamic neuronal development.

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms that include social interaction deficits, language difficulties and restricted, repetitive behavior. Early intervention through medication and behavioral therapy can eliminate some ASD-related symptoms and significantly improve the life-quality of the affected individuals. Currently, the diagnosis of ASD is highly limited.MethodsTo investigate the feasibility of early diagnosis of ASD, we tested extracellular vesicles (EVs) proteins obtained from ASD cases. First, plasma EVs were isolated from healthy controls (HCs) and ASD individuals and were analyzed using proximity extension assay (PEA) technology to quantify 1,196 protein expression level. Second, machine learning analysis and bioinformatic approaches were applied to explore how a combination of EV proteins could serve as biomarkers for ASD diagnosis.ResultsNo significant differences in the EV morphology and EV size distribution between HCs and ASD were observed, but the EV number was slightly lower in ASD plasma. We identified the top five downregulated proteins in plasma EVs isolated from ASD individuals: WW domain-containing protein 2 (WWP2), Heat shock protein 27 (HSP27), C-type lectin domain family 1 member B (CLEC1B), Cluster of differentiation 40 (CD40), and folate receptor alpha (FRalpha). Machine learning analysis and correlation analysis support the idea that these five EV proteins can be potential biomarkers for ASD.ConclusionWe identified the top five downregulated proteins in ASD EVs and examined that a combination of EV proteins could serve as biomarkers for ASD diagnosis.

We investigated whether a homozygous recessive genetic variant of NSF attachment protein beta (NAPB) gene inherited by monozygotic triplets contributed to their phenotype of early-onset epilepsy and autism. Induced pluripotent stem cell (iPSC) lines were generated from all three probands and both parents. The NAPB genetic variation was corrected in iPSC lines from two probands by CRISPR/Cas9 gene editing. Cortical neurons were produced by directed, in vitro differentiation from all iPSC lines. These cell line-derived neurons enabled us to determine that the genetic variation in the probands causes exon skipping and complete absence of NAPB protein. Electrophysiological and transcriptomic comparisons of cortical neurons derived from parents and probands cell lines indicate that loss of NAPB function contributes to alterations in neuronal functions and likely contributed to the impaired neurodevelopment of the triplets.

Oleate hydratases (OhyAs) catalyze the conversion of unsaturated fatty acids to 10-hydroxy fatty acids, which are used as precursors of important industrial compounds, including lactones and ω-hydroxycarboxylic and α,ω-dicarboxylic acids. The genes encoding OhyA and a putative fatty acid hydratase in Stenotrophomonas maltophilia were identified by genomic analysis. The putative fatty acid hydratase was purified and identified as an oleate hydratase (OhyA2) based on its substrate specificity. The activity of OhyA2 as a holoenzyme was not affected by adding cofactors, whereas the activity of the original oleate hydratase (OhyA1) showed an increase. Thus, all characterized OhyAs were categorized as either OhyA1 or OhyA2 based on the activities of holoenzymes upon adding cofactors, which were determined by the type of the fourth conserved amino acid of flavin adenine dinucleotide (FAD)-binding motif. The hydration activities of S. maltophilia OhyA2 toward unsaturated fatty acids, including oleic acid, palmitoleic acid, linoleic acid, α-linolenic acid, and γ-linolenic acid, were greater than those of OhyA1. Moreover, the specific activity of S. maltophilia OhyA2 toward unsaturated fatty acids, with the exception of γ-linolenic acid, was the highest among all reported OhyAs. IMPORTANCE All characterized OhyAs were categorized as OhyA1s or OhyA2s based on the different properties of the reported and newly identified holo-OhyAs in S. maltophilia upon the addition of cofactors. OhyA2s showed higher activities toward polyunsaturated fatty acids (PUFAs), including linoleic acid, α-linolenic acid, and γ-linolenic acid, than those of OhyA1s. This suggests that OhyA2s can be used more effectively to convert plant oils to 10-hydroxy fatty acids because plant oils contain not only oleic acid but also PUFAs. The hydration activity of the newly identified OhyA2 from S. maltophilia toward oleic acid was the highest among the activity levels reported so far. Therefore, this enzyme is an efficient biocatalyst for the conversion of plant oils to 10-hydroxy fatty acids, which can be further converted to important industrial materials. [ABSTRACT FROM AUTHOR]

Abstract 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, it is shown 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 are crucial for the reconstitution of physiological Ca2+‐dependent fusion, because vesicle‐mimicking liposomes fail to reproduce the cholesterol effect. Intriguingly, cholesterol has no effect on the membrane binding of synaptotagmin‐1, a Ca2+ sensor for ultrafast fusion. Cholesterol strengthens local membrane deformation and bending induced by synaptotagmin‐1, thereby lowering the energy barrier for Ca2+‐dependent fusion to occur. The data provide evidence that cholesterol depletion abolishes Ca2+‐dependent vesicle fusion by disrupting synaptotagmin‐1‐induced membrane bending, and suggests that cholesterol is an essential lipid regulator for Ca2+‐dependent fusion.

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.

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.

Mulberry (Morus alba) has been used in traditional oriental medicine since ages. Recently, it has been reported that mulberry produces hypotensive effects through the eNOS signaling pathway. However, the mechanism underlying the hypotensive effects of mulberry is not entirely clear. Moreover, the effects of mulberry on vascular remodeling events such as hyperplasia, an important etiology in the pathogenesis of hypertension and arteriosclerosis, are also ambiguous. Here, we hypothesized that an ethanolic extract of mulberry fruit (EMF) has beneficial effects on vascular remodeling and produces hypotensive effects. The effects of a 6-week oral administration of EMF were examined in spontaneously hypertensive rats (SHRs). The animals were divided into four groups: normotensive control (Wistar Kyoto rats), non-treated SHR, low-dose (100 mg/kg) EMF-treated SHR, and high-dose (300 mg/kg) EMF-treated SHR. Our results showed that the EMF-diet normalizes hypertension in SHRs in a dose-dependent manner, by preventing smooth muscle proliferation, thickening of the tunica media, and vascular hyper-reactivity. The endothelial functions were not substantially affected by the EMF diet in our experimental setting. In conclusion, we suggest that the mulberry fruit could act as a food supplement for reducing blood pressure in hypertensive subjects through its effects on smooth muscle proliferation and vascular contractility.

MK801 (dizocilpine), a phencyclidine (PCP) derivative, is a potent noncompetitive antagonist of the N-Methyl-D-aspartate receptor (NMDAr). Another PCP derivative, ketamine, was reported to block voltage-gated K+ (Kv) channels, which was independent of NMDAr function. Kv currents are major regulators of the membrane potential (Em) and excitability of muscles and neurons. Here, we investigated the effect of MK801 on the Kv channels and Em in rat mesenteric arterial smooth muscle cells (RMASMCs). We used the whole-cell patch clamp technique to analyze the effect of MK801 enantiomers on Kv channels and Em. (+)MK801 inhibited Kv channels in a concentration-dependent manner (IC50 of 89.1 ± 13.1 μM, Hill coefficient of 1.05 ± 0.08). The inhibition was voltage- and state- independent. (+)MK801 didn't influence steady-state activation and inactivation of Kv channels. (+)MK801 treatment depolarized Em in a concentration-dependent manner and concomitantly decreased membrane conductance. (−)MK801 also similarly inhibited the Kv channels (IC50 of 134.0 ± 17.5 μM, Hill coefficient of 0.87 ± 0.09). These results indicate that MK801 directly inhibits the Kv channel in a state-independent manner in RMASMCs. This MK801-mediated inhibition of Kv channels should be considered when assessing the various pharmacological effects produced by MK801, such as schizophrenia, neuroprotection, and hypertension.