Inactivation of Salmonella Typhimurium and Listeria monocytogenes on buckwheat seeds through combination treatment with plasma, vacuum packaging, and hot water.

AIMS: The objectives of this study were to evaluate the effect of combination treatment with cold plasma (CP), vacuum packaging (VP), and hot water (HW) on the inactivation of foodborne pathogens on buckwheat seeds, and determined the germination rates of seeds and the quality of sprouts following combination treatment. METHODS AND RESULTS: Buckwheat seeds inoculated with Salmonella Typhimurium and Listeria monocytogenes were treated with CP, HW, CP + HW, VP + HW, or CP + VP + HW. The germination rates of the HW-, CP + HW-, VP + HW-, and CP + VP + HW-treated seeds and the antioxidant activities and rutin contents of the CP + HW- and CP + VP + HW-treated sprouts were determined. HW, CP + HW, and CP + VP + HW were found to reduce the levels of the two pathogens to below the detection limit (1.0 log CFU g-1) at 70°C. However, HW and CP + HW significantly reduced the germination rate of buckwheat seeds. CP + VP + HW did not affect the germination rate of seeds nor the antioxidant activities and rutin content of buckwheat sprouts. CONCLUSIONS: These results indicate that CP + VP + HW can be used as a novel control method to reduce foodborne pathogens in seeds without causing quality deterioration.

Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion.

Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs), dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating adenosine triphosphate (ATP) and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stressors such as oxidative stress or in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure, induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde (GA) addition or oxygen supplementation, respectively. Finally, using pre-formed fibril α-synuclein treated neurons, we show fixative choice can alter experimental outcomes. Specifically, α-synuclein-induced mitochondrial remodeling could not be observed with PFA only fixation as fixation itself caused mitochondrial fragmentation. Our study provides optimized methods for examining mitochondrial morphology in neurons and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.

Saponins of Korean Red Ginseng May Protect Human Skin from Adipokine-Associated Inflammation and Pigmentation Resulting from Particulate Matter Exposure.

BACKGROUND: Exposure to airborne particulate matter (PM) is an ever-increasing concern worldwide. Strategies to counter the detrimental effects that follow cutaneous exposure to PM, such as induction of pigmentation, inflammation, and alterations in adipokine profile, need to be investigated further. Korean red ginseng (KRG) extracts and individual ingredients have been demonstrated to play an effective role in suppression of ROS, inflammation, and resultant skin aging. In addition, recent investigations revealed that Rg3 and Rf saponins work as antimelanogenic agents. In this study, we investigated whether saponins of KRG can protect against or reverse the PM-induced detrimental effects. METHODS: The biological effects of PM and saponins were evaluated both in vitro and ex vivo. Cell viability and intracellular ROS levels were determined in normal human epidermal melanocytes (NHMs), human epidermal keratinocytes (NHKs), and their cocultures. Experiments to demonstrate the protective properties of saponins against consequences of exposure to PM were performed. Melanin assay, quantitative real-time PCR, and Western blotting were carried out to determine the effects on melanogenesis and the implicated molecular signaling pathways. RESULTS: Exposure to PM resulted in decreased keratinocyte viability, which was coupled with augmented oxidative stress. These changes were attenuated by treatment with saponins. PM exposure resulted in increased expression of leptin, which was reduced by saponins. Moreover, PM exposure led to increased melanin production in a coculture model, which was mitigated by treatment with saponins. Treatment with saponins resulted in a decrease in matrix metalloproteinase (MMP) levels after exposure to PM. CONCLUSION: Saponins of KRG can protect the skin from the harmful effects of PM exposure by reducing levels of ROS, leptin, inflammatory cytokines, and melanin.

High-yield synthesis and purification of recombinant human GABA transaminase for high-throughput screening assays.

Many studies have focussed on modulating the activity of γ-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.

Transcriptome Profiling of Human Follicle Dermal Papilla Cells in response to Porphyra-334 Treatment by RNA-Seq.

Porphyra-334 is a kind of mycosporine-like amino acid absorbing ultraviolet-A. Here, we characterized porphyra-334 as a potential antiaging agent. An in vitro assay revealed that porphyra-334 dramatically promoted collagen synthesis in fibroblast cells. The effect of porphyra-334 on cell proliferation was dependent on the cell type, and the increase of cell viability by porphyra-334 was the highest in keratinocyte cells among the three tested cell types. An in vivo clinical test with 22 participants demonstrated the possible role of porphyra-334 in the improvement of periorbital wrinkles. RNA-sequencing using human follicle dermal papilla (HFDP) cells upon porphyra-334 treatment identified the upregulation of metallothionein- (MT-) associated genes, confirming the antioxidant role of porphyra-334 with MT. Moreover, the expression of genes involved in nuclear chromosome segregation and the encoding of components of kinetochores was upregulated by porphyra-334 treatment. Furthermore, we found that several genes associated with the hair follicle cycle, the hair follicle structure, the epidermal structure, and stem cells were upregulated by porphyra-334 treatment, suggesting the potential role of porphyra-334 in hair follicle growth and maintenance. In summary, we provided several new pieces of evidence of porphyra-334 as a potential antiaging cosmetic agent and elucidated the expression network in HFDP cells upon porphyra-334.

Improvement of the Antioxidant Activity, Water Solubility, and Dispersion Stability of Prickly Pear Cactus Fruit Extracts Using Argon Cold Plasma Treatment.

Microwave-powered cold plasma (CP) treatment was evaluated as a means to increase the antioxidant activity, water solubility, and dispersion stability of prickly pear cactus fruit (Opuntia ficus-indica (L.) Mill.) extract. The extract (2 g) was treated at various CP generation powers and treatment times at 25 °C to 28 °C. The antioxidant activity of the prickly pear cactus fruit extract increased by 1.8% and 1.7% after CP treatment at 750 W for 40 min and 856 W for 36 min, respectively. Both the water solubility and dispersion stability (delta backscattering) of the extract increased by 2.4% and 0.1%, respectively, following CP treatment at 644 W for 36 min. These results suggest the potential of CP treatment to increase the applicability of the prickly pear cactus fruit extract and possibly other insoluble natural antioxidant compounds in foods by improving their antioxidant activities and solubility in water. PRACTICAL APPLICATION: Prickly pear cactus fruit is a functional food with a high antioxidant concentration. This study demonstrated that cold plasma treatment improved the water solubility and dispersion stability of prickly pear cactus fruit extract without altering or improving its antioxidant activity. The obtained results suggested the potential of applying cold plasma technology to improve the applicability of the extract, which is difficult to solubilize in food systems, to various processed foods.

In vitro and in vivo hypoglycemic effects of cyanidin 3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside, an anthocyanin isolated from purple-fleshed sweet potato.

Anthocyanins are major components of purple sweet potatoes (PSP) with antioxidant, anti-obesity, and antidiabetic activity. In this study, we evaluated the hypoglycemic effects of 12 individual anthocyanins purified from PSP (Korean variety Shinzami). We separated the anthocyanins using liquid chromatography with diode array detection and electrospray ionization/mass spectrometry (LC-DAD-ESI/MS). Three anthocyanins were selected through a radical scavenging activity test. We examined whether individual anthocyanins inhibited glucose secretion in HepG2 cells (hepatic gluconeogenesis). Additionally, we determined the effect of each anthocyanin on fasting blood glucose levels in 6-week-old male C57BL/6J mice fed a 60% high-fat diet for 14 weeks. Mice were evaluated at 0, 1, 2, and 4 h after oral administration of anthocyanins (80 mg/kg), an anthocyanin-rich-fraction (80 mg/kg), positive control (metformin, 80 mg/kg), and distilled water (control). Cyanidin 3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside (PEAK9) was the main PSP anthocyanin that inhibited hepatic glucose secretion and reduced blood glucose.

Lack of the α1,3-Fucosyltransferase Gene (Osfuct) Affects Anther Development and Pollen Viability in Rice.

N-linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N-glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycans revealed that α1,3-fucose was lacking in the N-glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N-glycan modification and anther and pollen development in rice.

Andrographolide suppresses TRIF-dependent signaling of toll-like receptors by targeting TBK1.

Toll-like receptors (TLRs) play a crucial role in danger recognition and induction of innate immune response against bacterial and viral infections. The TLR adaptor molecule, toll-interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF), facilitates TLR3 and TLR4 signaling, leading to the activation of the transcription factor, NF-κB and interferon regulatory factor 3 (IRF3). Andrographolide, the active component of Andrographis paniculata, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the role of andrographolide in TLR signaling pathways. Andrographolide suppressed NF-κB activation as well as COX-2 expression induced by TLR3 or TLR4 agonists. Andrographolide also suppressed the activation of IRF3 and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. Andrographolide attenuated ligand-independent activation of IRF3 following overexpression of TRIF, TBK1, or IRF3. Furthermore, andrographolide inhibited TBK1 kinase activity in vitro. These results indicate that andrographolide modulates the TRIF-dependent pathway of TLRs by targeting TBK1 and represents a potential new anti-inflammatory candidate.

Prediction of key aroma development in coffees roasted to different degrees by colorimetric sensor array.

We developed a colorimetric sensor array (CSA) that is sensitive to highly contributory volatile compounds of coffee aroma for discrimination of coffee samples roasted to different roast degrees. Strecker aldehydes and α-diketones were significantly higher for the medium roast than the other roast degrees. The development of several sulfur compounds was pronounced in the medium-dark and dark roasts, except for dimethyl sulfide, which was only detected in the light roast. The CSA method coupled with principal component analysis or hierarchical cluster analysis successfully distinguished the roasted coffee samples according to roast degree. Partial least squares regression results showed that the CSA responses were well-correlated with the concentrations of volatile compounds in the coefficient of determination (rp2) range of 0.686-0.955. These results demonstrate that the CSA rapidly responded to coffee aroma compounds and was capable of predicting coffee aroma development.

Photoprotective Effect of Lotus (Nelumbo nucifera Gaertn.) Seed Tea against UVB Irradiation.

Lotus (Nelumbo nucifera Gaertn.) seed is widely used as a traditional medicine in countries of Asia. Among many functions of the lotus seed, one interesting activity is its skin protection from the sunlight and scar. In this study, we focused on the skin protective property of lotus seed tea against ultraviolet B (UVB) irradiation. Two groups of a hairless mouse model, water as control (water group) and lotus seed tea (LST group), were administrated a fluid drink water for six months. After 6 month of administration, UVB exposure was carried out to both groups for another 3 months. During and after the administration, the skin moisture content and the morphological and histopathological analyses through biopsy were carried out. Prior to UVB irradiation, no significant difference was discovered in the skin moisture content for the water group and LST group (P<0.05). However, drastic changes were observed after the UVB treatment. The LST group showed a clear evidence of skin protection compared to the control group (P<0.05). The moisture content, epidermal and horny layer thickness, and protein carbonyl values all revealed that the intake of the lotus seed tea enhanced protection against UVB exposure. As a result, the long-term intake of the lotus seed tea showed the effect of preventing loss of skin moisture, mitigating the formation of abnormal keratinocytes, and contributing to protein oxidation inhibition.

Hypopigmentary effects of ethyl P-methoxycinnamate isolated from Kaempferia galanga.

We isolated crystals from the chloroform fraction of an ethanol extract of Kaempferia galanga and identified it as ethyl p-methoxycinnamate through nuclear magnetic resonance analysis. In the present study, we found that ethyl p-methoxycinnamate significantly decreased melanin synthesis in B16F10 murine melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH). In a cell-free system, however, ethyl p-methoxycinnamate did not directly inhibit tyrosinase, the rate-limiting enzyme of melanogenesis. Instead, it inhibited tyrosinase activity in B16F10 cells in a dose-dependent manner. Furthermore, Western blot analysis showed that ethyl p-methoxycinnamate decreased microphthalmia-associated transcription factor and tyrosinase levels in α-MSH-stimulated B16F10 cells. These results indicate that the pigment-inhibitory effect of ethyl p-methoxycinnamate results from downregulation of tyrosinase. Ethyl p-methoxycinnamate isolated from K. galanga could be developed as a skin whitening agent to treat hyperpigmentary disorders.

Procyanidins from the stem wood of Machilus japonica and their inhibitory effect on LDL oxidation.

The stem wood of Machilus japonica Siebold & Zucc were extracted with 80 % aqueous MeOH, and the concentrated extract was successively partitioned with ethyl acetate (EtOAc), normal butanol, and water. From the EtOAc fraction, five procyanidins, procyanidin A1 (1), procyanidin A2 (2), procyanidin B7 (3), cinnamtannin B1 (4), and aesculitannin B (5), were isolated. Their chemical structures were identified through spectroscopic data analyses including NMR, MS, and IR. This is the first time any of these compounds have been isolated from this plant. The compounds were evaluated for inhibition activity on LDL oxidation. All of these compounds and the positive control, BHT, showed a very high inhibition effect with IC50 values of 0.94, 2.1, 1.8, 1.1, 1.0, and 1.9 µM, respectively.

A new phenanthrene derivative and two diarylheptanoids from the roots of Brassica rapa ssp. campestris inhibit the growth of cancer cell lines and LDL-oxidation.

Brassica rapa ssp. campestris (Brassicaceae) is a conical, deep purple, edible root vegetable commonly known as a turnip. We initiated phytochemical and pharmacological studies to search for biological active compounds from the roots of B. rapa ssp. campestris. We isolated a novel phenanthrene derivative, 6-methoxy-1-[10-methoxy-7-(3-methylbut-2-enyl)phenanthren-3-yl]undecane-2,4-dione, named brassicaphenanthrene A (3) along with two known diarylheptanoid compounds, 6-paradol (1) and trans-6-shogaol (2), through the repeated silica gel (SiO2), octadecyl silica gel, and Sephadex LH-20 column chromatography. The chemical structures of the compounds were determined by spectroscopic data analyses including nuclear magnetic resonance, mass spectrometry, ultraviolet spectroscopy, and infra-red spectroscopy. All compounds exhibited high inhibitory activity against the growth of human cancer lines, HCT-116, MCF-7, and HeLa, with IC50 values ranging from 15.0 to 35.0 µM and against LDL-oxidation with IC50 values ranging from 2.9 to 7.1 µM.

Discovery of small molecules that inhibit melanogenesis via regulation of tyrosinase expression.

5,6,7,8-Tetrahydro-4H-cyclohepta[d]isoxazole derivatives were synthesized and evaluated as a novel class of inhibitors for α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis in a mouse melanoma B16F10 cell line. Compound 8e (IC(50)=0.67 µM), 8h (IC(50)=1.01 µM) and 9b (IC(50)=0.99 µM) exhibited a potent inhibitory activity approximately 85- to 126-fold greater than kojic acid, a well-known potent inhibitor. A biochemical study indicates that the activity of this series should be displayed via down-regulation of the expression of tyrosinase.

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation.

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.