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10.3390/metabo8040058
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Ho, K.
Lei, Z.
Sumner, L. W.
Coggeshall, M. V.
Hsieh, H.
Stewart, G. C.
Lin, C.
Identifying Antibacterial Compounds in Black Walnuts (Juglans nigra) Using a Metabolomics Approach
Khanh-Van Ho
1,2 ,
Zhentian Lei
Lloyd W. Sumner
Mark V. Coggeshall
Hsin-Yeh Hsieh
George C. Stewart
Chung-Ho Lin
The Center for Agroforestry, School of Natural Resources, University of Missouri, Columbia, MO 65211, USA
Department of Food Technology, Can Tho University, Can Tho 90000, Vietnam
Metabolomics Center, University of Missouri, Columbia, MO 65211, USA
Department of Biochemistry, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
U. S. Northern Research Station, USDA-Forest Service, West Lafayette, IN 47907, USA
Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
Metabolites 2018, 8(4), 58; https://doi.org/10.3390/metabo8040058
Received: 1 September 2018 / Revised: 22 September 2018 / Accepted: 28 September 2018 / Published: 29 September 2018
(This article belongs to the Special Issue Mass Spectrometry-Based Metabolomics and Its Applications)
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Black walnut (Juglans nigra L.) is one of the most economically valuable hardwood species and a high value tree for edible nut production in the United States. Although consumption of black walnut has been linked to multiple health-promoting effects (e.g., antioxidant, antimicrobial, anti-inflammatory), the bioactive compounds have not been systematically characterized. In addition, the associations between different black walnut cultivars and their health-promoting compounds have not been well established. In this study, the kernels of twenty-two black walnut cultivars selected for nut production by the University of Missouri Center for Agroforestry (Columbia, MO, USA) were evaluated for their antibacterial activities using agar-well diffusion assay. Among the selected cultivars, four black walnut cultivars (i.e., Mystry, Surprise, D.34, and A.36) exhibited antibacterial activity against a Gram-positive bacterium (Staphylococcus aureus), whereas other cultivars showed no effect on the inhibition of this bacterium. The antibacterial compounds showing the strongest activity were isolated with bioassay-guided purification and identified using a metabolomics approach. Six antibacterial bioactive compounds responsible for antimicrobial activity were successfully identified. Glansreginin A, azelaic acid, quercetin, and eriodictyol-7-O-glucoside are novel antibacterial compounds identified in the kernels of black walnuts. The metabolomics approach provides a simple and cost-effective tool for bioactive compound identification.
black walnut; Juglans nigra; antibacterial; metabolomics approach; compound identification
Black walnut (Juglans nigra L.), known as eastern black walnut or America walnut, is economically valuable tree for hardwood and nut production [1,2], distributed throughout most of the eastern half of the United States [3]. The value of black walnut growing stock on timberland is estimated to be over one-half trillion dollars in the eastern United States [4]. This species is the second highest produced walnut nut in the United States and Missouri is the leading producer of black walnut [5]. Black walnut is often preferred in the food industry due to its unique flavor and aroma.
Black walnut has been identified and selected for propagation since late 1800s [6]. Currently, over 700 black walnut cultivars have been recorded and selected for either timber and nut production during the past century [7,8]. Selection traits for black walnut orchards include several characteristics such as yield, percent kernel, cultivar traits leafing date, flowering dates, growth habit, disease resistance, precocity, productivity, and shelling quality [9]. Several black walnut cultivars have been selected for nut production by the University of Missouri (MU) Center for Agroforestry (Columbia, MO, USA) [10].
Black walnut is an excellent source for phytochemical compounds including phenolic acids, flavonoids, and catechins [11] and monounsaturated fatty acids [5]. Our previous studies have revealed that the kernel of black walnuts contain several bioactive compounds such as quinic acid, gallic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, quercetin-3-d-glucoside, epicatechin gallate, rutin, naringin, and ferulic acid [11]. These compounds have been successfully identified from eleven black walnut cultivars using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis [11]. In fact, the kernel extraction of black walnuts has been linked to antibacterial properties [12,13,14,15,16,17,18]. The stem bark extraction of English walnut (Juglans regia L.) has been reported to exhibit the antibacterial activity against methicillin-resistant Staphylococcus aureus [19]. However, the antibacterial activities among the extracts from different black walnut cultivars have never been compared, and predominant bioactive compounds in black walnut have not been isolated or characterized.
Reverse-phase flash chromatography has been widely utilized to fractionate and separate the biomolecules [20]. In this technique, the powder resins collated with the sample extraction are loaded on chromatography column packed resin coated with hydrophobic functional groups, such as C18 or C18 Bondesil, and connected to a fraction collector. The phytochemical compounds from the plant extraction are separated based on differences in their physicochemical properties (e.g., hydrophobicity and affinity). The phytochemical compounds migrate through the column at different rates and then are automatically collected at different times. The identification of bioactive compounds can be facilitated via phytochemical screening assays. This technique provides several advantages (e.g., reproducibility) compared to the traditional normal-phase chromatography technique.
For the current paper, we first evaluated and compared the antibacterial properties among the extracts from twenty-two black walnut cultivars selected for nut production. We then isolated and identified bioactive compounds in the kernels of the best cultivar using bioassay-guided purification followed by a metabolomics analysis.
2.1. Antibacterial Activity from Twenty-Two Black Walnut Cultivars
The zones of inhibition caused by kernel extracts from twenty-two black walnut cultivars against a Gram-positive bacterium (S. aureus) showed significant differences (p < 0.0001, F22,75 = 200.19) (Figure 1). Four black walnut cultivars (i.e., Mystry, Surprise, D.34, and A.36) exhibited antibacterial activity against the Gram-positive bacterium whereas other cultivars (i.e., A4.1010, B.15, B.31, Bowser, C8.04.1003, D16.06.1036, Daniel, Davidson, Emma, Hay, Hare, Jackson, Kwik Krop, Schessler, Sparks, Sparrow, South Fork, and Thomas) had no inhibitory effects on this bacterium. Mystry had the greatest zone of inhibition (11.83 ± 0.75 mm) compared to other cultivars and the zones of inhibition of Surprise, D.34, and A.36 which were 7.42 ± 0.92 mm, 6.75 ± 0.42 mm, and 6.33 ± 0.52 mm, respectively, were significantly different.
2.2. Identification of Antibacterial Compounds Derived from the Kernel Extract of Mystry
2.2.1. Column Chromatography
The fractionation of kernel extract from Mystry by column chromatography yielded 46 fractions. The antibacterial activities of these fractions against the Gram-positive bacterium (S. aureus) were significant different (p < 0.001, F47,94 = 37.28) (Figure 2 and Supplementary Figure S1). The fraction numbers from 4 to 18 had antibacterial activities against S. aureus while other fractions had no inhibitory effects on S. aureus. The zones of inhibition of fraction 5 and 6 (11.17 ± 0.83 mm and 12.17 ± 0.44 mm, respectively) were significantly higher compared to that of other fractions. No significant difference was seen between the zones of inhibition of fraction 6 and the crude extract of Mystry whereas the zone of inhibition of fraction 5 was lower than that of the extract of Mystry (Figure 2).
2.2.2. HPLC Analysis
The further separation of fraction 6 by HPLC resulted in 27 sub-fractions (Figure S2). Only sub-fraction 14 exhibited the antibacterial activity against S. aureus while other sub-fractions had no effect on the inhibition of the Gram-positive bacterium (Figure 3). The zone of inhibition of sub-fraction 14 was significantly higher compared to other sub-fractions, but was lower compared to the crude extract of kernel from Mystry (p < 0.0001, F28,56 = 466.87).
2.2.3. UHPLC-QTOF-MS/MS Analysis to Identifying the Bioactive Compounds
Fraction 14 from HPLC fractionation that showed inhibition activity was subject to UHPLC-QTOF-MS/MS analysis in both negative and positive electrospray ionization modes. Six major peaks resolved bioactive compounds were identified tentatively by searching their MS/MS spectra with the metabolomics databases (Figure 4 and Figure 5, Table 1).
Compound 1 has retention time (rt) and m/z at 5.21 min and 463.0388, respectively. The MS/MS spectrum displayed fragment ions at m/z 301 [M − H − 162]−, 300 [M − H − 162 − H]−, 271 [M − H − 162 − 30]−, 255 [M − H − 162 − 46]−, 146 [M − H − 162 − 155]−, and 119 [M − H − 162 − 182]−. Loss of 162 suggested that it is glycosylated compound and fragment at m/z of 301 is the aglycone ion. The compound is not methylated as no loss of 15 Da was observed. The MS/MS was matched to quercetin-3-O-glucoside with a score of 834 (out of 1000) in the MassBank library. Thus, the compound is tentatively identified as quercetin-3-O-glucoside.
Compound 2 has rt and m/z at 5.77 min and 465.1414, respectively. The MS/MS spectrum displayed fragment ions at m/z of 301 [M − H − 164]−, 300 [M − H − 164 − H]−, 241 [M − H − 164 − 60]−, 169 [M − H − 164 − 132]−, 125 [M − H − 164 − 176]−, and 107 [M − H − 164 − 194]−. This information matches to agnuside, catechin-4-ol 3′-methyl ether 3-O-alpha-l-rhamnopyranoside, catechin 5-O-beta-d-glucopyranoside-4′-methyl ether, symplocoside (catechin 7-O-beta-d-glucopyranoside-3′-methyl ether) in Metlin database with the same mass tolerance (3.8 ppm). MS/MS similarity search showed that major fragments in MS/MS spectrum matched to those of epigallocatechin gallate with the score of 685, but the molecular weight did not match. Thus, it is possibly one of the catechin derivatives.
Compound 3 has rt and m/z at 6.15 min and 449.1102, respectively. The MS/MS spectrum displayed fragment ions at m/z 300 [M − H − 149]−, 299 [M − H − 149 − H.]−, 255 [M − H − 149 − 45]−, 200 [M − H − 149 − 100]−, 175 [M − H − 149 − 125]−, 151 [M − H − 149 − 149]−, and 135 [M − H − 149 − 165]−. The MS/MS was matched to eriodictyol-7-O-glucoside with a score of 872 (out of 1000) in the MassBank library. The ion 449.1102 m/z gave 151/135 as fragments, corresponding to the fragments previously described by Brito et al. [21] as key aglycone fragments. Thus, the compound is tentatively identified as eriodictyol-7-O-glucoside.
Compound 4 has rt and m/z at 6.26 min and 477.0941, respectively. The MS/MS spectrum displayed fragment ions at m/z 301 [M − H − 176]−, 300 [M − H − 176 − H.]−, 271 [M − H − 176 − 30]−, 255 [M − H − 176 − 46]−, 179 [M − H − 176 − 122]−, and 151 [M − H − 176 − 150]−. In MS/MS spectrum, the predominant ions at m/z 301 [M–H–176]− gave the proof of same glucuronyl unit loss and fragment at m/z of 301 is the aglycone ion. The characteristic product ions at m/z of 271, 255, 179, and 151 indicate the aglycone of quercetin. On the basis of the mass spectral data, compound 4 is tentatively identified as quercetrin with a score of 823 (out of 1000) in the metabolomics library developed by Lei et al. [22].
Compound 5 has rt and m/z at 6.58 min and 187.0977, respectively. The MS/MS spectrum displayed fragment ions at m/z 144 [M − H − 43]−, 125 [M − H − 43 − 19]−, 123 [M − H − 43 − 21]−, and 97 [M − H − 43 − 47]−. The MS/MS product ion spectrum is dominated by m/z of 125, which corresponds to the combined loss of a molecule water and CO2 (62 Da). Further fragmentation at m/z of 125 leads to ions at m/z of 123 and 97. The MS/MS was matched to azelaic acid in the MassBank library with a score of 813 (out of 1000). Therefore, compound 5 was tentatively identified as azelaic acid.
Compound 6 has rt and m/z at 7.18 min and 592.2043, respectively. The MS/MS spectrum displayed fragment ions at m/z of 283 [M − H − 309]−, 241 [M − H − 309 − 42]−, 223 [M − H − 309 − 60]−, 197 [M − H − 309 − 86]−, 181 [M − H − 309 − 102]−, 144 [M − H − 309 − 139]−, and 137 [M − H − 309 − 146]−. The ion 592.2043 m/z gave 241/197 as fragments, corresponding to the fragments previously described by Gómez-Caravaca et al. [23] to be glansreginins A. Thus, compound 6 is tentatively identified as glansreginin A with score of 1.0 (peaks: 13/21 annotated/matched) via the MetFrag library.
We demonstrated that the antibacterial properties of black walnuts against S. aureus were varied among tested cultivars. The four cultivars including Mystry, Surprise, D.34, and A.36 exhibited different antibacterial capacity against the Gram-positive bacterium (S. aureus RN6390). Other cultivars tested showed no effects on this bacterium. Several fractions of the kernel of Mystry from the column extraction showed the antibacterial activities (Figure 2), indicating the presence of multiple bioactive compounds and the possibilities of the synergy effects of these compounds that inhibited the bacterial growth. Vu et al. [11] reported differences in phenolic profiles of 11 different black walnut cultivars (e.g., Daniel, Davidson, Emma, Hay, Jackson, Kwik Krop, Mystry, Sparks, Sparrow, Schessler, Surprise, and Tomboy). Differences in the bioactive activity of black walnut cultivars are highly likely due to the differences in phytochemical profiles of these cultivars. This finding from this study clearly illustrated the differences in antibacterial properties among the black walnut cultivars, which can be used as a selection trait for improving the quality of nuts for nut production.
We found six bioactive compounds (i.e., quercetin-3-O-glucoside, a catechin derivative, eriodictyol-7-O-glucoside, quercetin, azelaic acid, and glansreginin A) responsible for the antibacterial activity of the kernels of Mystry against the Gram-positive bacterium (S. aureus) via the metabolomics approach combined with bioassay-guided fractionation strategy (Figure 6). The information on the biological activities of these compounds is summarized in Table 2. In addition to other bioactive compounds (i.e., quinic acid, gallic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, isoquercetin, catechin, epicatechin gallate, rutin, naringin, ferulic acid) in the kernels of black walnuts that have been linked to possess the antibacterial properties [11], our results reveal that glansreginin A, azelaic acid, quercetin, and eriodictyol-7-O-glucoside are the predominant antibacterial compounds in the kernels of black walnuts according to their hydrophobicity (retention times). Among the six bioactive compounds, glansreginin A was the most abundant in the purified bioactive fraction (Figure 4). However, the analytical standard for glansreginin A is not commercially available [24], making it difficult for the validation and qualification of this compound. Future research should focus on validation and characterization of these antibacterial compounds in black walnut cultivars when the authentic standards or purified reference standards are accessible. The antimicrobial agents identified black walnut extracts could be used to prevent growth of spoilage and pathogenic microorganisms in foods. They could also be used as the natural preservatives in the formulation of personal care product.
Quercetin-3-O-glucoside (known as isoquercitin), a flavonoid, is widely present in a variety of plants (e.g., medicinal herbs, fruits and vegetables) such as black walnut [11], English walnut (J. regia) [55], and buckwheat (Fagopyrum esculentum) [56]. In vitro, isoquercitin has also been documented to possess antibacterial activities against several bacteria such as S. aureus, S. epidermidis, and Propionibacterium acnes in disk diffusion assay [25]. Many reports revealed that isoquercitin has been linked to versatile biological properties in vitro and in vivo including antioxidant [26], anti-inflammatory [27], antifungal [28], antidiabetic [29], anti-allergic [30], antitumor [31,32], antiviral [33], anti-hypertensive [34], anti-apoptotic [35], and diuretic effects [36].
Catechin derivatives (e.g., (+)-catechin, (−)-epicatechin, (−)-epigallocatechin) are found as major flavonol components in beverages, vegetables, and fruits [39,57]. Catechin, epicatechin, and epicatechin gallate have also detected previously in the kernels of black walnuts [11]. This phytochemical group has been associated with a variety of biological functions including antibacterial, antioxidant, anti-inflammatory, antiviral, and antitumor effects [37,40,41,42,57].
Eriodictyol-7-O-glucoside is found as a major flavonoid component derived from a Chinese herb (Dracocephalum rupestre), and from several plants e.g., grapevine (Vitis vinifera) [58], pistachios (Pistacia vera) [59]. The biological property of eriodictyol-7-O-glucoside has not been well established. Information on the biological activity of this compound has focused on the neuroprotective effect against oxidative stress in vitro and in vivo via Nrf2/ARE activation [43]. In contrast to eriodictyol-7-O-glucoside, eriodictyol has been associated with a variety of biological properties in vitro and in vivo such as antioxidant [60], antimicrobial [61], anti-inflammatory [62], antineoplastic [63], and antinociception [64].
Quercetin, a flavonoid, and is commonly found in vegetables (e.g., onion, garlic). The antibacterial properties against several bacteria have been also reported for quercetin [25]. Quercetin exhibited stronger antibacterial effects against three bacteria (S. aureus, S. epidermidis, and P. acnes) compared to isoquercetin [25]. This compound has also been linked to multiple biological functions in vitro and in vivo, such as antioxidant [25], anti-inflammatory [44], anti-allergic [30], and antitumor [65] capabilities.
Azelaic acid is a naturally occurring saturated dicarboxylic acid derived from a variety of grains such as sorghum (Sorghum bicolor) [66], rye (Secale cereal) [67]. This compound has also been reported to exert a variety of biological activities in vitro and in vivo including antimicrobial [46] and antitumor [47,48] capabilities. The U.S. Food and Drug Administration (FDA) approved 15% gel formulation of azelaic acid for the treatment of rosacea in 2002 [68] and this compound is an excellent antimicrobial agent cosmetic for the treatment of comedonal and inflammatory acnes [69,70]. In vitro, azelaic acid exhibited the antibacterial effect against S. aureus, S. epidermidis, and P. acnes at pH of 5.6, but no antibacterial effect was seen at pH of 7.3 [46].
Glansreginin A, a dicarboxylic acid derivative, presented dominantly in the bioactive fraction might be mainly responsible for the antibacterial activity of the kernels of Mystry. Glansreginin A has been also found in the kernels of three other nuts including English walnut [23,49,54,55], pecans (Carya illinoinensis) [71], and hazelnut (Corylus avellana) [50]. Due to the presence of this compound in the kernels of these nuts, glansreginin A has been linked to multiple biological activities in vitro and in vivo such as antioxidant [49,50,72], anti-inflammatory [51], antiatherogenic effect [52], and antinociceptive effect [53], reduction of cholesterol absorption [54]. This is the first time, glansreginin A is reported to be associated with the antibacterial properties.
In recent years, the advancement in mass spectrometry, computation power, metabolomics algorithm and mass spectral libraries in metabolomics allows rapid identification of the bioactive molecules. The metabolomics approach combined with bioassay guided fractionation strategy in this study is a promising tool for putative identification of new bioactive compounds from natural sources. The high-resolution data generated from MS/MS offer the mass accuracy and specific fragmentation fingerprints needed for rapid identification of the antimicrobial molecules. Therefore, it eliminated the time consuming and labor-intensive large-scale purification procedure that is required in the traditional structural elucidation techniques. This approach is cost-effective compared to other approaches for compound identification e.g., nuclear magnetic resonance (NMR), which typically requires at least 1 mg of the purified crystal [73].
Black walnut is an excellent resource not only for nutrition but also medicinal values. Among twenty-two black walnut cultivars tested, four cultivars exhibited antibacterial activity against the gram-positive bacterium S. aureus and six antibacterial compounds in the kernels of Mystry were tentatively identified. With a growing global consumption of organic personal care products and diet supplements, through identifying the novel uses of the black walnut and its byproducts, this study will provide the opportunities to turn abundant, low-value, renewable materials from the black walnut and its byproducts into profitable value-added products for the industry. Future research should focus on exploring of other health-promoting properties (e.g., antioxidant, anti-inflammatory, antitumor) and industrial applications of bioactive compounds of black walnuts through utilizing the same metabolomics strategy.
4.1. Black Walnut Cultivars
The nuts of twenty-two black walnut cultivars (i.e., Bowser, Daniel, Davidson, Emma, Hay, Hare, Jackson, Kwik Krop, Mystry, Schessler, Sparks, Sparrow, South Fork, Surprise, Thomas, A4, A.36, B.15, B.31, C8, D16, and D.34) were collected at the University of Missouri, Horticulture and Agroforestry Research Center, New Franklin, MO, USA. The black walnuts were hulled mechanically and hang up to dry for 15 days in a dry and darkness place at 24 °C. The hulled nuts were then stored at −20 °C until analysis.
4.2. Extraction of Bioactive Compounds from the Kernels of Black Walnuts
The hulled nuts were manually cracked, and the kernels were shelled and homogenized using a coffee grinder (product # CBG100S, Black + Decker, Beachwood, OH, USA) prior to extraction. The phytochemicals in the kernels of each cultivar (3 g, 20–30 mesh) were extracted with sonication in 15 mL of methanol (HPLC grade, Fisher Scientific, Pittsburg, PA, USA). The extract was sonicated for 60 min followed by centrifugation for 10 min at 4000 rpm and the supernatant was collected. Subsequently, the supernatant was filtered through a 0.2 μm Whatman Anotop syringe membrane filter (Sigma-Aldrich, St. Louis, MO, USA). The aqueous extract was evaporated until dryness under a flow of nitrogen and the final extract was resuspended with dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) at concentration 0.12 g/mL for screening antibacterial activities using an agar-well diffusion assay.
4.3. Antibacterial Assay
The strain of bacteria used in this study was a Gram-positive bacterium (S. aureus strain RN 6390) [74]. S. aureus and Methicillin-resistant S. aureus (MRSA) are Gram-positive bacteria that are resistant to several antibiotics in the market. Identifying molecules or scaffolds that could inhibit the S. aureus and MRSA could help elucidating possible new antibacterial modes of action against these pathogens. In addition, many skin infections (acne) or gum disease often results from the infection of the Gram-positive bacteria. From the previous works, many antimicrobial compounds that inhibited the S. aureus could also use to treat skin infections and gum disease (e.g., totarol). Therefore, selecting this specific strain in the early stage screening study will help explore the future commercial application.
The antibacterial activities of black walnut extracts were determined using agar-well diffusion assay as described by Holder and Boyce [75]. The S. aureus RN6390 was streaked on Luria-Bertani (LB) agar plates incubated at 37 °C for 12 h. For preparation of 1 L of LB agar, 15 g of agar (Fisher Scientific), 10 g of tryptone (Fisher Scientific), 5 g of yeast extract (Fisher Scientific) and 10 g of NaCl (Fisher Scientific) were suspended in 1 L of distilled water. The mixture was homogenized and autoclaved at 121 °C for 1 h. It was then cooled to approximately 50 °C while mixing with a magnetic stirrer. The liquid LB agar was poured into Petri plates (150 mm diameter) and allowed to solidify at 25 °C. For preparation of an culture of RN6390, the bacteria were cultured in LB broth at 37 °C for 16 h and then diluted into 5 mL LB broth (to an OD600 of 0.02) and incubated in a shaker at 37 °C. Once the culture reached an OD600 of 0.1, it was swab-inoculated onto LB agar plates. For testing antibacterial activities of the extracts, wells (4.5 mm in diameter) were cut into the surface of the agar using a cork borer. The extracts (10 µL) were pipetted into the wells and the plates then incubated under aerobic condition at 37 °C for 16 h. A biocompatible solvent (DMSO) was used as negative control. The diameters of inhibition zones were measured by a ruler, with an accuracy of 0.5 mm. Each inhibition zone was measured three times and each extract was replicated at least three times in three different plates.
4.4. Identification of Bioactive Compounds Using a Metabolic Approach
The black walnut cultivar that exhibited the strongest antibacterial activities against the gram-positive bacterium was selected for bioassay-guided purification. The kernel extract of this cultivar was sequentially fractionated using column chromatography (CC) and then the bioactive fraction was further fractionated using high-performance liquid chromatography (HPLC). The agar-well diffusion assay was performed to identify the antibacterial activities of the bioactive fractions. The HPLC sub-fraction that had the strongest antibacterial activities against the Gram-positive bacterium was analyzed by high-resolution mass spectrometry and the mass spectra was analyzed to identify the bioactive compounds responsible for the antibacterial activities.
Kernels (25 g) were collected and the phytochemicals were extracted twice with methanol (100 mL:100 mL). The extract was homogenized thoroughly using a blender (Hamilton Beach, Inc., Glen Allen, VA, USA) and was sonicated for 60 min at temperature ≤ 30 °C. The extract was filtered through filter paper (125 mm in diameter, Whatman, GE Healthcare, Chicago, IL, USA) under SPE Vacuum Manifold (Visiprep™ SPE Vacuum Manifold, Sigma-Aldrich, Saint Louis, MO, USA) and then was concentrated by a rotary evaporator (BUCHI Rotary Evaporator R110, Buchi, Flawil, Switzerland) under a vacuum (Buchi), yielding a greenish yellow gum (4 g). The resultant gum (4 g) was dissolved in methanol (5 mL), impregnated with 4 g of sorbents Bondesil C18 (40 µm particle size; Agilent Technologies, Santa Clara, CA, USA) and placed in the hood for 12 h until all the methanol was evaporated, which yielded powder resins coated with the extract. The C18 resins with the extract were stored at 4 °C in darkness and used within a week.
The compounds were separated and fractionated by a Biotage FlashMaster II flash chromatography connected with an ISCO Foxy 200 fraction collector. The powder resins collated with the extract (8 g) were loaded on to the top of chromatography column (3.7 cm in diameter and 13.5 cm long) packed with 34 g of C18 Bondesil resin. The running time was 300 min at a flow rate of 0.5 mL/min. The mobile phase consisted of deionized water (A) and methanol (B) and a linear gradient was optimized as follows 25% B (0–40 min), 25–50% B (40–90 min), 50–75% B (90–120 min), 75% B (120–180 min), 75–100% B (180–240 min) and 100% B (240–300 min), respectively. The elution yielded forty-six fractions collected automatically by a fraction collector. All fractions were concentrated by nitrogen evaporator and re-dissolved in DMSO at 10× concentration for antibacterial activities testing using an agar-well diffusion assay.
The bioactive fraction from column chromatography that exhibited the strongest antibacterial activities against the Gram-positive bacterium was further fractionated by high-performance liquid chromatography (HPLC). The bioactive fraction (50 µL) was injected into a HPLC system consisting of a Shimadzu SCL-10Avp HPLC controller (Shimadzu Co., Columbia, MD, USA), a LC-10ADvp solvent delivery system, SIL-10ADvp auto-injector, a SPD-10Avp photodiode array detector, and a FRC-1500 HPLC micro fractionation collector (Shimadzu). The elution was performed with a Columbus C8 reverse-phase column (250 mm × 4.6 mm, 5 µm particle size; Phenomenex, Torrance, CA, USA). The running time was 30 min at a flow rate of 0.5 mL/min and the signals were monitored at both 254 nm and 220 nm. The mobile phase consisted of deionized water (A) and acetonitrile (B). The elution condition was optimized as follows 10–45% B (0–16 min), 45% B (16–16.2 min), 45–80% B (16.2–17 min), 80–98% B (17–18 min), 98% B (18–19 min), 98–20% B (19–20 min), and 20% B (20–30 min), respectively. The HPLC sub-fractions were collected automatically into fraction collection vials. The fractions having the same retention time window were pooled after 50-time injections of the bioactive fraction from column chromatography. The solvent of each separated fraction was evaporated under the hood and the samples were dissolved in DMSO at 100× concentration for testing antibacterial activities using an agar-well diffusion assay.
4.4.3. UHPLC-QTOF-MS/MS Analysis
The HPLC sub-fraction that exhibited the strongest antibacterial activities was concentrated 100X in 80% methanol containing an internal standard (umbelliferone) and then the sample was analyzed by UHPLC coupled to a maXis impact quadrupole-time-of-flight mass spectrometer (Bruker Daltonics, Gmbh, Bremen, Germany). The separation was achieved on a Waters Acquity UHPLC BEH C18 column (2.1 × 100 mm, 1.7 µm particles size) using a linear gradient of 95%: 5% to 30%: 70% eluents A: B (A: 0.1% formic acid and B: acetonitrile) in 30 min. From 30–40 min, a linear gradient was as follows 70–95% B (30–33 min), 95% B (33–35 min), 95–5% B (35–36 min), and 5% B (37–40 min), respectively. The flow rate was 0.56 mL/min and the column temperature was kept at 60 °C. Mass spectrometry was performed in both negative and positive electrospray ionization modes with the nebulization gas pressure at 43.5 psi, dry gas of 12 L/min, dry temperature of 250 °C and a capillary voltage of 4000V. MS/MS mass spectral data was collected automatically using following parameters including MS full scan from 100 to 1500 m/z, 3 precursors, threshold with 10 counts, active exclusion with three spectra released after 0.15 min, collision energy depending on mass such as 35 eV at 500 Da, 50 eV at 1000 Da and 70 eV at 2000 Da. The mass spectra were auto-calibrated using sodium formate after data acquisition.
The metabolite annotation in significant peaks in UV chromatogram of the MS/MS mass spectra was identified based on MS/MS fragmentation. The MS/MS fragments were referenced to the Metabolomics library developed by Lei et al. [20], as well as MetFrag (https://msbi.ipb-halle.de/MetFragBeta/), MassBank of North America (http://mona.fiehnlab.ucdavis.edu/spectra/), and METLIN (http://metlin.scripps.edu).
4.4.4. Statistical Analysis
In antibacterial experiments, zones of inhibition of black walnut extracts were analyzed as a randomized complete block design using PROC MIXED in SAS 9.4 (SAS Institute, Cary, NC, USA). If no inhibition was observed in the samples, a value of 4.5 mm, which was the diameter of the wells used in the agar-well diffusion assay, was assigned to these treatments prior to the analysis. The black walnut extract was the fixed effect and replication was the random variable. Differences between extracts were determined using Fisher's LSD.
The antibacterial properties of the kernels of black walnuts were successfully characterized. In fact, twenty-two black walnut cultivars (i.e., Bowser, Daniel, Davidson, Emma, Hay, Hare, Jackson, Kwik Krop, Mystry, Schessler, Sparks, Sparrow, South Fork, Surprise, Thomas, A4, A.36, B.15, B.31, C8, D16, and D.34) had shown differences in their antibacterial properties against the Gram-positive bacterium (S. aureus RN6390) and Mystry exhibited the strongest antibacterial activity. The antibacterial activity was also seen in Surprise, D.34, and A.36, but no antibacterial effect was seen in the other tested cultivars. Six possible antibacterial compounds in the kernels of Mystry were tentatively identified through the metabolomics approach combined with bioassay-guided purification. This approach is a promising tool for identifying the candidates of bioactive molecules from natural sources.
The following are available online at https://www.mdpi.com/2218-1989/8/4/58/s1, Figure S1: Zone of inhibition of 29 Mystry fractions (out of 46) from column chromatography. 1-29: fraction 1 through fraction 29, Figure S2: Shimadzu UV Chromatogram of HPLC sub-fraction F14 at 220 nm (A) and 254 nm (B) generated from SPD-10Avp photodiode array detector, Figure S3: Head-to-tail spectral comparisons between the experimental MS/MS spectra and the referenced library MS/MS spectra. (A) Peak 1: Isoquercitrin, m/z of 463.0888, retention time (rt) at 5.21 min; (B) Peak 2; (C) Peak 3: Eriodictyol-7-O-glucoside, m/z of 449.1102, rt at 6.15 min; (D) Peak 4: Quercitrin, m/z of 477.0941, rt at 6.26 min; (E) Peak 5: Azelaic acid, m/z of 187.0977, rt at 6.58 min; (F) Peak 6: Glansreginin A, m/z of 592.2043, rt at 7.18 min.
Conceptualization: C.-H.L.; methodology: K.-V.H. and C.-H.L.; formal analysis: K.-V.H., Z.L., and C.-H.L.; resources: C.-H.L., L.W.S., M.V.C., H.-Y.H., G.C.S., and C.-H.L.; data curation: K.-V.H.; writing—original draft preparation: K.-V.H. and C.-H.L.; writing—review and editing: all authors; investigation and funding acquisition: C.-H.L.
This research was funded by the Center of Agroforestry, University of Missouri under cooperative agreement 58-6227-1-004 with the USDA-ARS and Missouri Department of Agriculture Specialty Crop Block Grant (SCBGP) #16SCBGPMO0003. The Sumner lab and the MU Metabolomics Center have been graciously supported by several entities over the years for the development of natural products profiling and plant metabolomics. These specifically include support from the University of Missouri, The Samuel Roberts Noble Foundation, Bruker Daltonics Gmbh, Agilent Technologies, US National Science Foundation (NSF)-JST Metabolomics for a Low Carbon Society #1139489, NSF MRI DBI #1126719, NSF RCN #1340058, and NSF MCB #1024976.
The authors would like to thank the Center of Agroforestry, University of Missouri and Missouri Department of Agriculture Specialty Crop Block Grant Program for supporting this research.
The authors declare no conflict of interest. Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or the University of Missouri. Mention of specific companies, products, or trade names is made only to provide information to the reader and does not constitute endorsement by the USDA-Agricultural Research Service.
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Figure 1. Zones of inhibition of crude kernel extracts of twenty-two black walnut cultivars grown in Missouri for Staphylococcus aureus. If no inhibition was observed in samples, a value of 4.5 mm, which was the diameter of the wells in agar-well diffusion experiments, was assigned. Means within bars followed by different letters are significantly different (α = 0.05, ANOVA). Mean ± SEM.
Figure 2. Zones of inhibition of 46 Mystry fractions from column chromatography. 20–46: fraction 20 through fraction 46. If no inhibition was observed in samples, a value of 4.5 mm, which was the diameter of the wells in agar-well diffusion experiments, was assigned. Means within bars followed by different letters are significantly different (α = 0.05, ANOVA). Mean ± SEM.
Figure 3. Zones of inhibition of 27 Mystry HPLC sub-fractions of fraction 6 from column chromatography. 2–13, 16–27: fraction 2 through fraction 13 and fraction 16 through fraction 27, respectively. If no inhibition was observed in samples, a value of 4.5 mm, which was the diameter of the wells in agar-well diffusion experiments, was assigned. Means within bars followed by different letters are significantly different (α = 0.05, ANOVA). Mean ± SEM.
Figure 4. UV chromatogram of Mystry HPLC sub-fraction at 280 nm. 1–6: annotated metabolites, 1: quercetin-3-O-glucoside, 2: a catechin derivative, 3: eriodictyol-7-O-glucoside, 4: quercitrin, 5: azelaic acid, 6: glansreginin A, and IS: internal standard.
Figure 5. Head-to-tail spectral comparisons between the experimental MS/MS spectra and the referenced library MS/MS spectra. (A) Peak 1: Isoquercitrin, (B) peak 2: a catechin derivative; (C) peak 3: eriodictyol-7-O-glucoside, (D) peak 4: quercitrin, (E) peak 5: azelaic acid, (F) peak 6: glansreginin A. In each comparison, the experimental MS/MS spectra (upper) and the referenced library MS/MS spectra (lower). Since compound 6 was tentatively identified by MetFrag in silico interpretation of the MS/MS spectrum, head-to-tail of this spectral comparison did not show.
Figure 6. Chemical structures of tentative compounds from black walnut (Mystry).
Table 1. Putative bioactive compounds responsible for antibacterial activity in black walnut (Mystry).
Peak No.
(m/z)
Exact Mass
Δm
(ppm)
MS/MS Fragments, m/z, Intensity (%)
Putative
Identification *
1 5.21 463.0888 C21H20O12 464.0954 2.6 301.0308 (25.3), 300.0280 (100), 271.0260 (76.8), 255.0316 (34.3), 146.9262 (23.2), 119.9065 (21.1) Quercetin-3-O-glucoside
2 5.77 465.1414 C22H18O11 458.0849 3.8 301.0147 (2.7), 300.0280 (3.0), 241.0358 (4.2), 169.0146 (64.2), 125.0253 (100), 107.0132 (17.5) Agnuside Catechin-4-ol 3′-methyl ether 3-O-alpha-l-rhamnopyranoside Catechin 5-O-beta-d-glucopyranoside-4′-methyl ether Catechin 7-O-beta-d-glucopyranoside-3′-methyl ether
3 6.15 449.1102 C21H22O11 450.1162 4.0 299.9964 (12.4), 298.9994 (15.3), 255.0408 (12.4), 200.8817 (11.5), 174.9541 (17.1), 151.0039 (100), 135.0444 (77.5) Eriodictyol-7-O-glucoside
4 6.26 477.0941 C21H20O11 448.1006 1.3 301.0373 (43.2), 300.0282 (100), 271.0284 (90.3), 255.0264 (24.8), 178.9994 (29.1), 151.0032 (45.4) Quercitrin
5 6.58 187.0977 C9H14O4 186.0892 3.2 144.9013 (46.2), 125.0979 (100), 123.0804 (62.4), 97.0656 (47.8) Azelaic acid
6 7.18 592.2043 C28H35NO13 593.2108 2.1 283.1199 (8.5), 241.1080 (8.2), 223.0979 (19.9), 197.1185 (34.5), 181.0875 (30.8), 144.0456 (100), 137.0972 (12.1) Glansreginin A
* Tentative identification of compounds on the basis of MS/MS mass spectra and UV spectra reported in the referred databases.
Table 2. Bioactivities of six bioactive compounds responsible for antibacterial activity in the kernels of black walnut (Mystry).
Bioactivities
1 Quercetin-3-O-glucoside antimicrobial Wang et al. [25]
antioxidant Chang et al. [26]
anti-inflammatory Li et al. [27]
anti-fungal Yun et al. [28]
antidiabetic Zhang et al. [29]
anti-allergic Rogerio et al. [30]
antitumor Amado et al. [31], Chen et al. [32]
antiviral dos Santos et al. [33]
anti-hypertensive Junior et al. [34]
anti-apoptoti Zhu et al. [35]
diuretic effects Junior et al. [36]
2 Catechin derivatives antimicrobial Veluri et al. [37], Hara-Kudo et al. [38]
antioxidant Seeram et al. [39]
anti-inflammatory Mizushina et al. [40]
antitumor Cao et al. [41]
antiviral Song et al. [42]
3 Eriodictyol-7-O-glucoside antioxidant Jing et al. [43]
4 Quercitrin antimicrobial Wang et al. [25]
antioxidant Wang et al. [25]
anti-inflammatory Yang et al. [44]
antitumor Liu et al. [45]
5 Azelaic acid antimicrobial Charnock et al. [46]
antitumor Pan et al. [47], Breathnach [48]
6 Glansreginin A antioxidant Ito et al. [49], Slatnar et al. [50]
anti-inflammatory Papoutsi et al. [51]
antiatherogenic effect Berryman et al. [52]
antinociceptive effects Raafat [53]
reduction of cholesterol absorption Ren et al. [54]
© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Ho, K.-V.; Lei, Z.; Sumner, L.W.; Coggeshall, M.V.; Hsieh, H.-Y.; Stewart, G.C.; Lin, C.-H. Identifying Antibacterial Compounds in Black Walnuts (Juglans nigra) Using a Metabolomics Approach. Metabolites 2018, 8, 58. https://doi.org/10.3390/metabo8040058
Ho K-V, Lei Z, Sumner LW, Coggeshall MV, Hsieh H-Y, Stewart GC, Lin C-H. Identifying Antibacterial Compounds in Black Walnuts (Juglans nigra) Using a Metabolomics Approach. Metabolites. 2018; 8(4):58. https://doi.org/10.3390/metabo8040058
Ho, Khanh-Van, Zhentian Lei, Lloyd W. Sumner, Mark V. Coggeshall, Hsin-Yeh Hsieh, George C. Stewart, and Chung-Ho Lin. 2018. "Identifying Antibacterial Compounds in Black Walnuts (Juglans nigra) Using a Metabolomics Approach" Metabolites 8, no. 4: 58. https://doi.org/10.3390/metabo8040058
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Metabolites, EISSN 2218-1989, Published by MDPI


At The Printhouse we've been developing a prayer space that we've really been looking forward to opening up and offering to the city. We'd love to see people that live or work in the city centre, or those happy to travel from further afield, use the space to pray in, by night and by day.
The Sanctuary of the Bible is the place where God dwelt. It was a place of devotion, a place of worship, a refuge, a place of hope, as a shadow of what was to come and a witness to the nations.
In Ezekiel 47, the water flows out from the sanctuary and becomes a river that turns salt water fresh so that it teems with abundant life and nourishes the trees on its banks so that they produce fruit every month and their leaves bring healing.
We want this vision to inspire our prayer for the city from within the city; that it would be the kind of prayer that brings transformation to the people, communities and even to the nations which are represented in this city.


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Jimmy Pedro Judo Student And World Judo Champ Kayla Harrison Shows You The Ouchi Gari Major Inner Reaping Throw
Jimmy Pedro
judo technique
judo throw
September, 2013,
Kayla Harrison has studied judo with two-time Olympic medalist and 1999 world judo champ Jimmy Pedro since she was 16. At age 20, she stepped onto the World Judo Championships mat in Tokyo in September 2010. It had been more than a decade since the United States had produced a world judo champion and more than a quarter-century since that champion was a woman. While such pressure may have psyched out a lesser judo player, Kayla Harrison got in there and brought home the United States' fourth world-championship judo gold medal. And then, of course, she went on to win the United States' first gold medal in judo during the 2012 Summer Olympics in London! Before her historic Olympic win, Kayla Harrison visited Black Belt's photo studio in December 2010 and shared with us some of her winning judo techniques! In this segment, she demonstrates her usage of the ouchi gari, or major inner reaping throw.
JUDO TECHNIQUE VIDEO Kayla Harrison Demonstrates the Ouchi Gari (Major Inner Reaping Throw)
"In judo, there are three major parts to a throw," Kayla Harrison explains. "The first part, the kuzushi, or the off-balance, for ouchi gari is ... I'm going to snap my opponent forward. This is going to get him thinking that I'm trying to throw him forward, and in return, he's going to react by pulling backward. "The second part of the throw, or the entry -- the tsukuri -- I'm going to step with my right foot right in between his feet, followed by my left foot stepping right behind that foot. So it's going to be almost like a T.
Get inside the mind of Olympic judoka Ronda Rousey in this FREE download!
Ronda Rousey: An Exclusive Interview With the Gene LeBell Protégé, Olympic Judo Medalist and MMA Fighter
"The third step, for the finish — the kake — I'm going to push his elbow in and my forearm is gonna go right into his body. And as I do this motion, I'm going to hook his leg and make a circular motion." Kayla Harrison then proceeds to throw her somewhat unfortunate judo partner to the hard studio floor several times, but all can rest assured that he emerged from the ordeal unscathed.
To see more of Kayla Harrison, along with MMA champ Ronda Rousey, in action, purchase a copy of Winning on the Ground: Training and Techniques for Judo and MMA Fighters. The book is written by Dr. AnnMaria De Mars (1984 world judo champion) and James Pedro Sr. (coach of international judo medalists). Winning on the Ground includes detailed discussion of the following:
six secrets to better mat work (and mistakes to avoid)
coaching tips from the authors
a dozen quick ways to gain an opponent's submission
how to do the perfect armbar and half nelson
smarter training: drills for connecting techniques for the win
Kayla Harrison Shows You... Oct 2011
more here: Kayla Harrison Shows You Judo's Ouchi Gari (Major Inner Reaping … ← Full Contact Kickboxing Satin Trousers – Black With Stars |
BRUNO VADIGNO Oct 2011
O OUCHI GARI. Judo at it's finest simplicity. Excellent at the tournament seting if you are aware of the ambient situation. 1903. President THEODORE ROOSEVELT '' had a passion for physical and mental exercise.'' The East Room of the White House became the first Judo Gym in the United States. B.J.
STEPHEN HAMILTON Jun 2013
Beautiful technique, and I have a new respect to an art that was considered not "Combat Realistic" unless you were an almost expert before those movements can be used in real life fights. Thank You for an eye opening demo. Sincerly, Steve Hamilton, Fellow Martial artist

redpajama_set_name: RedPajamaCommonCrawl


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The Voice: Delta Goodrem breaks down in tears over agonising decision
Delta Goodrem had to leave the set and other coaches were in tears after an emotional performance on The Voice.
Andrew Bucklow@ACBucklow
Coaches break down in tears on The Voice (The Voice Australia)
A performance dedicated to the victims of the Christchurch shooting left three coaches in tears.
Emotions ran high on The Voice with several coaches and contestants in tears.Source:Channel 9
Three of the coaches were in tears and Delta Goodrem had to leave the set after an emotional performance on The Voice.
Kiwi trio The Koi Boys took on Daniel Shaw in "The Battles" round with a joint performance of The Beatles song, Let It Be.
The Koi Boys dedicated the song to the families and communities affected by the Christchurch shooting in March when a lone gunman killed 51 people and injured a further 49.
Daniel Shaw battled it out against The Koi Boys on The Voice.Source:Channel 9
Tears streamed down Goodrem's face as she watched the contestants battle it out to remain on "Team Delta" and advance to the next round of the competition.
After the moving performance, Kelly Rowland, who was also in tears, was unable to talk, and Guy Sebastian choked back tears as he praised the singers.
"As you were about to sing I could hear you guys sobbing, and then you've got to try and sing a song which is why … it's why we sing, because it's still a wonderful world," Sebastian said.
Kelly was crying.Source:Channel 9
Guy was welling up.Source:Channel 9
It was then up to Goodrem to decide who would be going through to the next round, but the Born to Try singer was visibly struggling.
"I just feel very strange about this moment because it's very beyond a competition," she said to the contestants. "Sorry, I'm just trying to find my words."
Goodrem then walked off stage and was shown in tears backstage where she told producers, "I just feel really unwell … I just don't want to upset any of them."
The decision proved to be a tough one for Delta.Source:Channel 9
Goodrem returned to the set after regaining her composure and announced, "I'm gonna go with the person I believe needs me more right now, I'm gonna go with Daniel."
But she didn't send The Koi Boys back to New Zealand empty-handed, with the Aussie singer inviting them to be her support act for a yet-to-be announced tour later in the year.
Daniel Shaw is through to the next round of The Voice.Source:Channel 9
The Voice returns next Monday night at 7.30pm on Channel 9
Anh Do brought to tears by Kylie Kwong's baby loss


The active regions across the suns surface contain material heated to several millions of degrees. The blue-white areas showing the NuSTAR data pinpoint the most energetic spots. During the observations, microflares went off, which are smaller versions of the larger flares that also erupt from the sun's surface. The microflares rapidly release energy and heat the material in the active regions.


Badgalriri made her Paris Fashion Week debut with her second Fenty Puma show at the Hotel Salomon de Rothschild yesterday. The singer and creative director of Puma was inspired by "the thought of Marie Antoinette at the gym". The Dior ambassador took elements from the 18th Century and turned them into modern day athleisure wear.
From satin corsets, lace fans, sweatshirts, pearl embellishments and brocade print on outerwear or just about everything else you could imagine the French Queen wearing on a treadmill.


Elevate your Kauai escape with this gorgeous 2-bedroom 2-bath vacation rental condo, which sleeps 8! With a bonus sleeping loft and sweeping views all the way up the Poipu coastline to Spouting Horn, you'll have the perfect island home-away-from-home. Watch dolphins dance and sea turtles snooze from the private oceanfront lanai, or relax by the community plunge pool while BBQing for the group. You'll have all the best Poipu attractions at your fingertips during your stay at this lovely condo.
This top-level Poipu Makai condo offers stunning coastline views that you won't find elsewhere! Wake early to enjoy your morning coffee on one of the private lanais, admiring the breathtaking ocean views that stretch all the way up to Spouting Horn.
On clear days, look beyond the crashing waves and you can see the Forbidden Island of Nihihau. Sea turtles and dolphins put on a show for you, and in season, the humpback whales are thrilling to watch from your lanais off the living room and one of the bedrooms.
This sleek condo is beautifully furnished and upgraded with modern touches, sure to add comfort to your stay. Prepare a tasty beach picnic in the fully equipped kitchen, which has a 4-person breakfast bar.
Embark on beach adventures with ease, as this property comes stocked with beach towels, umbrellas, beach chairs and boogie boards for your enjoyment! When you need a break from the saltwater, take the kids to the community plunge pool and fire up the Weber grill for an al fresco feast!
When night falls, both bedrooms host plush queen beds for your comfort. Upstairs, you'll find sleeping for 6 in the open loft with 2 twin-over-full bunk beds. There's even a great sitting area with separate TV and DVD/book library for entertainment!
Close to all the things Poipu is famous for - including top rated beaches, golf courses and great restaurants - this prime condo is the perfect getaway.
Begin your mornings with a tasty acai bowl from Anake's Juice Bar, and enjoy it along the pristine shores of nearby Poipu Beach. The calm waters and soft sands of this beach make it a family-friendly destination, well known for its fantastic snorkeling!
When you need a break from the head, walk just a few steps to Brennecke's Beach Broiler for a refreshing Mai Tai and fresh seafood overlooking the ocean.
Various other restaurants are found nearby, along with fun local shops and art galleries. If you're looking for a thrill, book a 4000-foot zipline flight, superman-style, over the Kipu Ranch - best known as the set for iconic scenes in films such as Jurassic Park and Tropic Thunder. They also offer 4x4 tours and other options to fit your style!
- Unbeatable Poipu location near beaches, restaurants, shopping and more!
Air Conditioned Oceanfront Updated Condo With Large Lanai And Spectacular Views!
Poipu Makai #C3 - OCEAN FRONT views - Close to Poipu Beach!
Kauai Kai: Oceanfront Privacy in Poipu with AC and Private Pool!
Spacious 5 Bedrooms in Poipu Kai for 14!


World Water Day will be celebrated at UBC Okanagan on Thursday, March 19, with a host of campus activities the week of March 17 to 20, to raise sustainability and water conservation awareness. World Water Day is a global event, supported by the United Nations, to raise awareness of the lack of clean drinking water around the world.
A panel discussion and film showing of The Paddler is on Tuesday, March 17, 5 p.m. at the University Centre Theatre UNC 106. Panel members include Clayton McCann, Curtis Volkle, Bob Purdy, Carey Missler, and Eileen Delehanty Pearkes.
Hundreds of political ecology posters by Assoc. Prof. John Wagner's students will be on display in the Arts building foyer on Wednesday, March 18.
On Thursday, March 19, day-long events kick off at noon, with ceremonies in the UBC central courtyard with live music, a talk by Union of British Columbia Indian Chiefs Grand Chief Stewart Phillip, remarks by UBC Okanagan Deputy Vice-Chancellor and Principal Deborah Buszard, performance by Okanagan Nation drummers and singer, and student presentations.
At 2 p.m. Thursday in the University Centre ballroom UNC200, a keynote address will be given by renowned novelist and natural history advocate Eileen Delehanty Pearkes, who recently completed a history of the upper Columbia River's natural systems and water policy.
The Splashdown Research Event in the University Centre theatre UNC 106 starts at 4 p.m. Thursday, with students making three-minute research presentations.
The Bluedrinks social event at the Well student pub starts at 5:30 p.m. Thursday.
On Friday, March 20, the Water Wars Student Games will be held in the central courtyard at 1 p.m.


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Spouse beneficiary meaning Form 2021-2023
Spouse beneficiary meaning Form
Spouse beneficiary meaning Form: What You Should Know
The first set of IRS Form 1099-MISC instructions were released to the public on April 24, 1990, by then Acting Commissioner of the IRS Richard Serena. The most common reasons why you would receive a Form 1099-MISC during tax year 2023 are to pay for: Income from investments. Income paid from sources other than a brokerage or a profit sharing plan. Interest on investment accounts. For other reasons, it is also possible for an employer to give you a Form 1099-MISC as a contribution for employee payroll deductions and some other deductions.
online solutions help you to manage your record administration along with raise the efficiency of the workflows. Stick to the fast guide to do Form I-130, steer clear of blunders along with furnish it in a timely manner:
How to complete any Form I-130 online:
Place an electronic digital unique in your Form I-130 by using Sign Device.
PDF editor permits you to help make changes to your Form I-130 from the internet connected gadget, personalize it based on your requirements, indicator this in electronic format and also disperse differently.
FAQ - Spouse beneficiary meaning
How can I find out if my husband has actually filed for a divorce?
Court will send summons to your address through their office server.Court will attempt few times to your address. If you are residing at that address and rejected it, the same will be noted and the Divorce case will proceed without your presence (ex-parte). If you donu2019t live at that address, Court will ask the Husband to advertise in major publications attempting to reach you. If you are not still reachable, Case will proceed ex-parte.One way to find out if your husband has filed for Divorce is to search in Home - eCourt India Services website. Browse to your district and select u201cCase Statusu201d. You can search based on your Husband name. Now, you have to make some intelligent guess about which court complex he would be filing in. Normally, that depends on where he resides. So, do some trial and error searches in this page. If he has indeed files for divorce, it will show up here.
How can I fill out a W-4 and taxes if my spouse has an expired visa (undocumented)?
First of all, someoneu2019s visa being expired does not mean they are undocumented. US visas are solely for entry, the visau2019s expiration date is the last day one can use that visa to enter the US, it has nothing to do with how long one can stay in the US. One can enter the US on the day the visa expires, and be admitted for months or years afterwards.Second, you fill out your W-4 withholding form based on your marital status and tax situation. Your spouseu2019s immigration status is not relevant to any part of the W-4 form. And generally, the tax laws apply the same way to people residing in the US, regardless of whether they are documented or undocumented. You and your spouseu2019s tax returns would be computed and filed the same way regardless of whether you and your spouse are documented or undocumented.
If my parents take out a life insurance policy on me in which they are the beneficiaries, does it affect my ability to take out a second policy where my spouse is the beneficiary?
Usually, when a parent takes out life insurance on their child, the amount of coverage is nominal - to cover final expenses - around $25,000 or less.However, you may purchase your own life insurance policy and get as much coverage as the insurance company agrees to offer you based on your own personal financial situation - income / financial responsibilities, etc.Many life insurers may allow you to be insured for up to 10 to 20 times your annual income (this is a total of all life insurance policies insuring your life).This amount may be higher based on your personal finances.
What are the documents my USA wife needs to fill out for a spouse visa for me outside of the US?
U.S. Citizen Petition for a SpouseEverything as mentioned in the link
How can I know whether my spouse, or intimate partner, is cheating?
So it all started back in the summer of 2023 my wife was just a few months into her new job. She had left the ems field for a 8 to 5 job. To be home with are two boys more. A little about us we meet in 2023 got married in 2023 had two bright and handsome boys. Back to what happened not to long after she started her new job about 5 month in her job site lost a co worker to a car accident. She she took it hard because she had talk to the co-worker that morning right before the accident. She called me at work and told me. Just like any lovey husband would do i told he ill be heading to pick her up. But she just told me no she going to stay at work. And come home. That night she did not come home tell 12am which was out of the ordinary for her. I asked her why she was late home. She told me she and her other coworkers had to help cook for the person family Gathering. So a couple of days past the same routine leave early come back late. Every day i try to ask if she was ok i even told her im there to comforter her and that she has someone there for her to cry on. But every day it was the same same answer she was ok. On the day of the the funeral she ran into one of her other coworkers and finally introduce me to him saying that this was her chump. The guy was as old as us he at first did not want to shake my hand or look me it the eyes. Then when she and him hug she started to cry on his shoulders. I found that very strange and that she never mentioned that she was going to all the Gatherings with him tell he had mentioned it when we first seen him and he finally shook my hand. Later that day i ask her what was her relationship what the guy I just met she said he was just a friend who took it hard and she needed to cheer him up along with her other coworkers. After that I started noticing she was texting him and calling him more often at work and after work she would say it was just work-related when I asked and I kept on asking if there was something going on between them. Fast forward two Christmas of 2023 I was told by my wife that she had to work late and that everyone had to work late too so I didn't question it. She didn't get home until 3 a.m. that morning. She slept till 11 a.m. by then I was up had breakfast cooked and the boys were fed she said it was it was very tough at work last night they had to do a lot of catching up. Life one on then till New Year's Eve she said she had to work late again and that she would be home late two. As usual I stayed home and watch the kids watch TV and cartoons with them the next day she was not home and she didn't get home until the afternoon she said she had to take a shower because she felt dirty from all the sweating and that the AC had stopped working in their building so as usual I didn't say nothing and let it be but deep down I knew she was with her male co-worker that had met only one. After that she changed are sex life died out completely. She would say she tired at night. She started to where g-strings that I had never seen to work. And then the end of January she called one night saying she had to work late the kids were at their grandparents house so the Curiosity got the best of me I went to her work to see if she wanted some dinner no one was there all the doors are locked even her car was not there. I called her cell it just went to voicemail every time i called it. So i gave up and went home then one day a couple of months later a good friend of my stop by to see me and ask how i was doing. He told me he had heard my wife was sleeping with a coworker from her job. And the he need to see if it was true. We talked for two hours. He left i was trying to get access to her cellphone accounts but it was inaccessisible to me. I had to contact a link i saw on quora DARK HAT on Strikingly to get the evidence i found out my wife text her coworker every minute everyday and it sexual at times. It got to a point where she even asked if he stop texting her because his wife was home. She even was sending him nude pictures of her to him. Now it was all adding up every time the company had dinner or cookouts he would avoid me every chance he got and she would avoid looking at him or talking to him while I was there. So after two more days of playing like i don't know what she up to. I called her at work and told her i was in her her neck of the woods and that if she would love to have lunch with me. She hesitate out first and said that she was supposed to go have lunch with her friend but eventually said she'll have lunch with me. At lunch we talk about are work. Then i final ask her about cheating with her man from work she kept denying it tell i told her i have seen every thing she text him. And that i was going to the head person to see what he thinks. She finally broke down and told me she was seen him. That it started that night there coworker died. And that it was just supposed to be a one-time thing but it continued and that her working late was just a cover for them. Because he was married too and thay where doing every time thay got. I was so disgusted by what I heard and was so mad and hated her so much that I left and went back home. She tried to call but i never wanted her calls. I just texted her telling her to stay at her mom house from the next couple of weeks. After two and a half months I finally decided that that my kids came first and that they needed their mother back home. I called her and told her that she could come home but under one condition that she could not talk see text any form of communication with her man from work. And she said she will keep her promise could not have anything to do with them.
What is the meaning of this sentence, u201cprinformation about the beneficiary's spouse and childrenu201d?
The statement is likely to be in relation to a Will, where a bequest has been detailed - the deceased person has left a sum of money or goods, or property to a person who becomes u201cthe beneficiaryu201d (because they will benefit). The beneficiaryu2019s wife or husband (spouse) and any children might also have been mentioned in the Will and the lawyer will need their details.


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Share Your Rainbow
Author: Various | Narrator: Catherine Ho
When the storm subsides and the sun comes out, don't forget to look for the rainbow!Inspired by kids across the country displaying rainbows in their windows, acclaimed illustrators come together to imagine everything these rainbows represent: caring ...
ABOUT THIS AUDIOBOOK
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I am Sacagawea
Author: Brad Meltzer and Various | Narrator: Tanis Parenteau
Sacagawea, the only Native American included in Lewis and Clark's historic expedition, joins the inspiring list of heroes whose stories are told in this New York Times Bestselling biography series. Sacagawea was the only girl, and the o...
Soul Looks Back in Wonder
Author: Various | Narrator: Cary Hite and Allyson Johnson
"The selections are uniformly uplifting, with affirming messages about the heritage, strength and dreams of African Americans."—Publishers Weekly In this compelling collection, thirteen major poets rise in response to the dazzling vistas a...
Nationally Recognized Features from Texas Monthly
Author: Various | Narrator: Various
In partnership with Texas Monthly, the following nationally recognized features are now available in a bundle as an audio download: * "Still Life" by Skip Hollandsworth* "Mothers, Sisters, Daughters, Wives" by Mimi Swartz* "The Innocent Man, Part One...
Texas Business and Culture from Texas Monthly
In partnership with Texas Monthly, the following articles highlighting Texas business and culture are now available in a bundle as an audio download: * "The Slow Life and Fast Death of DJ Screw" by Michael Hall, read by Matt Bull* "Dreaming of Her" b...
Social Studies from Texas Monthly
In partnership with Texas Monthly, the following articles focusing on Texas society are now available in a bundle as an audio download: * "Busting Out of Mexico" by Jan Reid, read by Bruce DuBose* "Showdown at Waggoner Ranch" by Gary Cartwright, read...
True Crime from Texas Monthly
In partnership with Texas Monthly, the following articles spotlighting true crime are now available in a bundle as an audio download: * "The Cheerleader Murder Plot" by Mimi Swartz, read by Pam Dougherty * "The Last Ride of Cowboy Bob" by Skip Hollan...
Meet Cute: Some people are destined to meet.
Author: Various, Nicola Yoon, Jennifer L. Armentrout and Sara Shepard | Narrator: Bahni Turpin, Kyla Garcia, Caitlin Davies, Sullivan Jones, Betsy Struxness and Dara Rosenberg
Whether or not you believe in fate, or luck, or love at first sight, every romance has to start somewhere. MEET CUTE is an anthology of original short stories featuring tales of "how they first met" from some of today's most popular YA authors.Listen...
Tortall: A Spy's Guide
Author: Tamora Pierce, Various and Timothy Liebe | Narrator: Marc Cashman and Ann Marie Lee
The must-have guide for any fan of #1 New York Times bestselling fantasy author TAMORA PIERCE! Enter the world of Tortall in this audiobook guide, brought to you by the author who Sarah J. Maas says "shaped [her] not only as a young writer but ...
Eat Pray Love Made Me Do It
Author: Various | Narrator: Cassandra Campbell, Marc Cashman, Mark Deakins, Robbie Daymond, Emily Rankin, Jorjeana Marie and Ariana Delawari
True stories inspired by one of the most iconic, beloved, bestselling books of our time In the ten years since its electrifying debut, Elizabeth Gilbert's Eat Pray Love has become a worldwide phenomenon, empowering millions of readers t...
The Declaration of Independence and the Constitution of the United States
Author: Various | Narrator: Boyd Gaines and Frank Langella
The two most treasured documents of American freedom together on one audiobook.We hold these truths to be self-evident, that all men are created equal…Drafted by Thomas Jefferson in 1776 as an act of rebellion, The Declaration of Independence p...
The Knopf National Poetry Month(TM) Collection
Since 1998, the Alfred A. Knopf Publishing Group has celebrated National Poetry Month™ by emailing a poem-a-day throughout the month of April to over 25,000 devoted poetry fans. In April 2006, Knopf introduced the Poem-a-Day podcast, which fe...


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Q: Handling multiple request in sequence generated by onchange event in javascript onchange event search() function is calling which is sending requests to GoodReadApi

I am new to Javascript and react js and I wanted to execute multiple requests in sequence as they are sending. I have tried my best but can't find best solution to the problem. anyone can tell me the way I can overcome this issue?

A: The best way is probably to have onChange update the field value, and to provide a 'Submit' or 'Search' button that calls the action creator that accesses GoodReads.

A: If I have got your opinion correctly, you need something like:
import React, {Component} from 'react';

class App extends Component {
  constructor(props) {
    super(props);
    this.state = {
      inputText: '',
    };
    this.handleOnChange = this.handleOnChange.bind(this);
  }

  handleOnChange(event) {
    this.search(event.target.value);
    this.setState({inputText: event.target.value});
  }

  search(text) {
    //call API here....
    console.log('call api....', text);
  }

  render() {
    console.log(this.state.inputText);
    return (
      <div>
        <input type="text" onChange={this.handleOnChange}/>
      </div>
    );
  }
}

export default App;

redpajama_set_name: RedPajamaStackExchange


More evidence that Obama and the Dems stole the 2012 elections.
More evidence that the United State of America has devolved into a Third World and third rate country.
That's the call to action from NC officials? Pathetic.
What this calls for is a class-action lawsuit to overturn and nullify the 2012 Election's supposed results — a lawsuit that should be appealed all the way to the Supreme Court.
Meanwhile, only the sound of crickets from the MSM on the massive 2012 voter fraud . . . .
"The dead can vote in NYC," Dec. 31, 2013.
"Obama poll worker and feminist nun charged with 2012 vote fraud in Ohio," Mar. 12, 2013.
"2012 Election was a two-party conspiracy," Nov. 30, 2012.
"Thousands of U.S. military votes uncounted or missing," Nov. 13, 2012.
"99% of Philadelphia wards voted for Obama!," Nov. 10, 2012.
"What You Can Do About Vote Fraud," Nov. 17, 2012.


text:
Card Index of Radar Sets - Weather Radars
as a list
as picture gallery
Print Versions
Propagation of Electromagnetic Waves
Characteristics of Transmission Lines
Radar Sets (Examples given)
Metric Measurement Units
alphabetical list of radars
EWIS II
Description of the radar set, tactical-technical characteristics
Figure 1: EWIS II
frequency: 5.61 GHz
(C-Band)
pulse repetition time (PRT):
pulse repetition frequency (PRF): 250, 900 or 1200 Hz
pulsewidth (τ): 0.58 or 2.0 µs
receive time:
dead time:
peak power: 250 kW
average power:
instrumented range: 480 km
range resolution:
beamwidth: 0.9°
hits per scan:
antenna rotation: 1 … 6 min⁻¹
EWIS Ⅱ is operating in C-Band weather radar.
It uses a magnetron-transmitter and an analog receiver. The polarization is oriented linear horizontal. A reflectivity volume scan contains 12 elevation angles (typical: 0.5°, 0.9°, 1.3°, 2.4°, 3.5°, 4.5°, 6.0°, 8.0°, 10.0°, 15.0°, 25.0° & 40.0°). Doppler scans contain 9 elevation angles (typical: 0.5°, 1.3°, 2.4°, 5.0°, 7.0°, 10.0°, 15.0°, 20.0° & 30.0°). The complete volume scan needs 15 minutes.
The first installations were in the late 1980's as a conventional Non-Doppler Radar. A modernisation changed the classification to Doppler-Radar in 2010 (since then the name EWIS Ⅱ). The radar is operational in Island near the airport Keflavík. It is located about 257 km from the Grímsvötn volcano, and 153 km from the Eyjafjallajökull volcano.
Publisher: Christian Wolff
Text is available under the GNU Free Documentation License, and the
Creative Commons Attribution-Share Alike 3.0 Unported license, additional terms may apply.


Sep. 15, 2017 5:30 p.m.
The inaugural jazz festival is all about getting the sweet sounds of jazz flowing around Nanaimo. Then, in the years to come, organizers will look at turning up the volume.
The Nanaimo International Jazz Festival is now underway in the Harbour City this weekend (Sept. 15-17) and organizers are excited about the weekend. There are nine events planned, five of them free and four of them ticketed.
Andrew Homzy, president of the festival association, said he had a vision for the festival almost from the first day he moved to Nanaimo in 2009, and always believed the downtown was the perfect location.
There's been a jazz community in Nanaimo for a long time, he said, but he didn't think there were enough opportunities for musicians to be heard.
He said organizers and sponsors recognize that starting a festival from scratch might take some time and they're committed to put in their best efforts in 2017 with the anticipation that the event will grow over the years to come.They've planned an inaugural event that they think will start to create that momentum. There are shows at the Port Theatre, dance and jam events at the Best Western Dorchester Hotel, and a parade, mardi gras party and free outdoor shows around the downtown.
For a schedule of events, visit www.nanaimojazzfest.ca.


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