text:
10.4236/ajps.2017.86091
Developing Stable Cultivar through Microspore Mutagenesis in ×Brassicoraphanus koranhort, Inter-Generic Allopolyploid between Brassica rapa and Raphanus sativus
Soo-Seong Lee1*, Byoung Ho Hwang1, Tae Yoon Kim1, Jeongmin Yang2, Na Rae Han2, Jongkee Kim2, Hyun Hee Kim3, Hadassah Roa Belandres3
1BioBreeding Institute, Sillyeong-Ro 153, Daeduk, Ansung, Gyeonggi, Korea
2Dept. of Integrative Plant Science, Chung-Ang University, Ansung, Korea
3Plant Biotechnology Institute, Department of Life Science, Sahmyook University, Seoul, Korea
Received: March 2, 2017; Accepted: May 23, 2017; Published: May 26, 2017
A stable progeny was developed through induced mutation, using microspore culture, of the hybrid (F1F1) produced by crossing a newly synthesized, unstable allopolyploid (F1) and a stable cultivar, BB#1(F1) in xBrassicoraphanus. An F1F1 plant was subjected to the induced mutation system established during production of BB#1. Morphological characteristics of the progeny such as color, and leaf number and length, differed from those of BB#1. The bolting time of the progeny in spring cropping was very late compared to BB#1, allowing it to be grown to an adult plant in spring. Genomic in situ hybridization analysis of pollen mother cells at prophase I identified 19 bivalents, 10 from Brassica rapa and 9 from Raphanus sativus. The glucoraphenin content was almost identical to that of BB#1. Two cultivars are available in the baemoochae crop now. These results indicate that induced mutation using microspore culture is a viable method of stabilizing inter-generic allopolyploids between B. rapa and R. sativus.
´Brassicoraphanus, Intergeneric Hybrid, Baemoochae, Brassica rapa, Raphanus sativus
Following Karpechenko's report in 1927 [1] , numerous studies have focused on the allopolyploid, inter-generic hybrid between Brassica and Raphanus, designated ×Brassicoraphanus [2] [3] . Most ×Brassicoraphanus hybrids were unstable; few have been used as breeding material [4] [5] [6] . Production of a stable line from 10 generations of mass selection of ×Brassicoraphanus as an inter-ge- neric allopolyploid from R. sativus (♀) and B. oleracea var. albogrobula (♂) was reported [7] . However, the line was not palatable and the oil content was low. Lee et al. (2011) [8] reported development of a stable inbred by microspore mutagenesis, using n-nitroso n-methyl urethane (NMU), applied to an unstable line, BB#4 of ×Brassicoraphanus, an allopolyploid from B. rapa (♀) and R. sativus (♂). This new inbred had high nutritional value and sulforaphene content, which has marked anticancer and antibacterial properties [9] . The inbred was very tender and crispy in texture, and sweet and pungent in taste [10] . The inbred was given the crop name "baemoochae" and released with the cultivar name BB#1 after registration for variety protection in 2009.
There has been interest in developing another stable line from different parental germplasm, thereby expanding the industrial use of baemoochae and confirming the effectiveness of microspore mutagenesis for speciation of allopolyploids from the cross-pollination of B. rapa and R. sativus. When a stable accession was recognized, it was named as BB#5 for conveniences in future's affaires, such as the cytological study, the analysis of content of glocosinolate and registration for variety protection.
2.1. Developing Stable Inbred
A unique landrace of kimchi cabbage, jombaechoo (B. rapa), and an accession from collections of the landrace gaetmoo (R. sativus), were used as parents to develop a new, stable baemoochae line. They were supplied by The Center of Germplasm, The Rural Development Administration in Korea with the introduction no. of PI204163 for B. rapa and IT216097 for R. sativus. They were known to be maintained high content of disulfide constituent of sulforaphene in an analysis. Jombaechoo is much similar to radish in the leaf morphology, an enlarged terminal lobe and smaller lateral lobes. Gaetmoo is a typical wild type in the root shape with numerous thin roots from the slender and little bit small root. Jombaechoo and gaetmoo were self-pollinated two and three times, respectively, to produce more uniform lines. Bud pollination was performed on jombaechoo pistils. Anthers from jombaechoo were removed 1 day before being pollinated by gaetmoo pollen [11] . Ten-day-old immature hybrid ovules were cultured in darkness at 25˚C on modified B5 medium (Duchefa Co.) [12] supplemented with 600 mg/L CaCl22H2O, 800 mg/L glutamine, 100 mg/L L-serine, 0.1 mg/L NAA, 0.1 mg/L BAP, 0.1 mg/L 2,4-D, and 30 g/L sucrose. The ovule-cul- ture derived plants were transplanted to soil for acclimatization. A 0.3% colchicine solution was applied to the growing points of established plants for 3 days, using absorbent cotton, to double chromosomes. The size and wave edge of leaves were different to both parents and flowers were white in color, as if the inter-generic hybrid. The F1 plants capable of producing pollen were self-polli- nated and cross-pollinated reciprocally to BB#1. Three to five fresh flowers and approximately 10 buds on the same flower branch were self-pollinated, and non-pollinated buds were removed. This procedure was repeated on several branches to acquire sufficient seed. Cross pollination to BB#1 was managed in the same way as the self pollination with flowers. The plant 07OV-127 was subjected to microspore mutagenesis. Liquid NLN 13 medium [13] , supplemented with 0.1 mg/L BA, was added to the microspore culture. A stock solution of n-nitroso n-methyl urethane (NMU) was added to the culture solution containing a microspore density of 104 per mL to a final concentration of 0.01 μM. Embryos derived from microspores were transplanted to MS2 medium supplemented with 2% sucrose to MS medium for plant regeneration [14] . Normal plants with well-developed shoots and roots were transplanted to pots for acclimatization. Abnormal plants were cut into pieces of about 5.0 ´ 5.0 mm and sub-cultured in MSk medium (1.0 mg/L BA, 0.2 mg/L NAA and 2% sucrose added to basic MS medium) to induce normal plants [14] . Acclimated plants were vernalized in a low-temperature greenhouse, in which the temperature was about 2˚C to 7˚C in the night time and 10˚C to 12˚C in the day time for 25 days, without colchicine treatment for chromosome doubling, followed by exposure to 12˚C - 30˚C for seed production.
Data collected from pollinations in earlier generations were abbreviated for shortening the volume of the manuscript and stated in the text for understand the flow of the story.
2.2. Cytology
Meiotic chromosome behavior of BB#5 from prophase I to anaphase II was observed as described by [15] [16] , with minor modifications. An unstable line, BB#4, and the stable line BB#1 were compared. Chromosome abnormalities were scored and analyzed in 500 pollen mother cells (PMCs) using a fluorescence microscope (Olympus BX53, Japan) equipped with a CCD camera (Leica DFC365 FS, Germany).
Genomic in situ hybridization (GISH) analysis, using blocking DNA of R. sativus and probe DNA of B. rapa, was applied to BB#5 to determine its meiotic chromosomal constitution [17] . The CTAB-extraction method was used to extract and purify total genomic DNA from young leaves of B. rapa and R. sativus [18] . Genomic DNA from B. rapa was labeled with dig-11-dUTP (Roche, Germany) by nick translation and used as a probe, while the unlabeled DNA of R. sativus was fragmented at 100˚C for 7 min and used as a block. During GISH analysis, the slides were pretreated with RNase A buffer (100 µg・ml −1 RNase A in 2´ SSC) at 37˚C for 1 h and then treated with 100 µg/ml pepsin (Sigma, USA) in 0.01 M HCl at 37˚C for 10 min. After washing for 5 min with 2´ SSC, the slides were post-fixed with 4% paraformaldehyde in 2´ SSC, washed with 2´ SSC, and dehydrated in an ethanol series (70, 90, and 100%) for 2 min each. A hybridization mixture (50% formamide, 10% dextransulfate, 2´ SSC, 5 ng・µl−1 unlabeled genomic DNA, and 50 ng・µl −1 of labeled genomic DNA as a probe) was prepared and then adjusted to 40 µl per slide with nuclease-free water. The GISH mixture was denatured at 90˚C for 10 min and immediately stored on ice for at least 5 min prior to mounting on slides. Chromosomes were then co-denatured at 80˚C for 3 min on a ThermoBrite (Fisher Scientific, USA), then hybridized overnight at 37˚C. The slides were washed in 2´ SSC for 30 min at RT, 0.1´ SSC for 30 min at room temperature and finally in 2´ SSC for 15 min at 42˚C. A dig-labeled probe was detected using an anti-dig-FITC conjugate (Sigma, USA) at 37˚C for 1 h. The slides were dehydrated in an ethanol series (70, 90, and 100 %) for 3 min each, air-dried, and counterstained with 1 μg/ml DAPI (Roche, Germany) in Vectashield (Vector Lab., Inc., USA). Images were captured with an Olympus BX53 fluorescence microscope equipped with a Leica DFC365 FS CCD camera, and processed using Cytovision version 7.2 (Leica Microsystems, Germany) software. Further image enhancements were performed using Adobe Photoshop CC.
2.3. Glucosinolate Analysis
Seed contents of total glucosinolate and glucoraphenin of BB#5, which is the precursor of sulforaphene, were measured according to the optimized official method, ISO 9167-1(1992) [19] . Two hundred milligrams of dried seeds were mixed with 10-ml boiled 70% methanol and incubated at 70˚C for 20 min to inactivate plant tissue enzymes. Following centrifugation at 3000 g for 15 min at 4˚C, the supernatant was concentrated to 3 ml under N2 gas. Residue was re- suspended with 1 mL of 20 mM sodium acetate buffer and loaded onto a DEAE Sephadex-A25 column. Purified sulfatase (75 µL, 150 U/mL) was also loaded onto a Sephadex-A25 column and stored at 37˚C for 12 h. Desulfo-glucosino- lates were eluted 1.5mL ddH2O (HPLC grade). The total eluted solution was concentrated to 0.5 mL for HPLC analysis. The sample was filtered through a 0.45-µm PVDF syringe filter before injection. The samples were analyzed on Varian LC-900 and separated on C-18 reverse phase column (Zorbax Eclipase XDB-C18, 4.6 ´ 250-mm i.d., 5-µm particle size, Macherey-nagel, Germany) at column temperature of 35˚C. Compounds were detected by UV detector set at 229 nm and separated by following program with a water-acetonitrile gradient at a constant 1 mL・min−1. A gradient of water (A) and acetonitrile (B) was employed to separate the compounds by increasing the proportion of solvent B from 2% to 20% over 25 min and then switched to an additional linear gradient of 20% to 100% of solvent B up to 35 min. The glucosinolate analysis was replicated three times.
Multiple plants were produced from the cultured hybrid ovules (F1) between jombaechoo and gaetmoo. Eight (F1) of 13 plants from 07OV-124 to 07OV-136 were transplanted into soil after acclimation, grown in the greenhouse, and treated with colchicine. Most plants produced large amounts of pollen. One plant, 07OV-125, produced only one seed (F2) from self-pollination and the other seven plants produced no F2 seed. However, 15 hybrid seeds from 1 to 4 of 9 mates designated F1F1 were produced by reciprocally cross-pollinating the plants with the cultivar BB#1. One plant, 07OV-127, subjected to microspore mutagenesis for stabilization, produced a few embryos and two plants (designated F1Mn1) were acclimated.
All seeds harvested from self-pollinating the F1 plants (one seed, F2) and cross- pollinating the F1 plants with BB#1 (15 seeds, F1F1) were planted and grown together with the two microspore-derived plants (F1Mn1). All plants produced large amounts of pollen. However, one F2 plant did not produce any seed from self-pollinations made on more than 100 flowers and cross-pollinations made on 45 flowers distributed over 10 branches over a period of 6 d. It was determined to be female sterile. Two plants (07OV-127Mn-1 and 07OV-127Mn-2) derived from microspore mutagenesis did not produce seed from self-pollinations despite a total of 1,099 pollinations. However, these plants produced some many seeds in reciprocal crosses with several accessions of ´Brassicoraphanus. These results indicated that both 07OV-127Mn-1 and 07OV-127Mn-2 were self sterile, and not stable. Only 5 of 15 F1F1 plants produced self seeds (F1F2). Seed yields were poor, at 1 - 3 seeds per plant, with the exception of (BB#1x07OV-128)-3, which produced 29 seeds. On the contrary, the yield of hybrid seeds (F1F1BC1) of three plants crossed with BB#1 was generally as high as 6 - 17 seeds per combination (Table 1). The reason that fewer seeds were produced from self-pollina- tion than cross-pollination is unknown.
Table 1. The number of seeds and plants obtained from pollination and microspore mutagenesis in 2nd generation and the number of seeds in 3rd generation of hybrid plants (F1) between land races of B. rapa (jombaechoo) and R. sativus (gaetmoo).
Mn: Microspore culture with mutagen NMU of 0.01 μM. Pollination was done to more than 15 flowers for each mate.
An F1F1 plant, (BB#1x07OV-128)-4, produced the greatest amount of seed from self-pollinations and was subjected to microspore mutagenesis. Twenty seven and 38 plants from plots treated with and without mutagen, respectively, were grown until flowering. Only 7 and 8 plants, among the 21 and 28 pollen- producing plants, respectively, produced seeds from self-pollination. However, only one plant derived from mutagen treatment and two plants derived without mutagen treatment in each plot were selected as being highly fertile, as they produced more than 10 seeds per plant and 1 seed per pollination on the highest branch (Table 2).
In the succeeding generation, two of the three high-seed-yielding plants were grown separately in a different net case of 1-m length ´ 1-m width ´ 2-m height and pollinated via bees for 20 d. From this trial, only one line originating from the NMU treatment produced an average of 35g (about 7.000 grains) of seeds per plant (Table 3). This seed yield was similar to that for BB#1. When grown in the field during the fall and spring seasons, the one line was highly uniform and different from BB#1 in morphology. Leaves were dark green and glossy, and longer and fewer in number than BB#1 leaves. Bolting time in spring was also much later than for BB#1 (Figure 1). This line was labeled BB#5 and designated for a cytology study, glucosinolate analysis and variety protection. Plant performance was stable for more than six subsequent generations. Microspore muta-
Table 2. The number of plants pollen-producing, fertile, sterile and high fertile in self pollination among microspore-derived plants with or without treatment of mutagen, NMU, to a hybrid (F1 F1) between 2 ×Brassicoraphanus accessions.
MC: Microspore culture. Mn: Microspore culture with mutagen NMU of 0.01 μM. The line, BB#1 is a stable cultivar and the plant 07OV-128 is a newly synthesized unstable allopolyploid in ×Brassicoraphanus. ※: Stabilized.
Table 3. Fertility of 3 plants selected for high seed yield among microspore-derived plants of an F₁F₁ hybrid in ×Brassicoraphanus.
MC: Microspore culture. Mn: Microspore culture with mutagen, NMU of 0.01 μM. Fertility in next generation: pollinated with bees in the net case of 1 m length ´1 m width ´2 m high for 20 d.
Figure 1. Morphology of BB#5 compared to BB#1. Right is BB#1 and left is BB#5 in each figure. Left: uniformity on the field in fall season. Right Length of flower stalk grown in spring season.
genesis with NMU was confirmed as a practical method of stabilizing allopolyploids between B. rapa and R. sativus. BB#5 was registered after 2 year's examination in 2015.
The unstable line BB#4 and stable cultivar BB#1 were compared cytologically with BB#5. Various irregular divisions in meiosis of chromosomes were observed in all lines. However, the numbers of cells undergoing abnormal division were different between stable and unstable lines. The stable BB#1 and BB#5 lines were 10% and 12%, respectively, in terms of irregularities from prophase to anaphase, whereas the unstable line BB#4 was 57.4% irregular from prophase to anaphase (Table 4). GISH analysis identified 19 chromosome pairs, 10 from the AA genome of B. rapa and 9 from the RR genome of R. sativus (Figure 2). These results indicated that the BB#5 of baemoochae undergoes diploid-like division during meiosis [2] .
The total glucosinolate content of BB# 5 was slightly lower than that of BB#1 (4.64 and 4.94 mg・g−1 dry weight [D.W.], respectively). The content of glucoraphenin, a precursor of sulforaphene, of BB#5 was similar to that of BB#1, at 1.57 and 1.52 mg・g−1 D.W., respectively (Table 5).
Inter-generic allopolyploids have not evolved into stable crops despite numerous attempts [2] . Chen and Wu (2008) [7] reported stable progeny of ×Brassicora- phanus, an allopolyploid between R. sativus and B. oleracea, which were derived from mass selection for 10 generations, although they were not developed into cultivated crops. Lee et al. (2011) [8] developed stable progeny of ×Brassicora- phanus, an inter-generic allopolyploid between B. rapa and R. sativus, through induced mutation using microspore culture. The result, "baemoochae", became a new vegetable crop [12] and was released as cultivar BB#1 [10] . Another stable
Figure 2. Genomic in situ hybridization on the meiotic chromosome pairs of BB#5 line in ×Brassicoraphanus. Meiotic chromosome pairs of Raphanus sativus (RR genome) are indicated with red arrows which show 9 bivalent chromosome pairs as expected to be derived from the RR genome (A) and those of Brassica rapa (AA genome) are shown as green signals indicated with blue arrows which show 10 bivalent chromosome pairs as expected to be derived from the AA genome. Scale bar, 5 μm.
Table 4. The number of irregular division cells out of 500 cells in 3 inbreds of ´Brassico- raphanus at different stage in meiosis.
BB#4 = unstable line. BB#1 and BB#5 = stable lines.
Table 5. Content of total glucosinolate and glucoraphenin in seed of baemoochae BB#5 and BB#1 (mg/g fresh weight).
line, BB#5, was also a commercial crop, confirming the effectiveness of microspore mutagenesis for stabilization of ×Brassicoraphanus. These results indicate that an evolution to speciation in allopolyploids of Brassica and Raphanus has been accomplished and that subsequent development of improved cultivars is feasible.
Diploid-like division in meiosis of allopolyploids was hypothesized to result from chromosomal rearrangement with genomic changes [20] [21] . The exchange of homoeologous chromosomes, and missing and duplicated homoeologous genes, without a differential change in the expression of homoeologous sets of genes, was required for this chromosomal rearrangement [22] [23] [24] . The morphological characteristics of BB#1 and BB#5 were similar to those of their respective wild accessions, making it difficult to distinguish them. It is presumed, therefore, that microspore-holding homoeologous chromosomes were rearranged in the stable accessions through microspore mutagenesis as haploids, followed by chromosome duplication without changes in whole expression of homoeologous sets of genes.
Based on these results, it seems that another ×Brassicoraphanus cultivar could be developed from the combinations R. sativus and B. nigra, one of the 3 basic genomes in Brassica crop, by either induced mutation or mass selection. A cubic triangle, with R. sativus on the apex of the U's plane triangle of Brassica species [25] , would be accomplished with three synthetic ×Brassicoraphanus on the three sides (Figure 3). This accomplishment would attract the attention of scientists aiming to produce improved Brassica cultivars.
Allopolyploids in plants are known to degenerate gradually by irregular division of chromosomes during meiosis [7] [26] [27] . Such unusual division should disturb the diploid-like division, which is a fundamental requirement in stabilizing allopolyploids [2] . The rates of abnormal chromosomes in meiosis were confirmed to be 10% and 12% in the stable lines BB#1 and BB#5, respectively, and 57.5% in the unstable line BB#4. These rates might be criteria for determining the stability of allopolyploids, although the true threshold has not been established. The rate of laggard cells in metaphase was as high as 31.0% (154 of 500 cells) in the unstable line, compared to around 3% (15 cells in BB#1 and 17 cells in BB#5 of a total of 500 cells) in the stable lines. The rate of laggard cells in metaphase may also be used as another standard to determine the stability of an allopolyploid.
Sulforaphene, a hydrolytic product of glucoraphenin, is a major isothiocya-
Figure 3. A cubic triangle with R. sativus on the apex of the U's plane triangle of Brassica species. The crosses between 3 basic genomes of the U's triangle and R. sativus would create 3 different species in ´Brassicoraphanus.
nate in baemoochae seeds [9] . Many studies have reported biological effects of sulforaphene, including antimicrobial, antifungal, antibacterial, nematocidal, and allelopatic properties [28] - [33] . The total glucosinolate content of BB#5 was slightly lower than that of BB#1, while the glucoraphenin content of the two lines was similar. This result indicated that baemoochae cultivars might perhaps be high levels in general of the anticancer and antibacterial substance glucoraphenin.
Two allopolyploid cultivars from the B. rapa and R. sativus cross and a stable allopolyploid line from the R. sativus and B. oleracea cross are currently available for field production and scientific studies. These cultivars differ in terms of their genomes and chromosome numbers. According to Williams' suggestions [34] , which are the capital letter for the cytoplasmic genome and the small letter for the nuclear genome in nomenclature, cultivars from the B. rapa and R. sativus cross have an Aaarr genome and the cultivar from the R. sativus and B. oleracea cross has an Rccrr genome. Their chromosome number are 2n = 38 in the former and 2n = 36 in the latter. Therefore, these cultivars should be allotted distinct scientific names to avoid confusion with ×Brassicoraphanus Sageret [3] . We would like to nominate ×Brassicoraphanus koranhort Sageret and Lee for the nomenclature of the baemoochae crop, an allopolyploid between B. rapa and R. sativus.
This work was supported by The Institute of Planning and Evaluation for Technology, Ministry of Food, Agriculture, Forestry, and Fisheries of Korea (506017-05-SB010) and AgriFood New Material Research Center, Gyonggi Regional Research Center, Chung-Ang University and The Next-Generation BioGreen21 Program, Rural Development Administration, Korea (NO. PJ009093).
Lee, S.-S., Hwang, B.H., Kim, T.Y., Yang, J., Han, N.R., Kim, J., Kim, H.H. and Belandres, H.R. (2017) Developing Stable Cultivar through Microspore Mutagenesis in ×Brassicoraphanus koranhort, Inter-Generic Allopolyploid between Brassica rapa and Raphanus sativus. American Journal of Plant Sciences, 8, 1345-1356. https://doi.org/10.4236/ajps.2017.86091
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29. Das, S., Tyagi, A.K. and Kaur, H. (2000) Cancer Modulation by Glucosinolates: A Review. Current Science, 79, 1665-1671.
30. Donaldson, M.S. (2004) Nutrition and Cancer: A review of the Evidence for an Anti-Cancer diet. Nutrition Journal, 3, 19.
31. Barillari, J., Cervellati, R., Costa, S., Guerra, M.C., Speroni, E., Utan, A. and Iori, R. (2006) Antioxidant and Choleretic Properties of Raphanus Sativus L. Sprout (Kaiware Daikon) Extract. Journal of Agricultural Food Chemistry, 54, 9773-9778.
https://doi.org/10.1021/jf061838u
32. Barillari, J., Iori, R., Broccoli, M., Pozzetti, L., Canistro, D., Sapone, A., Bonamassa, B., Biagi, G.L. and Paolini, M. (2007) Glucoraphasatin and Glucoraphenin, a Redox Pair of Glucosinolates of Brassicaceae, Differently Affect Metabolizing Enzymes in Rats. Journal of Agricultural Food Chemistry, 55, 5505-5511.
33. Pocasap, P., Weerapreeyakul, N. and Barusrux, S. (2013) Cancer Preventive Effect of Thai Rat-Tailed Radish (Raphanus Sativus L. Var. Caudatus Alef). Journal of Functional Foods, 5, 1372-1381.
https://doi.org/10.1016/j.jff.2013.05.005
34. Williams, P.H. and Heyn, F.W. (1981) The Origins and Development of Cytoplasmic Male Sterile Chinese Cabbage. Proceedings of the First International Symposium, Tainan, 23-27 March 1981, 293-300.


text:
KARL LUNTTA - LYDIA
Karl Luntta is the author of the novel "Know it By Heart" (Northwestern University Press/Curbstone) and short story collection "Swimming" (SUNY Press), and has published fiction in journals including International Quarterly, North Atlantic Review, Talking River Review, Baltimore Review, Scarlet Leaf Review, Northeast Corridor, and Toronto Review.
Lydia
He watched his wife shut the front door and walk to a waiting Uber, which took her to JFK, then to a flight with a stop in Chicago and on to San Francisco to the house of a man whose name he didn't know.
The day she left him trembling at the doorway, he resisted his more maudlin impulses, and he knew he had them -- the shuffling, the questioning. He thrust his hands deeply into his pockets, so deep his clenched fists nearly reached his knees, and he had this thought: I am blind, I'm an idiot, I am wrong in all ways.
Joe thought this over and over, standing still behind the door, staring at it as if it were alive and breathing, a flat, white animal. He stood there in the apartment's silence until he felt dizzy, and leaned into the door for support. The dog snuffed around his sneakers for a clue about what had just happened.
For a time after she left, he thought of himself as handicapped, somehow physically hurt. An image from his childhood surfaced, the one where he imagined what his soul looked like, its color and shape. Most often it was a matte silver, like mercury without the shine, a thick, viscous fluid that held no shape but permeated him internally, just below the surface of his skin, molding to his organs and bones. He'd imagined this soul's ability to instantly shrink to the size of a microscopic cell when his body was invaded by pain or fear, and he thought now his soul's hiding act was going to be permanent. It had secreted itself somewhere deep inside, possibly to never resurface.
He went from pain to bitter to numb in a familiar, slow-motion transition: first disheveled and confused, then forced-happy and mostly drunk at numerous Manhattan bars, first with friends and going-nowhere dates, and, inevitably, alone.
Two years and three days after she left him fist-clenched and dizzy at his breathing front door, he was introduced to the woman who might have been the last person on Earth to look at him had it not been for Jasper acting like the dog he was.
She was the new-agey pop psychologist and sometimes poet Lydia Johns, signing copies of her latest book at a Third Avenue bookstore, one which Joe frequented and liked when there were no crowds. He hadn't expected to see an author there and had to think for a moment before identifying her. He was helped by a poster on an easel by her book-laden table.
"Women With, Within, and Without" was her latest. The cover featured a photo of a young woman languishing under the low bough of a tree, facing a sunset, a pen and notebook in hand. A portrait of the artist as a young woman. He hadn't read anything Johns had written, but was aware of her work. The subtitle of the book said "Poetry by America's Leading Psychologist."
Psychology combined with poetry was a concept he hadn't thought much about. At all. Freudian Free Verse? Jungian Haiku? Why not. Overwrought poetry could not be any worse than overwrought self-help books.
He knew Johns was thought of as brilliant and accomplished, and she had a large following online, as well as for her podcasts and television appearances. Her books sold in the millions.
Her skin was alabaster, her wrists and hands frail and delicate. She wore a coal-grey suit and a medium-brimmed, black … was that a gaucho hat? Yes, yes it was. Her eyes were dancing, wet and bright and playing her crowd.
Jasper bolted for the table and the leash popped out of Joe's hand. The cocker spaniel mix immediately buried his snout under Johns's skirt and, from beneath, his tail wagged.
The writer's reaction was instantaneous and efficient. She said excuse me to the woman holding a book to be signed and grabbed Jasper's stump of a tail and pulled him from beneath the skirt, then turned and, almost imperceptibly, kicked his tail end. Jasper yelped and Joe was already on his way as she let the dog go and turned, smiling, back to her fans. Joe grabbed the leash and stood to the side of the line, wondering if he needed to say anything.
"So that's how you meet women?" she said, not looking at him.
"Well, I never wag my tail. I hope," he said. Heat rushed to his face.
"Good use of a wingman, though. Or pawman," Johns said, still not looking. "I'll give you that."
"I don't use him to meet women," Joe said. "I don't think he could take the punishment, frankly. But, I'm sorry."
"Oh, I'm not offended," she said. "I mean, he's germy and all, but at least he's attentive."
"He's got some good points," Joe said.
"Funny," she said. "I've always hated dogs in this city. The small ones are rats and the large ones drool rivers."
Joe tried to gauge her anger and felt none. "Jasper's a medium mutt," he said. "We saved him from elimination at the hands of murderous New York City canine death squads. He's a rescue. He's been grateful ever since."
"We?"
Right, and where had that come from? "He's just mine now, for the last couple of years."
She squinted. "Well, I'm sorry, he might not be feeling too well right now."
"He'll get over it."
"They always do, don't they. Men, I mean." She looked at Joe but held her hand out for the next book. "So, how do you, then?"
"How do I what?" Joe said.
"Meet women."
The air seemed to thicken and Joe looked at the line of people. "Good question. I don't really know."
"You don't know how to meet women or how you meet women?"
"You're going to make me say it out loud, aren't you. Okay, I don't really meet women."
She leaned back and smiled at the woman holding a book. Johns asked her name and signed it into the book.
"You don't meet women and why is that?" she said, not looking again. "To borrow your line, your dog is yours alone now, yes?"
"Yes, he is. We're both free men."
"Well, then, you've just met one," she said. "A woman, I mean." The crowd shifted and looked at Joe, heads turning at once like the audience at a tennis match.
Joe coughed and tightened his hold on the leash, conscious of his mouth working quicker than his thoughts. For some reason he wasn't completely sure of, he had to ask.
"Are you--?"
"A woman? Ask your dog the gynecologist." She smiled and drummed her pen.
"Got it," he said. "Alone, I meant."
"Am I married? Oh God, no," she said, then looked up as a middle-aged woman held out a book to sign. "No offense," she said, and the woman nodded, smiling.
As she signed the book, she said, "This is where you ask me what I'm doing for lunch." She looked up at the woman and winked, "Him, not you."
For a short second Joe actually thought about what he was doing for lunch, staggered by the sudden brilliance of the light around him. "I'll bite," he said, "what are you doing for lunch?" Jasper yelped at a squirrel out on the sidewalk, searching for Central Park, no doubt.
"Why, oddly, I'm free," she said.
"I'll be back at one," Joe said, and walked out to the sidewalk, pulling the dog behind. He realized he'd forgotten what he'd come there for. From behind came the faint sound of applause.
He returned to the bookstore at one, no more confident or less so than when he'd left. In fact, he felt a bit lost leaving Jasper at home. Where was the support animal when you needed him?
They took a cab downtown for lunch. He was surprised by what seemed like an undercurrent of nervousness from her. They were both that way, more or less, alone, with no crowd to cushion their banter. They had a giddy, expensive and drunken lunch, and Joe caught himself more than once in a babble, almost out of breath talking about growing up, living with a single mother, reading both Batman and Hemingway with equal vigor. "They're the same," she said, "if you think about it."
They avoided talking about relationships, and their own relationships. Lydia's eyes were half closed when she suggested they go back to his place to wash the dog. Joe said, "Is this a euphemism? First time I've heard that one."
She said, "I only use euphemisms when I talk to priests, or my mother."
They picked up another bottle of wine and wove their way through the city, laughing and walking, stepping over rubbish, kicking paper into the streets.
"My, my," she said when they got to his place on Second and Eighty-Third. "What do you do for a living?"
"I walk dogs," he said as they got into the elevator.
Jasper greeted them at the door, yapping and jumping at their legs.
He said, "He needs a treat."
"I need a drink," Lydia said.
"Wine?"
"Only when I don't get my way. Right now, I just need a drink."
"Let me get a corkscrew."
But she reached out and grabbed the arm of his coat. "Let me smell you," she said. The room moved slightly, shifting under him, as he stood with the bottle of wine in his hand. She lifted his coat and buried her head in his chest. She moved to his arm, his neck, his chin. Her eyes were closed and she leaned back, her nose inches from his lips.
"We need to let the wine breath for awhile, right?" she said.
"It's not uncorked," he said.
"But I am," she said.
They dropped to the couch.
Later, Lydia called the bookstore and apologized, saying she'd be out for the rest of the day but would be happy to come back tomorrow to make up for it. They fixed dinner together, a frozen pizza and ice cream, and watched, he couldn't believe it, Netflix. Then back to bed.
"Can I say something?" Lydia said. It was morning.
"Sure."
"You are a lovely man."
"Well, you are, too. Lovely, I mean, not a man."
"I get it. I understand words," she said. She exhaled, heavily it seemed. "So, you were married."
Joe stopped short. "I was. How did you know?"
"Because you can make love even when you're drunk, and you leave the toilet seat down. That's a married man."
"Or a considerate man? Maybe?"
She leaned over to find her phone on the nightstand, and Joe leaned back on the bed. Time? Yes, had to be. He took a breath.
"Look," he said, "I haven't done this in a long time, maybe forever. But I'm overtly up front about these things. I'm not sure I can be in, you know --."
She quickly turned over and said, a little too loudly, "A relationship? Love? Who said anything about that? I hardly know you, except for the better parts, so don't go sentimental on me. It's unseemly."
"Sorry," he said.
"Besides, I don't even know your name. For the record, I can't not love someone whose name I don't know."
"Joe," he said.
"I would have thought Joseph," she said. "Like Alan, not Al, or William, not Bill."
"I'm a nickname kind of guy, I guess," he said.
"Well, Mr. Joe I-already-don't-love-you," she said, "you've still been evasive about what it is you do to be able to take a day off to go get drunk with a famous writer and yet afford this rather fabulous two-bedroom flat in Yorktown."
"I'm afraid to say it," Joe said, and he was, because, really, right now?
"Try me, I'm tough. You sell sexual favors, am I right?"
"Nope, I'm a cheap date. I guess I want to write, like you. I mean, like you're a writer, not like a writer like you."
"It's clear. I understand words," she said.
"I'm just starting, got some ideas, that sort of thing."
Lydia leaned back on the bed and smells from beneath the sheets drifted out past their faces. She sighed and closed her eyes. "So you're a writer. That's swell."
"Like I said, I'm just starting."
"And is that why you said you can't love me, because I'm a writer?"
"I don't know why I said that. It was knee-jerk, like sneezing. I'm sorry."
"So what do you not yet write?" she said. "Not romance, I hope."
"I don't know. I've written a few tech articles over the years. Right now, it's short fictions. I've got half a novel down. I don't know if I should talk about it. Old fashioned, I don't want to jinx anything."
"Well stay away from romance," she said. "It's the highest and lowest form of literature, and no one does it well." She turned her head and closed her eyes, and they dropped it.
The stayed together for the rest of the day. They made food, coffee, and banter. They walked the dog.
"So what do you do if you haven't sold any of your writing?" she said. "Really."
"I have a company, I'm afraid to say," he said. He plunged forward, to get it out of the way. "I had luck with developing some apps, specifically one of the early apps about dogs. You know, tracking your pup's shots, weight, age, eating habits, lineage. Sharing photos and videos. Chats with vets. Anything to do with your dog. Jasper, we called it, after, you know. Google eventually licensed it, we all did really well, and here I am, a failed writer."
"I've heard of it. I have to ask, though, because not only am I nosy, I'm a bitch, did the app happen before or after your wife left?"
"Mostly after. I'd been developing it. That's half the reason she left me. We were going nowhere with it, long hours, no end in sight, no money."
"Half the reason?"
"The other half was the guy. Whatshisface. A dentist, apparently."
"So she left you, a soon-to-be-wealthy man with a writer's imagination and superior bathroom habits, for a dentist?"
"Her canines are in good hands all around," he said. "Look, can I admit something? You're famous, I've never read anything you've written."
"Neither have I," she said. "Anyway, don't bother, it's cliched and boring, like me. But you're not the academic psychology, self-improvement type, are you? And you don't strike me as a poet."
"Not often," he said. "Not gonna lie."
They walked out, and eventually made their way back to the bookstore. Joe was about to drop her off when he said, impulsively, "Can I see you again?"
Because he wanted to. That was clear.
She leaned back slightly, squinted, and said, "That's a twist. Sure, I'll swing by later today, after the signing. Say, about eight?"
"Today?" he said. He wondered if it was a test of some sort.
"We have unfinished business."
"I guess we do."
At eight, he pushed himself away from his laptop and checked on Jasper, who was dozing by the TV, where Happy Days was playing on an oldies channel. "Time for a walk, buddy. It's been an hour since you last peed on the carpet. In front of the door. Sorry about that. I was into something."
A buzz, the doorman announcing Miss Lydia Johns was here.
"I hate New York," she said at his doorway. "Your whole life here is about third-party intervention. First it's Jasper. Now your doorman."
She stepped into the foyer. Jasper looked up, and, recognizing something, stood up, stubby tail wagging, and started to snuffle at her shoes. "Look, Joseph. Joe. I want the dog."
"What?" he said.
"Jasper. I want Jasper." She smiled, tilted her head. Joe looked down at the little cocker spaniel, his tail moving like the prop of a toy airplane.
"Jasper?" he said. "My dog?"
"I've visited this apartment as recently as this morning," she said. "And unless there are a whole bunch of other Jaspers in this place, then, yes, my pretty, your little dog, too. Jasper." Still smiling.
"I don't get it," he said.
"I don't either," she said. "This is pure impulse, call it, say, knee-jerk. But let me explain the entirety of my feelings up to this very moment. This morning you told me you couldn't fall in love. You put up a border wall of emotive control."
"It's not what I meant," Joe said. "I meant--"
"Let me finish," she said. "I have finally met a man who puts down the toilet seat and loves his little dog. And, this is hard for me to say, so close your eyes and pretend it's not me saying it."
Joe blinked.
"I mean it, close your eyes."
He closed his eyes.
"I like you," she said. "A great deal. Most girls, when their boyfriend boots them out, will steal something, a t-shirt or a pair of cufflinks, little fetishes that remind her of him. Not me, I'm not even an official girlfriend. So I want a living thing, I want a heartbeat. I want Jasper. Now you can open your eyes."
He did. "But, Jasper's my … I love Jasper. I mean, he's my company."
"I will love him, too. I do already. He's the best good boy. And he can still be the face of your company."
As if by recognition, Jasper sat on his haunches and yapped once at Lydia.
"There, it's settled," Lydia said. "You'll have full visitation rights of course. You can walk him any day, even take him for weekends. And of course when I go out on a book tour you'll have to take him back for a while. Done. Does that work for you?"
Joe stood and turned over in his mind how this had occurred. He'd missed something, some word, some clue.
"How did we get to this?" he said.
"No matter, it's a done deal. Life comes at you fast, big boy. Now, I've got an Uber on the way, and you've got some food and a leash and his toys to bag up, so let's get to that part, stat."
He turned and looked at the scattered toys and the blankie, and Jasper, and knew it. This was the way it would be. Had to be. Inside, he sensed the mercury poke out to have a look around. "The squeaky bone is his favorite."
"I could say almost anything here that would be completely inappropriate," she said, "So I'll say this instead. What's your last name?"
"Whitman," he said. "Joe Whitman. I know."
"Oh, please," she said. "You cannot be serious. Can you?"
"I can."
"Then take that as a sign for your writing voice. Irony. It's you."
They packed up the rest in an old backpack with hardly a word, Jasper bouncing about between them as if he knew life was one big adventure and he was going to the mountaintop.
The intercom buzzed and the doorman let them know the Uber was here.
Lydia put her hand on Joe's elbow and smiled, but her eyes had misted slightly. "I am a highly trained, professional psychologist," she said. "I know what I'm doing. Exactly what I am doing. And I suspect you do, too. You have the other half of a book to write, so get to it. Jasper is in good hands."
"I understand," he said. And he did.
"I left a business card on the bedstand," she said.
"Saw it."
"Call me absolutely only during daylight or nighttime hours."
She held up Jasper and kissed the dog's face, while Joe leaned in and kissed the other side. Jasper licked his nose.
And they walked out as he shut the door, gently.
A. E. WILLIAMS
ALAN BERGER
ALEX WOOLF
ALFREDO SALVATORE ARCILESI
ALINA CVETKOVA
AMANDA BRADLEY
AMBER BRANDAU
AMBERLYNN BENNETT
ANITA G. GORMAN
ANITA HAAS
ANNA LINDWASSER
ARYTON WISE
AYAN DAS
BARRY VITCOV
BEK-ATA DANIYAL
BELINYA BANZE
BEN GILBERT
BIJIT SINHA
BILL CARR
BILL MESCE
BILL WILKINSON
BRAD SHURMANTINE
BRIAN YEAPLE
CAITLIN KILLION
CAITLIN MOORE
CAROLINE TAYLOR
CASSANDRA HOERRNER
C. C. KIMMEL
CHARLES CONLEY
CHERYL PENA
CHITRA GOPALAKRISHNAN
CHRISTINA REISS
CHRISTOPHER COSMOS
CINNAMON WARING
CRYSTAL "CRYS" LOPEZ-RODRIGUEZ
DAMIAN MAXIMUS
DAMION HAMILTON
DAVID LIGHTFOOT
DAVID POLSHAW
DAVID ROGERS
DESTANY TOLBERT
DOUG HAWLEY
DR. RICHARD AULT
ELLIE ROSE MCKEE
ENDA BOYLE
ERIC BURBRIDGE
ERNESTO I. GOMEZ BELLOSO
FRANCES KOZIAR
GABRIELLE SILVESTRE
GARRETT PRANGE
GARY P. PAVAO
GEOFFREY HEPTONSTALL
GEORGE LUBITZ
GLYNN GERMANY
HALEY GILMORE
HANNAH DURHAM
HARMAN BURGESS
HAYDEN MOORE
JAC0B AUSTIN
JACOB FROMMER
JACOB VINCENT
JAMES WRIGHT
JILL OLSON
JING "MICHELLE" DONG
JL WILLING
J. N. LANG
JOANNA ACEVEDO
JOHN F ZURN
JONATHAN FERRINI
JOSELYN JIMENEZ
JOSEPH R. DEMARE
JOSEPH SHARP
JULIA BENALLY
KARL LUNTTA
KATALINA BRYANT
KATE TOUGH
KEITH BURKHOLDER
KEMAL ONOR
KIARA MUNIZ
KIERAN J. THORNTON
LAYTON KELLY
LEISA JENNINGS
LOUISE WORTHINGTON
LUIS CASIANO
LYNDA SIMMONS
MATTHEW BARNINGER
MATTHEW MCAYEAL
MAXWELL STANNARD
MICKEY J. CORRIGAN
MICK MCCGRATH
NATALIE HAMPTON
NDABA SIBANDA
PATRICK NEENAN
PATRICK ROSCOE
PHYLISS MERION SHANKEN
PUSHPANJANA KARMAKAR BISWAS
R. G. ZIEMER
ROBERT P. BISHOP
RON RIEKKI
ROSS MAYO JR
R.R. UNDERSUN
RUDOLFO SAN MIGUEL
RUTH Z. DEMING
SAVONTE HICKS
SEAN FISHER
SEAN WAYMAN
SHAN BAE
SHELDON MCCARTHY
SHEVLIN SEBASTIAN
STEVEN PELCMAN
TINA STAGER
TOBIAS ROBBINS
TOM UKINSKI
TYHI CONLEY
VICTORIA ANN MALONEY
VICTORIA NAVA
VON BOLO
WENDY BENDLER
WU ALLAH-FERNANDEZ
XIAOCHEN SU
ZACK MURPHY

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Stamping Rules!: Free Magic Block Workshop!
Oooh! I'm so excited about this one! If you follow my blog, you can't have help but noticed that I absolutely am in love with the Rock the Block special running through the end of this month. For just $15 (with minimum purchase), you get this neat set of 9 paper cubes with a display tray PLUS an exclusive-only-available-this-month E-sized stamp set that is utterly adorable and versatile to boot! I've done a bazillion projects with the stamp set and blocks, but this one is making me giggle with GLEE!
The amazing Tresa Black from Fabulously Artsy did a fab video on using Rock the Block to make a magic block - an amazing, continuously flowing cube of photos (and stamped art if you want). I've wanted to make a Magic Cube forever., so of course I had to sit down and make one ASAP. So! Much! Fun!!
How much fun? So much that I am offering to teach you how to turn your blocks into a Magic Block just in time for Valentine's Day for FREE if you purchase the Rock The Block special this month! I'll be hosting a workshop to make your Magic Block in person (see details below), but I'll also do written instructions/hinge kits so that if you can't attend in person you can still whip up one of these babies at home. Soon you too will be flipping through your Magic Box photo cube...I need to make two, so that I can give one up to my hubby for his desk at work! Here's a video so you can see my cube in action. I chose to do it entirely in photos, since I had so many I wanted to fit on the cube!
CLICK HERE To order your Rock the Block today - or email me if you are local to ask how you can save on shipping!
What: Rock the Block Free Magic Block workshop!
When: Sunday, February 12th at 1 p.m.Bring: Your Rock the Block special kit, Bonding Memories Adhesive pen (Mono will not work), 20+ photos that are easy to trim into 4x4 size. Bring lots of pictures - you'll be amazed at how much you can get on this cube!
Cost: FREE with January Rock the Block purchase!


text:
Q: Permission being denied as I'm trying to write to a tsv file I am trying to write a series of dataframes to a series of seperate tsv files, but I am being met with 'PermissionError: [Errno 13] Permission denied'.
I can't attach an example code, as it is pretty hard to identify where the problem area would be, but I'm fairly certain I do not have the original tsv files open anywhere - the only places the original data files are accessed are in a basic 'read_csv', and if relevant the directories are accessed using os.walk & os.path.
Below is my code for handling output to tsv:
for key,value in Collection.items():
    for key1, value1 in value.items():
        foldername = r'C:\Users' + '\\' + key + '\\' + key1
        if not os.path.isdir(foldername):
            os.makedirs(foldername)
        headerInfo.to_csv(foldername,index=False)
        value1.to_csv(foldername,mode='a',index=False)

        base = os.path.splitext(filename)[0]
        os.rename(foldername, '.tsv')

I'm receiving the error at the lines concerning 'to_csv', so I'm not sure if at any point before that I'm leaving the output file open.
I'd appreciate any insights on my code, but would also like to ask is there another way to bypass the need for permission?

A: Replace the foldername in to_csv with a filename (tsvfile below):
for key,value in Collection.items():
    for key1, value1 in value.items():
        foldername = r'C:\Users' + '\\' + key + '\\' + key1
        if not os.path.isdir(foldername):
            os.makedirs(foldername)
        tsvfile = foldername + '\\filename.tsv'
        headerInfo.to_csv(tsvfile, index=False)
        value1.to_csv(tsvfile, mode='a', index=False)

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text:
Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus
John Kim, Mark Namchuk, Teodorica Bugawan, Qin Fu, Marc Jaffe, Yuguang Shi, Henk Jan Aanstoot, Christoph W. Turck, Henry Erlich, Vanda Lennon, Steinunn Baekkeskov
The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic β cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD6s in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD6s and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD6s molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD6s in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Thl subset of CD4+ T cells and cytotoxic T cell responses.
https://doi.org/10.1084/jem.180.2.595
10.1084/jem.180.2.595
Dive into the research topics of 'Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus'. Together they form a unique fingerprint.
Stiff-Person Syndrome Medicine & Life Sciences 100%
Autoantigens Medicine & Life Sciences 70%
Autoantibodies Medicine & Life Sciences 58%
Epitopes Medicine & Life Sciences 54%
T-Lymphocyte Subsets Medicine & Life Sciences 15%
Insulin-Dependent Diabetes Mellitus 11 Medicine & Life Sciences 14%
Diabetes Mellitus, Insulin-Dependent, 20 Medicine & Life Sciences 14%
Kim, J., Namchuk, M., Bugawan, T., Fu, Q., Jaffe, M., Shi, Y., Aanstoot, H. J., Turck, C. W., Erlich, H., Lennon, V., & Baekkeskov, S. (1994). Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus. Journal of Experimental Medicine, 180(2), 595-606. https://doi.org/10.1084/jem.180.2.595
Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus. / Kim, John; Namchuk, Mark; Bugawan, Teodorica; Fu, Qin; Jaffe, Marc; Shi, Yuguang; Aanstoot, Henk Jan; Turck, Christoph W.; Erlich, Henry; Lennon, Vanda; Baekkeskov, Steinunn.
In: Journal of Experimental Medicine, Vol. 180, No. 2, 01.08.1994, p. 595-606.
Kim, J, Namchuk, M, Bugawan, T, Fu, Q, Jaffe, M, Shi, Y, Aanstoot, HJ, Turck, CW, Erlich, H, Lennon, V & Baekkeskov, S 1994, 'Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus', Journal of Experimental Medicine, vol. 180, no. 2, pp. 595-606. https://doi.org/10.1084/jem.180.2.595
Kim J, Namchuk M, Bugawan T, Fu Q, Jaffe M, Shi Y et al. Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus. Journal of Experimental Medicine. 1994 Aug 1;180(2):595-606. https://doi.org/10.1084/jem.180.2.595
Kim, John ; Namchuk, Mark ; Bugawan, Teodorica ; Fu, Qin ; Jaffe, Marc ; Shi, Yuguang ; Aanstoot, Henk Jan ; Turck, Christoph W. ; Erlich, Henry ; Lennon, Vanda ; Baekkeskov, Steinunn. / Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus. In: Journal of Experimental Medicine. 1994 ; Vol. 180, No. 2. pp. 595-606.
@article{c5c0b35dbce94f79beef9800ec297364,
title = "Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus",
abstract = "The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic β cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD6s in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD6s and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD6s molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD6s in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Thl subset of CD4+ T cells and cytotoxic T cell responses.",
author = "John Kim and Mark Namchuk and Teodorica Bugawan and Qin Fu and Marc Jaffe and Yuguang Shi and Aanstoot, {Henk Jan} and Turck, {Christoph W.} and Henry Erlich and Vanda Lennon and Steinunn Baekkeskov",
doi = "10.1084/jem.180.2.595",
journal = "Journal of Experimental Medicine",
publisher = "Rockefeller University Press",
T1 - Higher autoantibody levels and recognition of a linear nh2-terminal epitope in the autoantigen gad65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus
AU - Kim, John
AU - Namchuk, Mark
AU - Bugawan, Teodorica
AU - Fu, Qin
AU - Jaffe, Marc
AU - Shi, Yuguang
AU - Aanstoot, Henk Jan
AU - Turck, Christoph W.
AU - Erlich, Henry
AU - Lennon, Vanda
AU - Baekkeskov, Steinunn
N2 - The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic β cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD6s in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD6s and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD6s molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD6s in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Thl subset of CD4+ T cells and cytotoxic T cell responses.
AB - The smaller form of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in two human diseases that affect its principal sites of expression. Thus, destruction of pancreatic β cells, which results in insulin-dependent diabetes mellitus (IDDM), and impairment of GABA-ergic synaptic transmission in Stiff-Man syndrome (SMS) are both characterized by circulating autoantibodies to GAD65. Anti-GAD65 autoantibodies in IDDM are predominantly directed to conformational epitopes. Here we report the characterization of humoral autoimmune responses to GAD6s in 35 SMS patients, of whom 13 (37%) also had IDDM. All SMS patients immunoprecipitated native GAD6s and the main titers were orders of magnitude higher than in IDDM patients. Furthermore, in contrast to the situation in IDDM, autoantibodies in 35 of 35 (100%) of SMS patients recognized denatured GAD65 on Western blots. Two major patterns of epitope specificity were identified on Western blots. The first pattern, detected in 25 of 35 SMS patients (71%), of whom 11 had IDDM (44%), was predominantly reactive with a linear NH2-terminal epitope residing in the first eight amino acids of GAD65. Nine of nine individuals who were HLA-haplotyped in this group carried an IDDM susceptibility haplotype and HLA-DR3, DQw2 was particularly abundant. The second pattern, detected in 10 of 35 patients (29%) of whom two had IDDM (20%), included reactivity with the NH2-terminal epitope plus strong reactivity with one or more additional epitope(s) residing COOH-terminal to amino acid 101. The second epitope pattern may represent epitope spreading in the GAD6s molecule, but may also include some cases of epitope recognition associated with IDDM resistant HLA-haplotypes. The principal NH2-terminal linear epitope in GAD65 distinguishes the reactivity of SMS and IDDM autoantibodies and may be a determinant of pathogenicity for GABA-ergic neurons. The greater magnitude and distinct specificity of the humoral response to GAD6s in SMS may reflect a biased involvement of the T helper cell type 2 (Th2) subset of CD4+ T cells and antibody responses, whereas IDDM is likely mediated by the Thl subset of CD4+ T cells and cytotoxic T cell responses.
U2 - 10.1084/jem.180.2.595
DO - 10.1084/jem.180.2.595
JO - Journal of Experimental Medicine
JF - Journal of Experimental Medicine


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text:
Surge in illegal dumping in Dundalk during lockdown
Calls have been made for the local authority to tackle the surge in illegal dumping in our communities since the pandemic.
Councillors highlighted the scourge of people dumping their rubbish on streets and alleyways around Dundalk and Louth.
They said that people in communities are carrying out clean ups in their areas, collecting the dumped litter, but within weeks rubbish dumped illegally has piled up again in public spaces.
Councillor Edel Corrigan, speaking at last week's Dundalk District Municipal monthly meeting, said: "There is a real issue with illegal dumping in our local communities.
"In the Faughart area, less than two weeks after a massive spring clean was carried out by households in the area, the litter warden on three sperate occasions had to come out to the area to address issues of illegal dumping.
"It is one of many areas that are having this problem and we don't seem to be getting anywhere with addressing this, it doesn't seem to be improving on the ground.
"I would urge that something more be done because what we are doing so far isn't working.
"We are coming back into the summer season and seeing restrictions start to ease and will see an even bigger surge in illegal dumping in the coming months.
"There is also illegal dumping in the Faughart graveyard."
Cllr Sean Kelly also highlighted the illegal dumping problem in the alleyways in Castleross estate and Marian Park.
"The litter warden has been here on numerous occasions to Castleross so I am hoping this can be marked as a blackspot to be checked on a regular basis because it is happening again and again" he told the local authority at the meeting held online last Tuesday.
"The site is somewhat secured as residents have gated off the back alleys, but people are still coming along and just throwing their rubbish over the gate and a rodent issue is going to arise if it persists.
"Also, the litter wardens have cleared a lot of rubbish in Marian Park, but there is the issue of overgrown trees and shrubbery in the alleyways which is encouraging dumping."
Cllr Liam Reilly added: "There is an issue with fly tipping and the increase in litter has been evident since the pandemic, especially in 2021.
"We did a Spring clean in Dromiskin two weeks ago and collected 60 bags of rubbish and unfortunately there is just as much litter on the roads again now."
Louth County Council said that fly tipping is a big problem, but they are "doing their best to deal with it as it arises".
"There is fly tipping going on everywhere with tons and tons of rubbish being dumped and we are dealing with it as best we can", the local authority's Mark Johnston said.
"We would like the laws to be more in our favour to put up CCTV cameras so we could identify the people dumping like we did in the past.
"But currently we are prohibited from putting cameras up unfortunately.
"That law might change later this year, we believe, so we might be back in a position to start getting cameras up in different places to deal with the fly tipping like we did before.
"We are collecting 10% extra tonnage of illegal dumped rubbish.
"We are dealing with it as best we can, the litter wardens are trying to identify the people doing it through letters or other forms of identification that they can find in the rubbish and we take those people to court and they are fined."
He said they are currently working on an anti-dumping initiative and have €150,000 revenue to promote anti-littering, to do clean ups and to do mattress and furniture collections.


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Criminal charges can be very intimidating: From traffic violations to murder, these charges can lead to fines and jail time that hurt your finances and limit your freedom. That's why it's important to work with an experienced attorney who can guide you through the legal process with confidence and competence.
At the Law Office of Robert D. Massey and Associates, we have been defending our clients from criminal charges for more than a decade. We will research your case thoroughly and design a defense strategy tailored to your specific needs. In some cases that can mean getting charges dropped; in others it may mean seeking reduced charges or a reduced sentence. No matter what charges you are facing, we offer aggressive, ethical advocacy for your interests.
In our years of experience, we have negotiated satisfactory settlements in cases which seemed indefensible, obtained not-guilty verdicts for clients facing the death penalty and exposed unconstitutional practices by law enforcement officers that have led prosecutors to dimiss cases against our clients. We cannot guarantee a particular result at the outset of any case, but we promise to work hard and stand up for your rights.
A criminal conviction in any of these areas can have severe consequences that can affect your ability to vote, seek employment, own a firearm and remain free from incarceration.
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text:
Winners Announced for MedVet's Shelter Surprise 2021
WORTHINGTON, Ohio (Oct. 27, 2021) – The votes are in for MedVet's Shelter Surprise contest which is awarding three deserving shelters cash prizes to support the important work they do to save pets. Out of more than 6,000 nominations, 10 animal shelter and rescue organizations were chosen, and more than 274,000 votes were cast to determine the winners.
The top prize of $2,000 goes to Shenango Valley Animal Shelter located in Hermitage, Pennsylvania, a community about an hour north of Pittsburgh. The shelter is a no-kill, unlimited stay shelter that takes in all breeds of animals and helps find them forever homes. They received 28 percent of the votes in the contest from their supporters.
The runner-up is Hospets which is in Columbus, Ohio. The organization will receive $1,250. And the third-place winner, which will receive $500, is Metairie Humane in New Orleans, Louisiana.
"Throughout the contest, we've loved learning more about the wonderful shelters and rescues that are working throughout the United States to save pets and help them find their forever homes. There are so many amazing organizations, and it has been an honor to support them in our shared mission of helping pets and their loving families," said Dr. Linda Lehmkuhl, Chief Executive Officer of MedVet. "We hope these organizations benefit not just from the additional funds but also from the raised awareness and community support as people rallied to vote for them."
The funds will be awarded to the winning organizations during National Animal Shelter and Rescue Appreciation Week which takes place Nov. 7-14. For more information about MedVet and the Shelter Surprise, visit http://www.medvet.com.
About MedVet:
MedVet is the leading veterinarian owned and led family of specialty and emergency hospitals dedicated to delivering exceptional care and a deeply supportive experience to pets and their loving families, referring veterinarians, and team members. For more than 30 years, MedVet's empathetic, insightful, and driven team of expert caregivers has helped the organization grow to be the preferred choice for high quality, compassionate care, proudly serving more than 400,000 patients each year in communities throughout the United States. To learn more about MedVet and its mission of Leading Specialty Healthcare for Pets, visit medvet.com.
By | Posted InNews Releases | TaggedShelter Surprise


With 2017 officially behind us, it's time to usher in 2018 with celebration, memories and most likely, umbrellas. And while Vancouver doesn't have the best Spring forecast, that doesn't mean you have to be indoors or strategically moving between puddles this season.
Whether you're the outdoorsy type or looking to try something new in the city, Vancouver boasts all kinds of activities for every occasion.
The following list includes the oldies but we're big fans of trying something new, and who knows, you might have a little fun!
Since the first plunge in 1920, the Polar Bear Swim continues to be a popular and chilling way to meet locals for a good cause this January. Costumes are optional, as are participants if you were just curious what your typical polar bear lover enjoys doing in January.
Plunging into English Bay requires you bring nothing but your favourite swimwear. Prizes are available for a wide variety contests plus swag can be bought for a good cause.
Click here for more scintillating info.
If jumping into English Bay during the coldest time of the year isn't your thing, don't worry. This January the annual Hot Chocolate Festival returns to Vancouver.
The Hot Chocolate Festival combines local and foreign chocolatiers, bakery chefs, pastry shops, gelato masters, and ice cream enthusiasts for an international experience unlike you've ever tasted.
A real life adventure game that incorporates puzzles, clues and other environmental elements within a time limit. Teams must solve the mysteries and escape before time's up!
It's classic team-building at it's finest. Each Escape Room around Vancouver hosts different atmospheres, puzzles, combinations and clues to keep you guessing.
Better teams are composed of positive individuals, coordinated leaders and logical thinkers who enjoy a challenge in a unique setting.
Tip: Clear headedness plays a critical factor as time winds down and you try and escape.
From Tuesday to Saturday you can get great seats at the Comedy Club. The club hosts Canada's best up and coming performers and comedians so you're sure to get a laugh.
Best nights are earlier in the week where tickets cost around 8-10 bucks. Booking is also incredibly easy from the website.
Just 20 minutes from the downtown core is the Capilano Suspension Bridge. The popular destination is an adventure-magnet during summer months for it's sublime natural backdrop.
Imagine suspended atop 230 feet while crossing a swaying bridge. Now magnify that with possible winds and dazzling lights and you'll have an unforgettable experience.
After your harrowing bridge journey, you will find yourself amongst Vancouver's treetop walks. Though it's Vancouver's darkest month of the year, the decorated Douglas Firs will have you captivated as you traverse through BC wilderness.
Why not top it all off by visiting the Ox Lounge this spring?
Located on Howe street for those refreshments after the Polar Bear Swim or maybe an adventure recap after the suspension bridge. Ox Lounge is the place to be this year and hosts live music, fresh food and iconic drinks.
This January, Vancouver is abundant with things to check out. You're sure to make some memories with locals from around BC. Last year alone, saw 2500 registered swimmer attending the Polar Bear Swim.
Anything we missed? What are you doing in January?


is it legal to bring second hand car parts in for personal use?
AliExpress - Anyone Used It?
Container shipping from Turkey to UK?
UPS - What Is Wrong With Them?
Sending things via Courier to Turkey.
Electronics from US to Turkey?!
Does Amazon Deliver to Turkey?
Next online delivery to Turkey!
Buying from eBay in Turkey?
Purchase on Gitti Gidiyor ship to UK. Will pay for your help!
Post Office and Custom new Rule?
Import electric bike into Turkey.
going to uk--what to return with?
Do you have to pay at customs?
Is this duty charge legit?
Niave Newby - Importing Furniture in 4 Days But !!!
Moving Goods from Istanbul to Bodrum suggestions please?


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Alfred de Zayas' Human Rights Corner
This blog is devoted to legal, historical and human rights matters, in which issues of general concern are addressed freely and spontaneously. It is intended to further an informal exchange of views in the democratic spirit of freedom of opinion and respect for the opinions of others, in an effort to understand rather than condemn, to propose constructive solutions rather than grandstand. The perspective is both from inside and outside the box and the added value lies more in the questions than in the answers.
Former UN Independent Expert
Professor of International law
November 24, 2017 alfreddezayas2 Comments on Wales Online: Peaceful Catalans bullied by Madrid
Wales Online: Peaceful Catalans bullied by Madrid
Link: http://www.walesonline.co.uk/news/local-news/western-mail-letters-wednesday-november-13941518
Peaceful Catalans bullied by Madrid
Many people were shocked to see the brutality of the Madrid police, pulling people by their hair out of a polling station in an effort to stop them voting for independence.
Spanish people see nothing wrong in the brutality of the bullfight, when the bull is tormented for a long time and then wounded in the neck and shoulder such that it cannot fight any more, so we have an unfair fight. In this country we stopped bear-baiting 200 years ago.
Catalan people have turned their backs on bullfighting. They are a civilised people with their own language, and wish to govern themselves and achieve this by non-violent means. It is Madrid that resorts to violence. In Madrid the police supporters were shaking their clenched fists in a threatening way. But in Barcelona the Catalans were waving their open hands in a friendly way.
The Catalan leaders have been arrested and face charges which may detain them in prison for 30 years for peacefully advocating Catalan independence.
European countries have been painfully silent about the injustice suffered by Catalan people, especially when we remember that these European countries (including Spain) signed a treaty giving humanitarian rights to all their citizens and the right of small nations to democratic elections to decide how they should be governed. Our Government has failed to criticise Spain. It is therefore necessary for individuals who are concerned about the injustice in Catalonia to take action by not buying anything from Spain.
In Geneva (October 25, 2017) the UN independent expert on the promotion of a democratic and equitable international order, Alfred de Zayas, issued the following statement: "I deplore the decision of the Spanish government to suspend Catalan autonomy. This action constitutes retrogression in human rights protection, incompatible with Articles 1, 19, 25 and 27 of the International Covenant on Civil and Political Rights (ICCPR). Pursuant to Articles 10 (2) and 96 of the Spanish constitution, international treaties constitute the law of the land and, therefore, Spanish law must be interpreted in conformity with international treaties."
Geraint Jones
Llanfyllin, Powys
Published by alfreddezayas
Alfred Maurice de Zayas {born 31 May 1947 in Havana, Cuba), also known as Alfred de Zayas, is an American lawyer, writer, historian, a leading expert in the field of human rights and international law. From 2012 until April 2018, he was the United Nations (UN) Independent Expert on the Promotion of a Democratic and Equitable International Order (also known as Special Rapporteur), appointed by the United Nations (UN) Human Rights Council. View all posts by alfreddezayas
Categories UN, United Nations (UN)Tags Alfred de Zayas, Catalan, Catalonia, UN Independent Expert on the promotion of a democratic and equitable international order, Wales
2 thoughts on "Wales Online: Peaceful Catalans bullied by Madrid"
Li Fontrodona November 25, 2017 — 10:05 pm
Reblogged this on INVISIBLE FORMS and commented:
Thanks heartily to Mr. Alfred de Zayas from UNO for sharing this article, as well as posting many other personal judgements and statements in favour of our national rights, so severely violated by Spain – as usual from centuries ago.
martingugino December 11, 2017 — 6:50 am
you say in your Guantanamo lecture of 19 Nov 2003 in Vancouver the following:
"The 1903 agreements were further supplemented by a new treaty, signed in Washington on May 29, 1934, in which the United States agreed to increase the amount of the lease from $2,000 to $4,085 per annum" page 289.
The 1934 lease is available online at Yale/Avalon project.
That change in the lease amount is not part of the text – ie is unilateral.
and was made in 1974 not 1934. See "The Leasing of Guantanamo" / Michael J Strauss / Praeger Press.
The US was obligated by the Treaty of Paris to protect Cuba, and that obligation is reiterated in the 1903 lease document. (for its own defense, the government of Cuba shall…) The lease is void, because this benefit, of the essence, isbeing withheld.
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text:
Q: Python - merge items of a dynamic nested list in a single list I have a code which takes input of the users and creates a nested list.
List = [list 1, list2, list 3,.....]

The list 1, list 2, list 3 and so on are dynamic and can keep increasing or decreasing.
So far my code output is:
All_data = [[a,b],[c,d],[1,2],[3,4],.....]

The output that I want (for the dynamic nested list):
All_data = [a,b,c,d,1,2,3,4,......]

Can anyone suggest me a solution to solve this?

A: You can use itertools.chain:
itertools.chain(*All_data)

Or:
itertools.chain.from_iterable(All_data)

This creates an iterator, if you want to get a list:
list(itertools.chain.from_iterable(All_data))

A: You can use numpy.concatenate(). It will condense the nested list into a single list.
If your nested list is dynamic and can grow exponentially, then I will suggest to use itertools.chain.from_iterable() from itertools library. It will be faster because it will not convert the list into a numpy array like the first option.


Delegates from 18 countries attended the 22nd Latin American Conference at The Presidente Intercontinental Hotel in Mexico City from 6 – 8 June.
The agenda included presentations covering Cryptocurrencies; Anti-corruption; Tax Strategies, Transfer Pricing and Brand Development. Andrew Collier, Kreston's Director of Quality and Professional Standards updated all on Kreston's strategy and the Quality Review process.
"This successful event again brought members together from across the region and from the wider network, offering opportunities to build new and develop existing business relationships".
Prior to the conference, all members of the Kreston Board met and were introduced to Kreston CEO Jon Lisby's successor Liza Robbins. Liza offered delegates an insight into her vision and plans for Kreston's future strategy during a presentation in the main session.
"It was excellent that Liza was able to join us for this important conference and had the opportunity to meet with the Board and colleagues from around the world. Kreston Latin America is dedicated to and continues to support the development of the Kreston brand across the region which will lead to more collaborative business opportunities".


It will be easy to decide not to. You will make hundreds of decisions this week and you probably won't even be aware you decided. It just happens. Time gets away from you and suddenly another week has gone by and good intentions are swallowed up by stronger desires and the tyranny of the urgent.
This week can be different.
Decide now. What are you going to do this week to help the children you love get to know God a little better?
Now that the decision is made, take a few more minutes to decide when, where and how. Put it in the day planner and then do it.
It's best if you can do a little each day but if you think you only have time for it once this week…do it once…one seed is better than none. One seed can grow. The garden of their hearts will be bare if the seeds aren't planted at all.
If I can help you in any way, please let me know on our Facebook page, Discover God 4Kids.
Have fun planting some seeds!! You can do it!
This entry was posted in Kathy Brights Korner on September 21, 2015 by Kathy Bright.


Comprehensive cover: In addition to third party, fire and theft, it covers accidental and malicious damage to the vehicle. It covers the insured against loss or damage to the motor vehicle and its accessories and spare parts therein.
In any case, the insurance contain an imposed excess. An excess is the first part of any claim which is borne by the insured.