CDC4, which encodes an F-box protein that is a member of the Skp1-Cdc53/Cul1-F-box (SCF) ubiquitin E3 ligase, was initially identified in the budding yeast Saccharomyces cerevisiae as an essential gene for progression through G1-S transition of the cell cycle. Although Candida albicans CDC4 (CaCDC4) can release the mitotic defect caused by the loss of CDC4 in S. cerevisiae, CaCDC4 is nonessential and suppresses filamentation.
To further elucidate the function of CaCDC4, a C. albicans strain, with one CaCDC4 allele deleted and the other under the repressible C. albicans MET3 promoter (CaMET3 p) control, was made before introducing cassettes capable of doxycycline (Dox)-induced expression of various C. albicans Cdc4 (Ca Cdc4) domains. Cells from each strain could express a specific Ca Cdc4 domain under Dox-induced, but CaMET3-CaCDC4 repressed conditions. Cells expressing domains without either the F-box or WD40-repeat exhibited filamentation and flocculation similarly to those lacking CaCDC4 expression, indicating the functional essentiality of the F-box and WD40-repeat. Notably, cells expressing the N-terminal 85-amino acid truncated Ca Cdc4 partially reverse the filament-to-yeast and weaken the ability to flocculate compared to those expressing the full-length Ca Cdc4, suggesting that N-terminal 85-amino acid of Ca Cdc4 regulates both morphogenesis and flocculation.
The F-box and the WD40-repeat of Ca Cdc4 are essential in inhibiting yeast-to-filament transition and flocculation. The N-terminal region (1–85) of Ca Cdc4 also has a positive role for its function, lost of which impairs both the ability to flocculate and to reverse filamentous growth in C. albicans.
Candida albicans is a natural diploid without a complete sexual cycle and exists as yeast, pseudohyphal, and hyphal cells . It is capable of a morphological switch induced by environmental stimuli , essentially via cAMP-mediated and MAPK signaling pathways . Importantly, its ability to alter morphology among cell types is associated with virulence to humans . Many cell cycle regulators including cyclins are also known to control morphogenesis in C. albicans.
Recently, an F-box protein encoded C. albicans CDC4 (CaCDC4) has been shown to play a role in filamentous development [6, 7]. Cdc4, originally identified in the budding yeast Saccharomyces cerevisiae, encodes ubiquitin E3 ligases, which belongs to a member of the Skp1-Cdc53/Cul1-F-box (SCF) complex. This complex is known to play a role in ubiquitin-proteasome dependent degradation of regulatory proteins in eukaryotes . A specific SCF complex is designated by its associated F-box protein. This protein is variable with two interacting domains of F-box for Skp1 and WD40-repeat (or LRR) for specific substrates , such that Cdc4 can be named SCFCdc4. To progress through the G1-S transition in S. cerevisiae, SCFCdc4 is required to degrade Sic1  and Far1 , which are the cyclin-dependent kinase inhibitors. Therefore, S. cerevisiae CDC4 (ScCDC4) is essential in S. cerevisiae.
Although Ca Cdc4 is a structural homolog of S. cerevisiae Cdc4 (Sc Cdc4) and is capable of rescuing the mitotic defect caused by the loss of ScCDC4 in S. cerevisiae, the functions of Ca Cdc4 and ScCdc4 are dissimilar as the null Cacdc4 mutant is viable and the depletion of Ca Cdc4 causes the accumulation of Sol1 (Sic1 like) for hyphal development rather than initiation of cell cycle arrest . This verifies that CaCDC4 is nonessential and suppresses filamentation and suggests that controlling the degradation on Sol1 in C. albicans by Ca Cdc4 is important for inhibition of filamentation. Therefore, while C. albicans Sol1 is likely a substrate of SCFCaCdc4, which can be demonstrated by the reduction of Sol1 when Ca Cdc4 is overexpressed , there has not been any direct evidence to support this hypothesis. Additionally, the filamentous properties for mutants of Cacdc4 null and Cacdc4 sol1 double null were comparable. This refutes the idea that Sol1 is the sole target of Ca Cdc4. Indeed, with an affinity-purification approach, we have isolated at least two novel Ca Cdc4-associated proteins  that are potential substrates of Ca Cdc4.
To further elucidate the role of CaCDC4 and its mediation through a characteristic F-box protein of SCF ubiquitin E3 ligase in C. albicans, we have sought to dissect the Ca Cdc4 domains associated with filamentation. In this study, we made a C. albicans strain with one deleted CaCDC4 allele and repressed the other by CaMET3 promoter (CaMET3 p) using methionine and cysteine (Met/Cys). We used this strain to introduce plasmids capable of inducing expression of various Ca Cdc4 domains with doxycycline (Dox). We observed the roles of F-box and WD40-repeat for Ca Cdc4 function and the possible role of the N-terminal 85-amino acid for morphogenesis. We also showed that C. albicans cells that lacked Ca Cdc4 triggered flocculation. Moreover, we found that N-terminal 85-amino acid of Ca Cdc4 is required for inhibition of both filamentation and flocculation.
E. coli strain DH5α was used for the routine manipulation of the plasmids. They were grown at 37°C in LB broth medium  or on plates containing 1.5% agar (Difco, BD Biosciences), with 50 μg/ml ampicillin or 30 μg/ml kanamycin. All C. albicans strains (Table 1) were derived from auxotrophic strain BWP17 (arg4/arg4 his1/his1 ura3/ura3) . They were grown at 30°C in either yeast extract-peptone-dextrose (YEPD) or supplemented minimal synthetic defined (SD) medium with 2% glucose with or without 2% agar . While Ura+ prototrophs were selected on SD agar plates without uridine, His+ prototrophs were selected on SD plates without histidine. Selection for the loss of the C. albicans URA3 (CaURA3) marker was performed on plates with 50 μg/ml uridine and 1 mg/ml 5-fluoroorotic acid (5-FOA, MD Bio). To repress the CaCDC4 expression that was controlled by CaMET3 p, strains were grown on SD medium or on plates with 2.5 mM Met/Cys, which has been shown to optimally switch off the expression of the CaMET3 p-driven downstream gene . To induce gene expression under the Tet-on system, 40 μg/ml Dox (Sigma) was added to YEPD or SD media.
Plasmid DNA was extracted routinely from E. coli cultures using Gene-SpinTM MiniPrep purification Kit-V2 (PRO TECH, Taipei, Taiwan) and the instructions provided by the manufacturer. E. coli was transformed with plasmid DNA by using CaCl2. The DNA cassettes were introduced into C. albicans by the lithium acetate method as described previously .
Initially, a strain with repressed CaCDC4 expression was made. A mini-Ura-blaster cassette, flanked with 60-bp sequences homologous to CaCDC4, was PCR-amplified using a template of plasmid pDDB57 and long primers of CaCDC4-URA3-F and CaCDC4-URA3-R (Table 1). BWP17 was transformed by integration of the cassette into the CaCDC4 locus to generate Ura+ strain JSCA0018. The plasmid pFA-HIS1-MET3p-CaCDC4, with a partial CaCDC4 coding sequence for N-terminal Ca Cdc4 (1–563), was linearized with Bsp EI and used to transform JSCA0018 to generate His+ JSCA0021 (Figure 1A; Table 1). Cells of JSCA0021 were plated with 5-FOA to induce recombination between two copies of dpl200 flanking the mini-Ura-blaster for a loss of CaURA3 to generate JSCA0022.
Construction of a C. albicans strain for repressibly expressing CaCDC4 . (A) Strain construction (detailed in the Methods). The first CaCDC4 allele on BWP17 was deleted by mini-Ura-blaster to obtain JSCA0018. Plasmid pFA-HIS1-MET3p-CaCDC4 containing partial CaCDC4 coding sequence was linearized at a unique site for introducing into strain JSCA0018 to generate JSCA0021. 5-FOA was used to counter-select CaURA3 removal to obtain JSCA0022 for re-introducing the Tet-on plasmid with a CaURA3 marker. (B) Verification of constructed strains by Southern blotting analysis. Organization of the CaCDC4 locus with respect to Nde I sites is shown. The relative positions of the probe used and the predicted Nde I-digested pattern of the CaCDC4 locus are indicated. Two Nde I-fragments of 14 kb and 8.5 kb, specific to CaCDC4, could be detected in genomic DNA from BWP17 digested with Nde I; two Nde I-fragments of 8.5 kb and 4.5 kb, specific to CaCDC4 and Cacdc4::URA3-dpl200, respectively, could be detected in JCSA0018; two Nde I-fragments of 4.5 kb and 7.4 kb specific to Cacdc4::URA3-dpl200 and Cacdc4::P MET3 -CDC4:HIS1, respectively, could be detected in JSCA0021; and two Nde I-fragments of 13.5 kb and 7.4 kb specific to Cacdc4::dpl200 and Cacdc4::P MET3 -CDC4:HIS1, respectively, could be detected in JSCA0022. A non-specific Nde I-fragment is indicated as "*" can be detected in all strains tested.
To allow the expression of cassettes encoding assorted Ca Cdc4 domains in C. albicans, a Tet-on plasmid, pTET25M , which is derived from pTET25  for inducing gene expression with Dox, has been developed. To regulate CaCDC4 expression by the Tet-on system, the coding sequence of CaCDC4 was PCR-amplified using plasmid CaCDC4-SBTA bearing CaCDC4 (Lai WC, unpublished results), primers CaCDC4-SalI and CaCDC4-BglII (Table 2), and Pfu polymerase (5 U/μl, MD bio), digested with Sal I and Bgl II for cloning into pTET25M, from which pTET25M-CaCDC4 was generated. Moreover, CaCDC4-6HF, which encodes 6×histidine and FLAG (6HF) tags at the C-terminal of Ca Cdc4, was PCR-amplified with primers CaCDC4-6HF SalI and CaCDC4-6HF BglII (Table 2), followed by digestion with Sal I and Bgl II and cloning into pTET25M to obtain pTET25M-CaCDC4-6HF.
a Restriction enzyme sites are in italics.
b Sequences complementary to those encoding 6×His and FLAG are in lower case letters. The italics has been used for restriction enzymes as in note "a". The underline is new replaced with lower case letters.
To define the function of the distinct Ca Cdc4 domains (Figure 2A), different CaCDC4 portions were used to replace the full length CaCDC4 coding sequence on pTET25M-CaCDC4-6HF. By using the primer sets listed in Table 2, the following constructs were made: pTET25M-ΔNCaCDC4-6HF (with primers CaCDC4 ΔN AatII and CaCDC4 ΔN XhoI), which encodes the N-terminal truncated Ca Cdc4; pTET25M-F-6HF (with primers CaCDC4 F-box AatII and CaCDC4 F-box XhoI), which encodes the F-box domain with flanking regions; pTET25M-WD40-6HF (with primers CaCDC4 WD40 AatII and CaCDC4 ΔN XhoI), which encodes eight copies of WD40-repeat; and pTET25M-ΔNF-6HF (with primers CaCDC4 ΔN AatII and CaCDC4 F-box XhoI), which encodes truncated N-terminal Ca Cdc4 and the F-box domain. All inserts of the constructs were released with Aat II and Xho I to replace the full-length CaCDC4 on pTET25M-CaCDC4-6HF. Consequently, plasmids bearing those CaCDC4 segments flanked with common C. albicans ADH1 (CaADH1) sites were digested with Sac II and Kpn I, each of which was transformed into C. albicans for integration at the CaADH1 locus. All strains were verified by colony PCR with specific primers before subjecting to Southern blotting analysis.
Morphological analysis of the constructed CaCDC4 repressible strains. Cells of strains JSCA0021 and JSCA0022 were grown on SD medium or plates with (+) or without (-) Met/Cys. Colonies were photographed with MEIJI stereoscopic microscope EMZ5 at 40× magnification (top panel). Cells in liquid culture were visualized and recorded with a Nikon 50i microscope at 400× magnification (bottom panel). Bars represent 10 μm.
Genomic DNA from the C. albicans strains was isolated by the MasterPure™ Yeast DNA Purification Kit (Epicentre®, an Illumina company) according to the manufacture's instruction. Southern blotting was performed with the aid of the Rapid Downward Transfer System (TurboBlotter™, Whatman) using 10 μg of the restriction enzyme-digested genomic DNA. The DNA on the blot was hybridized with a probe amplified by the PCR DIG probe synthesis kit (Roche) with the primers CaCDC4_Probe_F and CaCDC4_Probe_R for CaCDC4 locus or CaADH1 Probe_F and CaADH1 probe_R for ADH1 locus (Table 2) using DIG Easy Hyb (Roche). To reveal the structure of gene locus, the DIG Luminescent Detection Kit (Roche) was used after hybridization, and the luminescent images of blot were captured with the imaging analysis system (ImageQuant LAS4000 mini, GE Healthcare Life Sciences).
Cultured cells were collected, and the total protein from each sample was extracted as described previously . The proteins were resolved by 10% SDS-PAGE and transferred to PVDF membranes (PerkinElmer, Boston, USA). Proteins on the membranes were probed with polyclonal antibody to FLAG (Sigma) in 1:2000 dilution and detected using the SuperSignal West Pico Chemiluminescent Substrate Kit (PIERCE). These were recorded with the Luminescent Image Analyzer (FUJIFILM LAS-1000) and analyzed by ImageGauge 3.46 and L Process v 1.96 (FUJIFILM).
The cells of strains were streaked on YPD agar plate for 3 days and colonies were picked and inoculated into SD medium with required supplements for 48 hrs. Next, the cultures were diluted into fresh SD medium to 0.1 of an initial OD600 with required supplements. To simultaneously repress the expression of CaMET3 p-driven CaCDC4 and to induce the expression of various CaCDC4 segments encoding series of Ca Cdc4 domains, 2.5 mM Met/Cys and 40 μg/ml Dox were also added into the SD medium. After 48 hrs, the cultures were spun down for 1 minute at 500 rpm, and the suspensions of the cultures were sampled to determine their optical density at OD600. Three independent assays were conducted and each sample was assayed in duplication. A paired Student t test with p < 0.05 was considered significance.
The FLO-encoded flocculins are known to be essential for flocculation in S. cerevisiae. Functional homologues of FLO genes have been found in C. albicans. In particular, the important S. cerevisiae gene FLO11 responsible for flocculation has C. albicans functional counterpart ALS1. Since FLO11-associated flocculation is dependent on the presence of Ca2+, we adopted an alternative flocculation assay in which the rate of flocculation is initiated by Ca2+ and the optical density was assessed within a short time-frame . Briefly, to initiate flocculation, an aliquot of 800 μl deflocculated cell suspension was transferred into a 1-ml cuvette, followed by addition of 200 μl of 100 mM CaCl2. The cuvette was mixed robustly by pipetting and the absorbance (OD600) was assessed instantly at 30-s intervals for 5 minutes using a spectrophotometer (DU800, Beckman Coulter, Inc.). All assays were conducted in triplicate.
To establish C. albicans strains capable of expressing CaCDC4 and its domains solely controlled under a Tet promoter directly in C. albicans, BWP17, with both alleles of CaCDC4 deleted, was constructed to accommodate Tet-on plasmid cassettes capable of expressing assorted Ca Cdc4 domains induced by Dox. The first allele of CaCDC4 was deleted in BWP17 by mini-Ura-blaster  to generate the JSCA0018 strain (Figure 1A; Table 1). This strain was used to delete the second CaCDC4 allele to obtain a Cacdc4 null mutant. However, Cacdc4 null mutant cells growing as filamentous form with toughened cell walls obstructed transformation.
To overcome this problem, the strain JSCA0021 (Figure 1A; Table 1) was created that had one CaCDC4 allele deleted and the other under CaMET3 control that was Met/Cys repressible. To allow the introduction of Tet-on cassettes with the same CaURA3 selectable marker as the mini-Ura-blaster on JSCA0021, 5-FOA was used as a counter-selection agent to remove CaURA3 from JSCA0021, from which JSCA0022 was obtained (Figure 1A; Table 1). The strains were PCR-confirmed with specific primers before subjecting to Southern blotting analysis. The CaCDC4 locus from BWP17 strain could detect two Nde I-digested fragments with size of 14 kb and 8.5 kb, respectively (Figure 1B). The size shifting of Nde I-fragment flanking CaCDC4 from 14 kb to 4.5 kb demonstrated that one CaCDC4 allele was integrated with the mini-Ura-blaster cassette as in strain JSCA0018 (Figure 1B). The size shifting of Nde I-fragment flanking CaCDC4 from 8.5 kb to 7.4 kb demonstrated that the other CaCDC4 allele integrated with the MET3-diven CaCDC4 plasmid as in strain JSCA0021 (Figure 1B). Strain JSCA0021 could be further popped out the mini-Ura-blaster cassette to obtain strain JSCA0022 in which the size shifting of Nde I-fragment flanking CaCDC4 from 4.5 kb to 13.5 kb (Figure 1B). These results indicate that all strains constructed have expected organizations in their genome.
It has been shown that Ura— auxotrophic mutants are avirulent  and other virulence-associated features can be influenced by the level of CaURA3 gene expression . To assess presence of CaURA3 having effect on yeast-to-filament transition, the yeast-to-filament transitions between strain JSCA0021 and JSCA0022 were compared, cells of those strains were assessed under CaMET3 p repressed or de-repressed conditions. Cells of both strains on SD plates without Met/Cys grew as circular colonies with smooth surfaces (Figure 2). By contrast, cells on plates with Met/Cys formed irregular colonies with filaments (Figure 2). Under the microscope, these strains exhibited equivalent filamentous forms, suggesting a comparable ability to deplete CaCDC4 for expression and inability of CaURA3 interfering with yeast-to-filament transition in C. albicans. Subsequently, JSCA0022 was used as a parental strain to introduce the Tet-on cassettes (with CaURA3 marker) that encoded assorted Ca Cdc4 domains.
The filamentous development of JSCA0022 under CaMET3 p-CaCDC4 repressed conditions, with Met/Cys and the Tet-on system, allows us to study the function of the Ca Cdc4 domains. A set of Tet-on cassettes (obtained from pTET25M-CaCDC4-6HF, pTET25M-ΔN-6HF, pTET25M-F-box-6HF, pTET25M-WD40-6HF, and pTET25M-ΔNF-6HF) that encoded each of the assorted domains of Ca Cdc4 (Figure 3A) were used to transform JSCA0022 (which contained a CaMET3 p-repressible CaCDC4) to Ura+ by integration at the CaADH1 locus (Figure 3B). The correctness of the strains was confirmed by yeast colony PCR with specific primers before Southern blotting analysis. The CaADH1 locus from strain JSCA0022 could detect a Spe I-digested fragment with size of 3.3 kb (Figure 3C). The CaADH1 locus from strains JSCA0023 and JSCA0024 detected an increased Spe I-digested fragment of 9.4 kb due to the integration of Tet-on cassettes of either pTET25M-CaCDC4 or pTET25M-CaCDC4-6HF (Figure 3C). The CaADH1 locus from other strains also showed expected alteration in size according to the size of different CaCDC4 domains (Figure 3C). These results confirmed the correctness of the strains.
Construction of C. albicans strains for Dox-inducing the expression of assorted CaCDC4 domains. (A) Schematic representation of Ca Cdc4 domains expressed from the Tet-on system. The strains with which they are derived and predicted Tet-on cassette size are shown (B) Generation of Tet-on cassettes for expressing assorted CaCdc4 domains. Different portions of CaCDC4 were PCR-generated with primer sets (Table 2) containing common Aat II and Xho I sites for replacing full-length CaCDC4 on pTET25M-CaCDC4-6HF as described in the Methods. By digestion with Sac II and Kpn I, each cassette was used to transform C. albicans strain JSCA0022 for integration into the CaADH1 locus. (C) Verification of Tet-on cassettes being integrated into CaADH1 locus by Southern blotting analysis. Organization of the CaADH1 locus with respect to Spe I sites is shown in Figure 3B. The relative positions of the probe used and the predicted Spe I-digested pattern of the CaADHI locus are indicated in Figure 3B. One Spe I-fragment of 3.3 kb specific to CaADH1 locus could be detected in genomic DNA from strain JSCA0022 and its derivatives digested with Spe I. The correctness of integration of the cassette into the CaADH1 locus of various strains was confirmed by alteration of the Spe I-fragment from size of 3.3 kb to 9.4 kb (Figure 3B) or various sizes as indicated in Figure 3A.
The JSCA0022 strain, which expressed the non-tagged and repressible Ca Cdc4, was used as a negative control. The sample obtained from JSCA0022 contained two prominent proteins of approximately 55 kDa and 72 kDa (Figure 4A) which were presumably a result of cross-reactivity to the anti-FLAG antibody. Those two proteins were used as an internal control. The F-box and WD40-repeat proteins from strains JSCA0026 and JSCA0027 migrated to their expected positions of approximately 19 kDa and 43 kDa (Figure 4A), respectively. However, the full-length Ca Cdc4 and the N-terminus truncated Ca Cdc4 (ΔN) from strains JSCA0024 and JSCA0025 exhibited signals at positions corresponding to 100 kDa and over 100 kDa (Figure 4A), respectively, as opposed to 86 kDa and 77 kDa, respectively. Three distinctive signals (Figure 4A) were observed for strain JSCA0030 expressing ΔNF of Ca Cdc4, but none of them matched the expected size of 34 kDa; however, the signal at the lowest position could be meaningful. These patterns of expression were similar to strains expressing each of the domains, with either BWP17 or JSCA0021 as a parental strain (Lai WC, unpublished results). Therefore, even though some of the strains expressed domains with unexpected size, they were unique from the negative control of JSCA0022. We concluded that the Tet-on system functions in JSCA0022 and that Ca Cdc4 might be undergoing undefined modifications.
Morphological analysis of C. albicans strains capable of Dox-inducing the expression of assorted CaCDC4 domains. Cells were grown initially in SD medium without Met/Cys to saturation and were diluted to the same initial concentration. Cells were grown exponentially in SD in the absence of 2.5 mM Met/Cys, with or without 50 μg/ml Dox (-Met/Cys + Dox or -Met/Cys), or in the presence of 2.5 mM Met/Cys, with or without 50 μg/ml Dox (+Met/Cys + Dox or + Met/Cys). (A) The Dox-inducibly expressing assorted Ca Cdc4 protein domains under CaMET3-CaCDC4 repressed conditions was verified by Western blotting with polyclonal antibody to FLAG. The non-specific signals between 72 and 55 kDa, and between 55 and 40 kDa are served as a loading controls. (B) The images were visualized and recorded with a Nikon 50i microscope at 400× magnification. The arrow in white indicates filamentous cells. Bars represent 10 μm. The designations of strains are the same as in Table 1.
To determine the function of the assorted Ca Cdc4 domains, JSCA0022-based strains capable of repressing CaCDC4 and inducing expression of assorted Ca Cdc4 domains were grown in SD medium with or without Met/Cys and in the presence or absence of Dox. Cells from strains in SD medium without Met/Cys grew as yeast in the presence or absence of Dox (Figure 4B). By contrast, cells from strains in medium with Met/Cys grew with filaments (Figure 4B). As expected, cells of JSCA0023 and JSCA0024 growing on medium with Met/Cys and Dox and that expressed the full-length Ca Cdc4 with or without tag grew as yeast. Disregarding the full-length Ca Cdc4, cells from all strains, except JSCA0025 expressing assorted domains, still grew as filaments (Figure 4B). Under Met/Cys and Dox conditions, cells from JSCA0025 expressing the N-terminal 85-amino acid truncated Ca Cdc4 seemed to have an ability to suppress filamentation but not complete back to the yeast form (Figure 4B). This is in consistent with our previous observation in which, comparing with cells capable of expressing the full-length Ca Cdc4 under the CaMET3 p repressible control, those cells expressing the N-terminal 85-amino acid truncated Ca Cdc4 lagged behind in reaching exponential stage (Additional file 1: Figure S1) and converted to filamentous form earlier (Additional file 2: Figure S2) in the repressed condition.
Significant differences in the ability among strains to form suspensions (to resist flocculation) were observed. The extent of flocculation among strains was observed after resuspending the cells in cuvettes, where they remained for 30 seconds. When cells were grown under the Met/Cys and Dox conditions, only those from JSCA0023 and JSCA0024 were somewhat easier to maintain as a suspension. To exclude the possibility that this was a result of increases in cell density, cells from all strains were initially grown to saturation, and the cultures were subsequently diluted to the same initial optical density and grown exponentially to similar optical density. The extent of flocculation among strains was observed after spinning the cells for 1 minute at 500 rpm. The suspended cells were sampled for determination of their optical density. Cells resisted in flocculation would remain in suspension upon centrifugation. Under the CaMET3 p de-repressed condition and in the presence or absence of Dox, all strains exhibited a similar degree of suspension. However, under the CaMET3 p repressed condition, JSCA0026, JSCA0027, and JSCA0030 displayed flocculation similar to JSCA0022 regardless of the presence or absence of Dox (Figure 5A). While more cells of strains JSCA0023, JSCA0024 maintained as suspension, those of JSCA0025 with some filamentous cells, showed comparable extent of flocculation to JSCA0022 under CaMET3 p repressed but Tet-on induced conditions (Figure 5).
Analysis of cell flocculation by low-speed centrifugation. Cultures of the indicated strains were grown in SD medium with histidine, arginine, uridine for 2 days before diluting into the SD medium to an initial OD600 = 0.1 with addition of 2.5 mM Met/Cys to repress the expression of CaMET3 p-driven CaCDC4 and 40 μg/ml Dox to induce the expression of Ca Cdc4 domains tested for 48 hrs to OD600 ≈ 1.6. Cultures were photographed before and after centrifugation. (A). A representative of the cultures. Upper panel: two-day culture. Bottom panel: cultures being spun down with 500 rpm for 1 minute. (B). Quantitative results. Data are represented as means with standard deviation from three independent experiments, each sample was in duplication. The data from JSCA0022 were compared with those of other strains. **: P < 0.01. The designations of strains are the same as in Table 1.
To solidify our observations, an alternative flocculation assay where flocculation is initiated by addition of Ca2+ to the culture medium being depleted with Ca2+ beforehand was used . Only cells of JSCA0023 and JSCA0024 remained resistance in flocculation during the time-frame of 5-minute assay compared with those of the rest of strains (Figure 6), which were consistent with the results shown in Figure 5. However, both strains JSCA0025 and JSCA0027 exhibited greater ability to resist flocculation than that of JCSA 0026 and JSCA0030 when considering the differences in OD600 from the initial to the end points.
Analysis of Ca 2+ initiated cell flocculation. The strains were grown as described in Figure 5 to saturation at OD600 ≈ 1.4. The cultures were harvested and washed twice with deflocculation buffer, followed by initiation of flocculation as described in the Methods. The assays were conducted in triplicate. The name of each strain shows only the last two numbers.
In this study, we aimed to dissect the function of Ca Cdc4 domains by introducing a Tet-on system with cassettes that encoded for a variety of Ca Cdc4 domains in a C. albicans mutant of Cacdc4 null. However, the Cacdc4 null mutant with a filamentous form could not be easily used to introduce the Tet-on cassettes; therefore, we constructed the JSCA0022 strain, where CaURA3 was released from the strain JSCA0021, and CaCDC4 expression was repressible. Under repressed conditions, the JSCA0022 strain showed similar filamentous morphology (Figure 2) to those from previous reports of cells with CaCDC4 repressed strain [6, 7] and of cacdc4 null mutant  (Tseng TL, Hsu WH, and Shieh JC, unpublished results). We confirmed that the JSCA0022 strain under repressed conditions was equivalent to a strain that had completely lost CaCDC4 function. Hence, by introduction of the Tet-on cassettes into JCSA0022 strain, each of the strains was capable of expressing individual Ca Cdc4 domains in the presence of Met/Cys and Dox for functional comparisons.
To verify the ability of the Tet-on cassettes in C. albicans, each of the cassettes encoding various Ca Cdc4 domains was transformed into BWP17 and JSCA0021 before introducing them into JSCA0022 at the CaADH1 locus. Individual Ca Cdc4 domains from relevant strains were all detectable, suggesting that the Tet-on system functions in C. albicans. However, while cells expressing the F-box and the WD40 repeat could be detected as their expected sizes, those expressing the full-length Ca Cdc4, the N-terminus truncated Ca Cdc4 (ΔN), and the ΔNF of Ca Cdc4 could be detected at positions higher than anticipated (Figure 4A). In particular, the sample from strain JSCA0030 expressing the ΔNF could be detected three signals (Figure 4A), all of which were greater than the predicted sizes. These results suggest that the N-terminal Ca Cdc4 from residue 85 to 241 (Figure 3A) might be undergoing post-translational modification during the Tet-on-induced expression, although its functional significance is unknown. Interestingly, the region between residue 85 and 241 of Ca Cdc4 contains abundant serine and threonine residues, the majority of which are homologous to S. cerevisiae Cdc4 . This implies possible phosphorylations or other modifications on these residues that is specific to C. albicans. However, the genuine nature of these residues remains to be determined, and their functional significance of this N-terminal Ca Cdc4 requires further study.
With regards to integration of CaADH1 locus by the Tet-on cassette, it is known that C. albicans adh1 homozygous null mutant gains the ability to form biofilm both in vitro and in vivo, suggesting a possible role of CaADH1 in flocculation. However, the heterozygous CaADH1 null mutant with which the homozygous adh1 null mutant is reintegrated a functional copy of CaADH1 to the CaADH1 locus appears to be similar in biofilm formation as its isogenic wild-type strain. In addition, disruption of CaADH1 has no consequence of morphology alteration in C. albicans (Lai WC, unpublished results). Therefore, the possible effect of Tet-on cassette on flocculation and filamentation by integration, hence disruption of a copy of CaADH1 locus can be excluded.
Under the Met/Cys and Dox conditions, cells expressing F-box, WD40 repeat, and the ΔNF of Ca Cdc4 exhibited filamentous forms similar to those of JSCA0022, whose CaCDC4 was repressed, compared to those expressing the full-length Ca Cdc4 without or with tag (JSCA0023 and JSCA0024), which exhibited yeast forms (Figure 4B). These results suggest that both the WD40 repeat and F-box are essential to suppress the yeast-to-filament transition. Cells from strain JSCA0025 expressing the ΔN of Ca Cdc4, which were grown in the presence of Met/Cys and Dox, were only partially able to reverse filamentous cells to yeast cells, suggesting that the N-terminal 85-amino acid of Ca Cdc4 plays a role in the yeast-to-filament transition in C. albicans. The role of the N-terminal 85-amino acid of Ca Cdc4 for growth was observed previously, in which cells expressing N-terminal 85-amino acid truncated Ca Cdc4 lagged slightly in proliferation during the exponential stage (Additional file 1: Figure S1), and repression of the expression of the N-terminal 85-amino acid truncated Ca Cdc4 resulted in prominently lagging behind in growth, which was presumably due to the morphological alteration of cells to filaments in advance (Additional file 2: Figure S2) that delays proliferation as compared to those of yeast cells. Since the N-terminal 85-amino acid of Ca Cdc4 is unique compared to that of the S. cerevisiae Cdc4 , our finding reveals a role of N-terminal 85-amino acid of Ca Cdc4 on morphogenesis, which is unknown previously.
Importantly, cells of all JSCA0022-based strains exhibited flocculation in medium with Met/Cys, but the strains JSCA0023 (CaCDC4) and JSCA0024 (CaCDC4-6HF) exhibited less flocculation by adding Dox simultaneously (Figure 5). Unlike cells of JSCA0023 and JSCA0024, those of JSCA0025 expressing N-terminal 85-amino acid truncated Ca Cdc4 were unable to totally overturn filamentous-to-yeast cells, suggesting that N-terminal 85-amino acid is required for full activity of CaCDC4 function in C. albicans to inhibit filamentation. However, if flocculation is tightly associated with filamentation, we expect to see the extent of flocculation in JCSA0025 (ΔN 6HF) being greater than that of JSCA0022 but less than that of JSCA0023 and JSCA0024 in the presence of Met/Cys and Dox. This was not revealed by the low speed-centrifugation method but by the Ca2+-initiation assay. Importantly, both JSCA0025 and JSCA0027 expressing Ca Cdc4 lacking N-terminal 85-amino acid (Figure 3A) exhibits similar extent of flocculation. Moreover, JSCA0025 that expressing Ca Cdc4 lacking N-terminal 85-amino acid could only partially suppress filamentation yet JSCA0027 that expressing Ca Cdc4 lacking N-terminal 85-amino acid and F-box with flanking regions completely lose the ability to inhibit filamentation (Figure 3A and Figure 4B). These results imply that N-terminal 85-amino acid of Ca Cdc4 has a role in inhibition of cell flocculation in C. albicans and that the F-box and its flanking region in addition to the N-terminal 85-amino acid of Ca Cdc4 might be associated with proper control of both morphogenesis and flocculation.
Therefore, we conclude that F-box and WD40-repeat are important in suppressing yeast-to-filament transition and flocculation and that the N-terminal region (1–85) has a positive role in CaCDC4 function, lost of which impairs reverse of filament-to-yeast and reduces the ability to flocculate in C. albicans. Moreover, the function of Ca Cdc4 for suppressing flocculation that is related to cell-cell adhesion  implies a role of CaCDC4 in biofilm formation  that is under investigation.
The authors thank Dr. A. Mitchell (Columbia University) for C. albicans strain BWP17 and plasmid pDDB57, Dr. J. Wendland for pFA-HIS1-MET3p (Friedrich-Schiller-University, Jena, Germany), and Dr. J. Morschhäuser for pTET25 (Universität Würzburg University, Würzburg, Germany). We gratefully acknowledge the financial support for this work provided by a grant of a collaborative agreement between the Chung Shan Medical University (Taichung, Taiwan) and the Antai Tian-Sheng Memorial Hospital (Pingdung, Taiwan) (CSMU-TSMH-097-005) and grants from the National Health Research Institutes (NHRI-EX100-9808SI) and National Science Council (NSC 101-2629-B-040-001-MY3 ) of Taiwan, R.O.C.
CC, WCL, JCS, TLL conceived and designed the experiments. CC, WCL, and TLT performed the experiments. CC, WCL, JCS, and TLT analyzed the data. WCL, TLL, and TLT contributed reagents and materials. JCS wrote the paper. All authors read and approved the final manuscript.


The Governing Body is responsible for securing the "efficient and effective management of College activities and it's property". It is also the employer of all managers and staff of the College.
Mr Hetherington is a qualified accountant and member of the Chartered Institute of Public Finance and Accountancy. He worked in economics and public policy for over 10 years as a management consultant. More recently, Mr Hetherington took up the position of Associate Director in the Northern Ireland Centre for Economic Policy, based in the Ulster Business School at the University of Ulster. He brings to the Governing Body particular strengths in public accountability, analytical thinking and a detailed understanding of the role of further education in the economy. He does not hold any other public appointments.
Ken has a wide and varied professional background, including working in Central Government, Local Government and the Private Sector. His extensive roles have included leading and managing diverse areas from PPP/PFI projects, through national advertising campaigns to international sales in Agri-food industry and IT consultancy. Ken is a former student of the FE College in Lisburn, a qualified teacher, and Chartered Accountant. He holds a number of further qualifications from Queen's University Belfast, University of Ulster and Warwick University Business School.
Mrs Reid qualified as a teacher in 1984 and worked in a large urban secondary school. During a period of 9 years she held the position of Head of Year, Head of Department, Head of Junior School and Teacher Governor. In 1995 she was appointed Vice Principal of a rural comprehensive school. She was appointed Vice Principal of a Post Primary School from February 1998 – September 2004 and Principal of a Post Primary School from September 2004 – August 2009. She has also been a member of the South Belfast Area Learning Community and was appointed Chair in 2007. In this role she was involved in activities to encourage a more collaborative approach between schools and Further Education Colleges, to provide a more relevant curriculum and make better use of available resources. Mrs Reid lives in Jordanstown. She has carried out no political activity in the last five years and does not currently hold any other public appointments.
Barbara Larkin joined SERC as a Member of the Governing Body in August 2013. In her career to date Barbara worked in banking and financial services in the UK and Ireland since 1986. A native of Newry, she began her career at Northern Bank working in Retail and Business Banking. She studied at the, University College, Dublin, Michael Smurfit Graduate Business School, graduating from there in 1995 with an Honours Degree in Financial Services. Moving to England to work within National Australia Group, Barbara gained further skills in business banking, managing and growing new business across sectors as diverse as manufacturing, retail, services, logistics, technology, property and export. Her skill set then grew to incorporate business analysis and planning as well as supporting with HR issues. Moving back to Northern Ireland in 2003 she took up a role with Ulster Bank working with existing and new business and corporate clients With her career in banking Barbara has gained widespread experience in a variety of industry sectors working with companies at varying stages in their development.
Christine has worked at senior management level in BT for over a decade and is currently employed in an Ireland-wide lead Strategic Resourcing role. She has a number of tertiary level and professional qualifications in marketing, human resources, finance, business improvement and project management. A full member of the Chartered Institute of Personnel and Development (CIPD), she previously served on the Northern Ireland Executive Committee as Treasurer and Secretary for the Strategy and Policy Sub Committee. She has also served as a director on the Lanyon Place Management Company and with the Beat Carnival, a local carnival company. Ms Goodwin holds one other public appointment.
Steve Pollard is a Lecturer in Management Development with The Business Institute Ulster University Business School. An experienced Non-Executive Director and Business Adviser Steve has held senior roles in the public, private and third sectors. This includes experience in vocational education and training in the further education sector as well as running privately held training organisations. Steve has a long standing interest in community economic development with a particular focus on enterprise and skills. Steve has recently joined the Board of Supporting Communities NI.
Mr Bodger is currently a Trustee Member of the Audit Committee and Member of the Capital Development Committee of National Museums, Northern Ireland. He does not receive any remuneration for this position. He is also Chair of the Audit Committee of Stranmillis University College, for which he also receives no remuneration. He is a member of the NI Social Care Council for which he receives £6242 per annum. He has held various Board positions during the last 10 years including Chair of the Appeals Tribunals for the Southern Education & Library Board. Mr Bodger is retired and lives outside Rathfriland. He has carried out no political activity in the last five years.
Professor Alan Woodside serves as Emeritus Professor in Highway Engineering at the University of Ulster. He has a background in civil engineering and higher education and is the current chair of the Waste Industry Safety and Health Forum for Northern Ireland and a public director on the Board of the Lagan Canal Trust. Professor Woodside is an elected member of council of the Chartered Institution of Highways and Transportation and acts as the national vice-chair of its Education and Membership Committee. He is actively involved in the promotion of STEM in schools as an Ambassador for Sentinus and W5 and serves on the Board of Governors of both a local primary and grammar school. He is credited with more than 200 technical publications and holds an OBE for services to highway engineering and higher education.
Mrs Fraser has 14 years' experience working in pedagogic research and educational development. She started academic life as a Teaching Fellow in the School of Computing & Mathematics at the University of Ulster & is currently an Educational Developer at Queen's University in Belfast. Mrs Fraser has a research background in student engagement and student retention and has extensive experience designing, developing and delivering academic enhancement projects & workshops in employability, assessment & feedback, students as partners, MOOCs and getting started in pedagogic research. She also manages research projects and has a particular interest in Science, Technology, Engineering and Mathematics (STEM) education. Mrs Fraser is a Principle Fellow of the Higher Education Academy (PFHEA), a professional member of the British Computer Society, a Fellow of the Staff and Educational Development Association, a professional member of SIGCSE (Special Group Computer Science Education), a member of the JISC Learning and Teaching Practice Experts Group (Curriculum Design) and a Member of Computing Science Graduate Employment Steering Group (HEFCE & BIS).
Mr John Mackell graduated in law from the Queens University of Belfast in 2001 and went on to complete his legal studies prior to admission to the rolls of Solicitor for Northern Ireland in 2004 and the roll of Solicitors in the Republic of Ireland in 2008. While at QUB he was President of Queen's Student Union and latterly a member of the University Senate as well as an Officer Board Member for the Union of Students' in Ireland. Mr Mackell has been the lead solicitor on a number of high profile complex criminal cases. He is currently employed as a solicitor in the public sector. Prior to this Mr Mackell worked for 10 years in private practice, specialising in Criminal, Litigation and Public Law. An experience Court Advocate, Mr Mackell is also an accredited mediator. He was appointed by the Minister for Justice as Chairperson of the Northern Ireland Civil Legal Services Appeals Panel for the period 1st April 2015 until 31st March 2020, with remuneration of £350 per sitting to include preparation required and attendance at each sitting of the Appeal Panel. He is also an appointed Legal Chairman of the Health and Social Care Board's Disciplinary Committee.
Carolyn King is the Corporate Services Staff representative on the Governing Body. She worked in various roles in the private and public sectors in the UK and Europe and joined the college in 2001, moving into her current role as Work Placement Officer in 2008. Carolyn actively promotes a more collaborative approach to placements in FE and conceived, developed and implemented the Business Engagement & Student Tracking System (BEST) across HE, FE and the Training Organisation. Her continued personal and professional development has led to her co-authoring two work placement papers and presenting at HE Work Placement Conferences in the Universities of Leicester and Greenwich. Her exploration of work placements continues through participation in a two-year funded project in collaboration with European partners. Carolyn is a former student of the FE College in Bangor and is a Chartered Manager with CMI.
Andrew studied architecture at the University of Edinburgh before returning to take up a position with a Design Consultancy in Belfast. He established his own consultancy soon afterwards and combined this work with part time lecturing. He was appointed an Associate Lecturer and subsequently became a full time lecturer with SERC. He is currently a Deputy Head of School in the School of Performing and Creative Arts and represents the Lecturing Staff on the Governing Body.
Any correspondence to the Governing Body should be sent to the Secretary to the Governing Body, Claire Williamson cwilliamson@serc.ac.uk or Tel: 07919597703.
The Governing Body publishes minutes of all its meetings and those of its committees soon after they are approved by members.


text:
May 1991 - Volume 67 - 787
Alimentary tract granulomas. (1 May, 1991) Free
D. G. James, R. E. Pounder
Palliation of oesophageal cancer--endoscopic intubation and laser therapy. (1 May, 1991) Free
J. D. Urschel, J. S. Cockburn, A. V. Foote
Positive evidence for P6 acupuncture antiemesis. (1 May, 1991) Free
J. W. Dundee, C. McMillan
Medicine in the elderly. (1 May, 1991) Free
P. Diggory, A. Homer, J. Liddle, C. F. Pratt, S. Samadian, R. Tozer, C. Weinstein
Granulomatous enteropathy in common variable immunodeficiency: a cause of chronic diarrhoea. (1 May, 1991) Free
N. Mike, T. T. Hansel, J. Newman, P. Asquith
Low dose danazol in the treatment of the premenstrual syndrome. (1 May, 1991) Free
M. Deeny, R. Hawthorn, D. McKay Hart
Misoprostol does not alter the pharmacokinetics of propranolol. (1 May, 1991) Free
P. N. Bennett, G. C. Fenn, L. J. Notarianni, C. E. Lee
Multilocular bone tumour. (1 May, 1991) Free
L. Kreel, M. A. al-Kutoubi
Hypoglycaemia associated with anorexia nervosa. (1 May, 1991) Free
V. Fonseca, S. Ball, V. Marks, C. W. Havard
Giant thymic carcinoid. (1 May, 1991) Free
L. C. John, P. Hornick, S. Lang, J. Wallis, S. J. Edmondson
Shrinkage of thyrotrophin secreting pituitary adenoma treated with octreotide. (1 May, 1991) Free
S. M. Orme, J. T. Lamb, M. Nelson, P. E. Belchetz
Primary hyperparathyroidism presenting as unilateral visual loss. (1 May, 1991) Free
D. Chappel, K. Farrington
Congenital central alveolar hypoventilation syndrome (Ondine's Curse): effectiveness of early home ventilation for normal development. (1 May, 1991) Free
Y. L. Chang, P. W. Meerstadt
Atrial septal defect presenting as recurrent primary amoebic lung abscess. (1 May, 1991) Free
M. Sharma, H. Mehta, S. K. Sharma
Vogt-Koyanagi-Harada syndrome, a rare association of Hodgkin's disease. (1 May, 1991) Free
S. Y. Chuah, A. J. Lyne, M. W. Dronfield
Massive gastrointestinal haemorrhage associated with ileal lymphoid hyperplasia in Gaucher's disease. (1 May, 1991) Free
D. R. Jones, J. Hoffman, R. Downie, M. Haqqani
Localized primary sclerosing cholangitis mimicking a cholecystectomy stricture relieved by an endoprosthesis. (1 May, 1991) Free
R. Oren, E. Goldin, N. Harats, E. Libson, D. Shouval
European Medical Research Group (1 May, 1991) Free
Helicobacter pylori--our knowledge is growing. (1 May, 1991) Free
Testicular teratoma and peripheral neurofibromatosis. (1 May, 1991) Free
N. Oakley, B. Todd
Occult spontaneous intramuscular haemorrhage as a complication of heparin for unstable angina. (1 May, 1991) Free
S. R. Johnston, H. M. Mather
Venepuncture for calcium assays: should we still avoid tourniquet? (1 May, 1991) Free
B. E. Walker, R. B. Payne
Biliary peritonitis, due to rupture of intrahepatic duct. (1 May, 1991) Free
H. Raza, A. M. Hussain
Hyperimmunoglobulinaemia D and periodic fever mimicking familial Mediterranean fever in the Mediterranean. (1 May, 1991) Free
R. Topaloğlu, U. Saatçi
Multiple pulmonary infarctions caused by giant cell arteritis. (1 May, 1991) Free
M. Radhamanohar
Serial neopterin concentrations in disseminated T-cell lymphoma. (1 May, 1991) Free
C. Carter, R. J. Leeming, A. Pollock
Helicobacter pylori - our knowledge is growing (1 May, 1991) Free
C. Holcombe
The photic sneeze (1 May, 1991) Free
D.S.C. Rose
Clinical Medicine (1 May, 1991) Free
D.G. Beevers
The Courts and the Doctor (1 May, 1991) Free
D.R. Chambers
Synopsis of Ophthalmology (1 May, 1991) Free
Jeffrey S. Hillman
ABC of Resuscitation (1 May, 1991) Free
R. Mayall
A Researcher's Guide to Scientific and Medical Illustrations (1 May, 1991) Free
A. Rollason
Roundsmanship '90. A Yearbook Guide to Clinical Medicine (1 May, 1991) Free
J.C. Ferguson
Year Book of Family Practice 1990 (1 May, 1991) Free
A.J. McDonald
My Heart's Health: A New Revolutionary View (1 May, 1991) Free
Bernard Clarke
Advances in Pineal Research (1 May, 1991) Free
Ehud Ur
Erratum (1 May, 1991) Free
Cover (PDF)
Admin (PDF)

redpajama_set_name: RedPajamaCommonCrawl


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Adaptation / Agriculture/Agroforestry / ASEAN / Cambodia / CRP6-Forests, Trees and Agroforestry / CRPs / Laos / Myanmar / Natural Resource Management / SDG13-Climate change / Thailand / Viet Nam
Vice-president of Chiang Mai University calls for integrated research in the Greater Mekong Sub-region
by Rob Finlayson · November 5, 2014
There are manifold problems facing the hundreds of millions of people who rely on the Mekong River and its watersheds. Dealing with the problems starts with coordinated research
Associate Professor Doctor Sermkiat Jomjunyong, the vice-president for research and academic services at Chiang Mai University, Thailand, is calling for multidisciplinary, cross-border research collaboration throughout the Greater Mekong Sub-region, beginning in 2015, to meet the multiple challenges of ensuring food security, improving livelihoods and adapting to climate change.
The year marks the beginning of integration for the member states of the Association of Southeast Asian Nations (ASEAN) and represents a perfect opportunity for coordinating research, he said.
'Given my responsibility for research at the university, I have been talking with many groups about climate change, agroforestry, land use and natural resources management', said Dr Jomjunyong. 'These issues have to be worked on by a network of researchers, not one institution alone, because they affect all countries in the sub-region.
The Chiang Mai University senate, which is a policy body, wants to position the university as a leader in research in northern ASEAN and sees the renewed partnership with the World Agroforestry Centre as strategically important given both research bodies' focus on multidisciplinary research, their experience in uplands with high biodiversity threatened by land degradation and links with the other countries of the upper Greater Mekong Sub-region.
'The World Agroforestry Centre is fully supportive of Dr Jomjunyong's plans', said Dr Prasit Wangpakapattanawong, the Centre's Thailand program coordinator and a director of the Knowledge Support Centre for the Greater Mekong Sub-region, a joint research centre established with Chiang Mai University. 'We see the same need for coordinated research—especially in remoter areas of Myanmar, Lao, Cambodia and Viet Nam—to understand the common and unique problems throughout the sub-region. The Centre has been conducting regional projects with international and national partners for many years, and national projects in Thailand and Viet Nam, and now is the time to bring all our expertise and knowledge together. We are currently undertaking a scoping study in Lao and Cambodia to better understand how we can contribute our specialist technical expertise, such as our negotiation-support methods, land-use planning and agroforestry system technologies'.
Dr Prasit Wangpakapattanawong (l) and Associate Professor Doctor Sermkiat Jomjunyong
Chiang Mai University, similarly, is in the process of setting up national and regional networks, including a closer collaboration with the World Agroforestry Centre.
'I want to see more multidisciplinary research with our neighbours in Lao, Cambodia, Viet Nam and Myanmar, in particular', said Dr Jomjunyong, 'because a strength of the university is our diverse research groups—for example, humanities and social sciences, health sciences, and science and technology—that can share much with our colleagues to really understand transboundary issues. Working in an integrated fashion will enable us to solve problems locally, nationally and regionally to make society and the environment better'.
Last year, the World Agroforestry Centre celebrated 20 years of research in Thailand and this year Chiang Mai University marks its 50th. Both organizations will hold a celebratory seminar, Greater Mekong Sub-region Seminar on Climate Change and Sustainable Resources Management, 3-4 December 2014, in Chiang Mai that will bring together other research organizations, development agencies and governments to establish a common approach to the land-use challenges facing the region.
This work is linked to the CGIAR Research Program on Forests, Trees and Agroforestry and the CGIAR Research Program on the Humid Tropics
Tags: Chiang MaiGreater Mekong Sub-regionICRAFMekongresearchThailanduniversityWorld Agroforestry Centre
Robert Finlayson is the Southeast Asia program's regional communications specialist. As well as writing stories for the Centre's website, he devises and supervises strategies for projects and the countries in the Southeast Asia region, including scripting and producing videos, supervising editors and translators and also assisting with resource mobilization.
Closing the gap between man and nature – The Satoyama Initiative
More than just cooking: linking energy and food security
New report says forests and trees could be major factor in efforts to end global hunger
Next story REDD+: From Euphoria to Reality
Previous story Meeting discusses formation of International Union for Agroforestry


In this exhibition, different teachers' graduates are brought together for the first time.
Three of the photographers are mentored by Bertram Kober and address social positions in the area of photojournalism and architecture photography: Ciro Caltana tells stories about people for whom work is about far more than making a living; Anatol Kowalewski, who found his material on a remote coast in Brazil, also shares this passion. In contrast, Kichun Park investigates how space is perceived and understood.
Two of the graduates studied with Eva Bertram: in her series, Chiara de Maria explores the grey zone between consensuality and molestation, while Antonia Pischke focuses on borderline situations of the physical and on deformation.
The work mentored by Eva Maria Ocherbauer explore intimacy: MagdaTheGreat plays with her own sexuality in a performative, gender-crossing way.


Enhancing your presence on the web is important, regardless of what industry you are in or how large your company is. The fact is though, developing an effective website is no small task. What's more, a top quality site needs to be carefully created, and in the best interest of your visitors should only be done so by someone with many years of web design experience. As the need for websites has increased, so too has the number of firms and freelancers available to design your website. How do you know who to choose for a professional Iowa City web design company? Below, we have helped make the decision of choosing a web design firm easier by listing out some things to consider.
Be sure you contact multiple web designers in Iowa City and view their work before making a decision. Price is also a key point in your decision, so be sure to request quotes from each business you speak to.
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By keeping your goals and objectives as our highest priority as we offer web design in Iowa City, IA, we will do everything in our power to make sure they are met.
Our company doesn't stop until you have a completed website and we understand the significance of having a finished site as soon as possible.
Finally, keep in mind that you were able to connect with us just now by performing your own search of the internet. We know what we're doing on the subject of SEO and helping people make meaningful connections on the web. With the proper website, folks in your neighborhood can get your services on the internet by simply doing a web search. Call us right now to discuss options for your company, or simply complete the form on this page. You can get additional information about our expertise by taking a look at our case study of the overhaul of the website for a local cleaning company. It reveals how the project translated into more traffic (the free, organic kind), leads and sales for their business. It may be downloaded here.


Elko, NV real estate appraisals M.S. Mitchell and Associates Inc.
Looking for an appraiser in the Elko area?
When you're looking for a real estate appraiser in Elko, NV, your search should start and end here at M.S. Mitchell and Associates Inc. We've been in the real estate appraisal business for over 35 years! Elko real estate values are unique and you need an appraiser with experience completing appraisals in Elko and surrounding areas.
We are experts in providing mortgage appraisals on real estate in Elko. We perform full residential appraisals on Fannie Mae form 1004, drive-bys with exterior photos, interior inspections, single family, multi-family, condo, manufactured housing, HUD/FHA appraisals, purchased properties, or refinancing.
We offer pre-listing appraisal services in Elko, as well as a full range of appraisal services to support your clients' home buying or home selling needs.
A listing appraisal can be a powerful tool for a for-sale-by-owner home seller. And give yourself peace of mind that your largest investment — your Elko real estate — is really worth what you think it is by obtaining a professional appraisal.
Official website for Elko, NV.


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Why the Picture of Dinosaur Sex Bouncing Around the Internet Is Insane
Sometimes scientific illustrators get a bit too creative.
Ben Guarino
On Thursday, a group of dinosaur experts from the University of Colorado, Denver, made a plausible case that they'd found evidence of theropod mating rituals. Based on the shapes of fossilized footprints and scrapes, the scientists write in the journal Scientific Reports, large meat-eaters like Acrocanthosaurus may have performed a courtship dance akin to the one seen among modern-day puffins. What seems less likely is the theory suggested by the art accompanying the press release: Theropods had raucous, rough, and roaring sex.
Paleontologist Twitter, which is a great place to lurk if you have an interest in dinosaurs, noticed something a little familiar with the image of large predators caught in the act:
The tyrannosaurs painted in flagrante on the left, as University of Southampton vertebrate paleontologist Darren Naish pointed out, come from a museum exhibit in Spain.
The boning bones at Jurassic Museum of Asturias aren't actually fossils, but replicas; the posture displayed here is, charitably, a guesstimate. There are several factual hurdles: It's hard to tell male and female tyrannosaurs apart, and we don't really have a reference for bipedal animals that get so big. Based on living crocodiles and birds, there is good evidence that T. Rex had a penis, as science writer Brian Switek told Slate in 2015. Extrapolating from a duck's corkscrew penis, paleontologist John Long estimates a Tyrannosaurus penis could have stretched past the two meter mark.
There's much less evidence for dino sounds. And because we don't have any Cretaceous-era MP3s, we have a hard time knowing exactly what noises they make. Though the Jurassic Park roar is impressive, it's also mostly mammal cries, leaning heavily on screams of a baby elephant.
To paint the animal kingdom with a sweeping brush, the act of sex itself tends to be a quiet affair. Primates like bonobos may produce "copulation calls", but observations of ejaculating birds such as the buffalo weaver displayed nothing more than shivers and clenched feet. (This author once watched elephants copulate at the Berlin Zoo, and though the visuals are seared into his brain there wasn't much in the way of accompanying sounds.)
If today's wolves are vulnerable during sex, it doesn't take much to imagine that the top predators of the Cretaceous would want to bone softly, too.


Photo courtesy of King Cardinal.
Every year for the past three years, Denver band King Cardinal has released a new holiday song paired with a benefit show. The annual show, titled King Cardinal's Annual Holiday Spectacular, will donate proceeds to The Joshua School (TJS), one of the top schools for autistic youth in the country. King Cardinal said in a press release, "TJS makes such a revolutionary difference in the lives of so many individuals in and around Denver. It's a worthy cause and one that we feel could use more attention." This year the show is at Syntax Physic Opera on December 14 with additional performances from Kyle Emerson and Grayson County Burn Ban.
In 2015 King Cardinal released "Alone On Christmas Eve" and last year they followed it up with "Christmas from O'Hare." Outside of holiday songs, King Cardinal released a 10-song album just over two months ago titled Great Lakes. Since then, they've been traveling the country on their album release tour but are finally returning home for the holidays.
King Cardinal's newest Christmas song is titled "Winter Waltz" and can be streamed exclusively below before its official release on Tuesday, December 5. Tickets to King Cardinal's Third Annual Holiday Spectacular start at $10 and are available here.


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Q: Adding conditions at the WHERE clause gives more results I use SqlServer. I have a table with lots of columns the importants of which are:
· User_name
· Partition - Date in xxxx-xx-xx format
· Game - a string that works as an ID
· Credits - A number
· Bet - Another number
· Prize - Another number
· Num_Spins - Another number
I wrote a query to select of those the ones that interest me given a specific date.
Select distinct CONCAT(User_Name, DATALENGTH(User_Name)) as User_name, Partition, Game, Bet, Num_spins, Credits, Prize
            from ***
            where Partition>='2019-09-01' and Partition<'2019-11-17' and Bet>0 and credits is not null
            and User_Name IN (Select distinct userName from *** where GeoIpCountryCode='ES')

I wish I could make that a view or something, but unfortunately I don't have the privileges to do so. Therefore, I do a subquery from it:
I want to find out of those rows, the ones whose numbers follow a certain math result: (Credits+Bet-Prize) > 100000 and num_spins>5
Select user_name, partition, count(Game) as difMachines
        FROM
        (
            Select distinct CONCAT(User_Name, DATALENGTH(User_Name)) as User_name, Partition, Game, Bet, Num_spins, Credits, Prize
            from ***
            where Partition>='2019-09-01' and Partition<'2019-11-17' and Bet>0 and credits is not null
            and User_Name IN (Select distinct userName from *** where GeoIpCountryCode='ES')
        ) as A
        where
        (Credits+Bet-Prize) > 100000 and num_spins>5
        group by User_Name, Partition;

Now, I got all the information I need. I run the last query, to group_by date these results so I can analyze them:
Select  datepart(week,Partition) as Week, count (distinct user_name) as Users
    from (
        Select user_name, partition, count(Game) as difMachines
        FROM
        (
            Select distinct CONCAT(User_Name, DATALENGTH(User_Name)) as User_name, Partition, Game, Bet, Num_spins, Credits, Prize
            from ***
            where Partition>='2019-09-01' and Partition<'2019-11-17' and Bet>0 and credits is not null
            and User_Name IN (Select distinct userName from *** where GeoIpCountryCode='ES')
        ) as A
        where
        (Credits+Bet-Prize) > 100000 and num_spins>5
        group by User_Name, Partition
    ) as B
    Where difMachines=1
    group by datepart(week,Partition)
    order by Week asc;

I know the query can be optimized, but that's not what troubles me. The problem is that when running this query, I obtain at week 36 17050 users. If I change this line (Credits+Bet-Prize) > 100000 and num_spins>5 for this one (Credits+Bet-Prize) > 100000 (so, I purely remove the num_spins>5 part), I get 16800 users instead.
To sum up, I get more results by being more restrictive in my query. That does not make sense to me. Someone please can help? Head me to the right direction or something?
Thank you

A: You are trying to get the count of result set with this filter diffmachine=1,isn't?. but if you remove the filter num_spins>5 then count will increase for diffmachine greater than 1.here i give an example like yours
Declare  @t table
(
    [user_name] varchar(5), [partition] date, Game varchar(10),num_spins int
)

insert into @t
select 'a','01nov19','g1',1
union all
select 'a','01nov19','g1',2
union all
select 'a','01nov19','g1',3
union all
select 'a','01nov19','g1',4
union all
select 'a','01nov19','g1',5
union all
select 'a','01nov19','g1',6
union all
select 'b','01nov19','g1',7

select * from
(
    select [user_name],[partition],count(game) cnt
    from  @t
    where num_spins>5
    group by [user_name],[partition]
)a
where cnt=1

redpajama_set_name: RedPajamaStackExchange


Basil, especially sweet basil or Genoa basil so popular in Italian dishes and used in pesto, is notoriously tricky to keep fresh. It turns black and wilts at an astonishingly quick rate. There are methods, however, of keeping the leaves fresh once the stems have been picked. The fresher the basil you buy, of course, the longer it will keep in any case.
Hand's down the best way to store basil in to put the bunch in a glass or jar filled with water, just like flowers.
Trim the cut ends, put the bunch in a glass, jar, or vase of water that will fit in the fridge, and cover the whole thing, or at least the basil leaves, with a plastic bag. Put the whole in in a refrigerator. Basil kept that way will stay vibrant and green for up to a week.
Is it a bit of a pain? Sure. Not super-duper convenient? Fair point. Takes up a lot of fridge space? No one could argue against argue that point. Does it also keep basil as fresh as possible for as long as possible? Absolutely it does.
We've established that the very best method isn't always as practical as one might like. Luckily, there is a second-best-but-still-completely-serviceable choice.
The second best way to store basil is to store it like salad greens: pick the leaves off the stems and lay them in more or less a single layer on layers of paper towels or a clean kitchen towel, roll up the towel around the leaves, and put the roll in a plastic bag. This method keeps the leaves hydrated but not wet or damp. For step-by-step photo details, see How to Store Greens.
Want to keep basil around for more than a few days? Freeze it! There are several ways to do it effectively.
For the greenest, freshest seeming basil: quickly blanch the basil in boiling salt water for 15 seconds, drain it, and dunk it into ice water to cool it quickly (this sets the green color; see How to Blanch for details), and then whirl it in a blender with just a bit of olive oil. You can pour the purée in ice cubes trays, freeze them, and transfer the cubes to re-sealable plastic bags where they keep for up to a year.
Admittedly, that method requires a touch of work. Alternatively, you can simply lay clean, dry basil leaves on a baking sheet, freeze them until frozen (about an hour will do it), and transfer them to resealable plastic bags. Basil frozen this way will turn dark, almost black, but retain plenty of basil flavor. To keep it green, you'll need to blanch it, squeeze it dry, then lay it flat to freeze. Once you bother blanching the basil, though, it's worth making the purée and freezing it that way, since it retains so much more flavor with that method.
See How to Freeze Basil for more details.


The first day with my Sony WH-1000XM2.
I have avoided over the ear or bulky headphones for years. I have relied on Etymotic, Shure, RHA and even Bose QC i20 to handle the noise while I travel. It's undeniable that my new XM2 are bulky, but they are also slick(in a geek kind of way) and premium. The way this headset feel in my hands put my concerns about the quality of the product to rest.
I had previously compared this headset and the Bose QC 35II and side by side, the latter feel lighter and at first glance more comfortable. For an fair comparison, one would need to do a scientific test. That aside, the XM2 do not feel uncomfortable after a few hours of use. I'm sure, I will easily get used to them after a couple of long trips.
The moment you pair the headset to your phone, it will suggest downloading the app.
Before testing the noise canceling capabilities of my XM2, I decided to test its ability to handle different generes. In my initial test, they handled everything from classical music to hip hop quite nicely. Call it the bliss of not being an audiophile if you wish, but to my humble ears the Sony WH-1000XM2 sound as good or better than anything else I have ever had. I paired the XM2 to my Apple TV and watched the Counterpart season finale. The show felt more immersive than when I watch it straight from the tv. I was concerned about latency, however, I did not notice any at all.
In a way, the great sound and additional controls are added bonus because noise canceling was the primary reason to get this product. Their many features over the competition are just added value.
On the next post, I will share my take on the Sony 1000XM2's wired performance and noise canceling.


text:
ViralityToday
Thousands Are Sticking Thin Blue Lines To Their Cars To Support Police Officers
Jan Jan
When we were kids, the police were the safe place. "If you ever feel
threatened," they said, "find a police officer, they'll help you."
Recently, we've seen headlines that highlight some of the "bad apples"
of policing. Every batch has one or two and lately, they have been
getting all of the attention. This is not a fair representation of the
men and women who keep peace and order, often at the expense of family
relationships or worse. The time has come to remember the good cops who
don't get enough recognition for the job they're doing.
When Inspiration Meets Action
April Killingsworth is a manager of a Tangent Signs, a printing company in Texas.
April is also an appreciative citizen who wanted to show her support for local law enforcement in her own special way.
She felt compelled to make a statement that police officers and members of the public would notice and appreciate.
This Dad Took A Selfie Instead Of Watching His Daughter's In...
17 Stars Who Have Siblings We Didn't Even Know About
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Copyright © 2021 ViralityToday.com | All Rights Reserved


text:
Source: GAIKINDO Indonesia International AUTO SHOW (GIIAS)
25th GAIKINDO Indonesia International Auto Show (GIIAS 2017) Officially Opens
BSD City, INDONESIA, Aug 14, 2017 - (ACN Newswire) - The GAIKINDO Indonesia International Auto Show (GIIAS 2017), Indonesia's premiere international automotive event, opened the doors to a 25th anniversary edition on Thursday, August 10. Endorsed by OICA (International Organization of Motor Vehicle Manufacturers) and organized by GAIKINDO (Association of Indonesian Automotive Industries), GIIAS 2017 runs from August 10 - 20 at the Indonesia Convention Exhibition (ICE) in BSD City, near Jakarta.
GIIAS 2017 is presenting the latest range of vehicles, products and technologies from the Indonesian, and international, automotive industries. With "Rise of the Future Mobility" as its theme, GIIAS 2017 embodies the spirit of GAIKINDO - that of building and growing the Indonesian automotive industry into the future.
Jusuf Kalla : The Government is Prepared to Face Global Challenges
Indonesian Vice President Jusuf Kalla, opening the 12th Gaikindo International Automotive Conference (GIAC) on August 11, said, "The Indonesian automotive industry is critical, as it has a direct and continuing impact on other industries; if the car industry in Indonesia continues to grow then there will be a few hundred thousand other industries that will also grow. In addition, it will create new employment opportunities for millions of people."
Kalla stressed that if the government was ready to improve and work on infrastructure and technology, then that goal would be achieved more easily. "The hope is that we can increase car exports. Efforts are in setting up an auto terminal at Tanjung Priuk, and later there will be a larger terminal, which is currently being built near Karawang. Furthermore, infrastructure must be prepared in advance so that automotive exports can be rapidly increased."
Indonesian Minister of Industry Airlangga Hartarto said, "Congratulations to the GIIAS exhibition, which has been going on now for 25 years. We hope this exhibition will have a positive impact in introducing new products and technology trends." The Minister also stressed hopes that GIIAS would provide the public with the latest information and technology developments happening within the Indonesian motor vehicle industry.
GIIAS 2017 Presents Three World Premieres
Yohanes Nangoi, the General Chairman of GAIKINDO, hopes that GIIAS becomes an event that builds and enlarges Indonesia's automotive industry into the future. "We hope that the GIIAS auto show will help to develop Indonesia's automotive industry, as it is an industry having a significant role in the Indonesian economy, and continue the spirit and initiatives of GAIKINDO in building and developing the Indonesian automotive industry."
GIIAS 2017 is supported by the 32 GAIKINDO-member brands, including 24 passenger vehicles brands - Audi, BMW, Chevrolet, Daihatsu, Datsun, Dodge, Honda, Hyundai, Isuzu, Jeep, KIA, Lexus, Mazda, Mercedes-Benz, MINI, Mitsubishi Motors, Nissan, Renault, Suzuki, Tata Motors, Toyota, Volvo, VW and Wuling; as well as 8 commercial vehicle brands - DFSK, FAW, Hino, Hyundai Bus and Truck, Isuzu, Mitsubishi FUSO, Tata Motors, and UD Truck. Support also comes from more than 300 automotive industry players in Indonesia.
"This year we have invited the exhibitors, including APMs, to present and exhibit new products and new technologies so that the public in Indonesia, and industry players worldwide, have a better understanding of the rapid progress in the automotive industry both in Indonesia and in other developing countries," said Nangoi. GIIAS 2017 includes more than 40 new vehicle launches, including world premieres, Asian premieres, Indonesian premieres and concept car launches.
Three APMs chose Indonesia and GIIAS 2017 for the world premieres of new vehicles or concept vehicles. From the passenger vehicle segment, Mitsubishi launched its small MPV flagship Mitsubishi Xpander in a world premiere, while Daihatsu helped maximize its participation in GIIAS 2017 by exhibiting two concept vehicles for the first time in the world, namely the DN F-Sedan and DN Multisix.
UD Trucks became the first commercial vehicle participant to present a world premiere, the Kuzer Truck, its latest product in the light duty segment. UD Truck explained that it chose to launch light truck products in Indonesia because the commercial vehicle market had such great potential, and the growing need was the reason for choosing GIIAS 2017 as the location for launching the Kuzer Truck.
GAIKINDO Chairman Yohanes Nangoi, further said, "We hope that exhibitors who are also members of GAIKINDO continue to play an active role in developing the Indonesian automotive industry. One way they can do this is by presenting more and more world premieres in Indonesia. This effort will certainly help advance the Indonesian automotive industry, which has a strategic role, with a double impact in contributing to the national economy."
Seven Events
Diah Putri
E: [email protected]
W: www.indonesiaautoshow.com
Sectors: Automotive
GIIAS 2017, Indonesia's Premier International Automotive Event, Concludes
August 21 2017 22:00 JST
GAIKINDO Indonesia International Auto Show (GIIAS 2017) Hosts Three World Premieres
Forty New Vehicles Launching at the GAIKINDO Indonesia International Auto Show (GIIAS) 2017
July 27 2017 15:30 JST
GAIKINDO Indonesia International Auto Show (GIIAS 2017) Presents a World Class Auto Show with Latest Products and Technology
June 19 2017 17:00 JST
Road to GAIKINDO Indonesia International Auto Show (GIIAS 2017)
March 27 2017 22:00 JST
GAIKINDO Indonesia Auto Show (GIIAS 2016) a Window on Growth of Indonesian Automotive Industry
GAIKINDO Indonesia Auto Show (GIIAS 2016) Enlivened by New & Concept Automobiles and Commercial Vehicles
24th GAIKINDO Indonesia International AUTO SHOW (GIIAS) Opens Amid Renewed Optimism in Local Market


Indian brands must now navigate this awkward transitionary period of soggy paper straws, and rapidly adapt to a new, eco-friendly, reality, or risk being left behind. If you are here, reading this, then you are already poised to take a step in the right direction. Embracing eco-friendly packaging will be a critical step for many Indian brands, but what does eco-friendly packaging actually mean?
Here are the 3 key ways in which brands are changing how they package their products to be more environmentally friendly.
The first, most straightforward step that any brand can take is to minimize the unnecessary use of plastic in their packaging. The fact that many products are over-packaged makes this an easy port-of-call. According to reports, PET bottles are already 30% lighter than they were 15 years ago. Proctor & Gamble has been working behind the scenes to reduce the amount of plastic in the packaging for many of their products over the last few years. Gillette Fusion ProGlide redesigned its pack to use 57% less plastic.
Through structural and design innovation, brands can reduce the impact of their packaging. In the long run, this also has the added benefit of lowered material costs. However, this offers only an improvement, not a truly sustainable solution, as new plastic is still being introduced to the ecosystem.
Recycling of materials is perhaps the practice most associated with sustainability, as it is a consumer-facing process. We have seen a number of brands make bold strides in this, blending advanced recycling technologies and material collection schemes. Evian announced plans earlier this year to make their bottles out of 100% recycled plastic, using new technology to 'depolymerize' waste plastic and turn it into the same base materials used in making virgin plastic.
Coffee giant, Costa Coffee, has pledged to recycle an equivalent number of cups to the ones it sells (including rival cups) by 2020, in a 'cup cycling revolution' scheme that financially incentivises collection of the cups. Nespresso encourages consumers to return their coffee pods for recycling by providing them with labelled return envelopes.
Method launched a special range of eco-friendly packaging made from ocean plastic. By teaming up with volunteers to collect plastic from beaches of Hawaii, and tagging the recycled bottles, the brand includes the consumer in the process and story.
Recycling is a great approach to eco-friendly packaging which also has a positive rub-off effect on the brand in the eyes of increasingly conscious consumers. The key to achieving success in recycling is to seek out and put in place the right processes for the collection and processing of materials.
Anyone who has grown up in India has likely encountered returnable glass bottles for soft drinks. According to Coca-Cola, 50% of their sales take place through these returnable glass bottles, of which all are recovered (minus breakages). This is a classic example of reusability, making this truly eco-friendly packaging.
Biodegradable materials offer a different avenue for sustainable packaging innovation. In the news, PepsiCo has declared it will launch the first-ever 100% compostable, plant-based packaging for Lay's and Kurkure snacks products this year, with India as one of the first countries to pilot the new sustainable option.
The products themselves can be reimagined to achieve more eco-friendly packaging. P&G developed a 2.5x concentrated version of their product, Mr. Clean Liquid Muscle, leading to a smaller package that used 45% less plastic and 64% less water.
Solid bars of shampoo is a reality found in the perfumed halls of Lush. With products formulated to live packaging-free, Lush's fresh and handmade cosmetics take a step towards eco-friendly packaging by eliminating the packaging altogether. With the option of reusable tins, they offer a far more attractive alternative for conscious consumers.
While the task of moving towards eco-friendly packaging may seem daunting, there is much we can learn from the bold steps that brands are already taking towards a more sustainable and conscious future of consumption. With a plethora of new materials, processes and technology available to us, the question that any designer, brand custodian, or decision maker must now ask themselves is 'what opportunity do I have to make a change and steer our collective consumption in a better, more sustainable direction?


text:
package com.amazonaws.services.securitytoken.model.transform;

import javax.xml.stream.events.XMLEvent;
import javax.annotation.Generated;

import com.amazonaws.services.securitytoken.model.*;
import com.amazonaws.transform.Unmarshaller;

import com.amazonaws.transform.StaxUnmarshallerContext;
import com.amazonaws.transform.SimpleTypeStaxUnmarshallers.*;

/**
 * AssumeRoleResult StAX Unmarshaller
 */

@Generated("com.amazonaws:aws-java-sdk-code-generator")
public class AssumeRoleResultStaxUnmarshaller implements Unmarshaller<AssumeRoleResult, StaxUnmarshallerContext> {

    public AssumeRoleResult unmarshall(StaxUnmarshallerContext context) throws Exception {
        AssumeRoleResult assumeRoleResult = new AssumeRoleResult();
        int originalDepth = context.getCurrentDepth();
        int targetDepth = originalDepth + 1;

        if (context.isStartOfDocument())
            targetDepth += 2;

        while (true) {
            XMLEvent xmlEvent = context.nextEvent();
            if (xmlEvent.isEndDocument())
                return assumeRoleResult;

            if (xmlEvent.isAttribute() || xmlEvent.isStartElement()) {

                if (context.testExpression("Credentials", targetDepth)) {
                    assumeRoleResult.setCredentials(CredentialsStaxUnmarshaller.getInstance().unmarshall(context));
                    continue;
                }

                if (context.testExpression("AssumedRoleUser", targetDepth)) {
                    assumeRoleResult.setAssumedRoleUser(AssumedRoleUserStaxUnmarshaller.getInstance().unmarshall(context));
                    continue;
                }

                if (context.testExpression("PackedPolicySize", targetDepth)) {
                    assumeRoleResult.setPackedPolicySize(IntegerStaxUnmarshaller.getInstance().unmarshall(context));
                    continue;
                }

                if (context.testExpression("SourceIdentity", targetDepth)) {
                    assumeRoleResult.setSourceIdentity(StringStaxUnmarshaller.getInstance().unmarshall(context));
                    continue;
                }
            } else if (xmlEvent.isEndElement()) {
                if (context.getCurrentDepth() < originalDepth) {
                    return assumeRoleResult;
                }
            }
        }
    }

    private static AssumeRoleResultStaxUnmarshaller instance;

    public static AssumeRoleResultStaxUnmarshaller getInstance() {
        if (instance == null)
            instance = new AssumeRoleResultStaxUnmarshaller();
        return instance;
    }
}

redpajama_set_name: RedPajamaGithub


The vehicle is substantially designed or equipped, or materially altered from its original construction, for the purpose of agricultural use.
The vehicle was designed and manufactured primarily for highway use.
Unless the vehicle was manufactured prior to 1970, the vehicle was manufactured to meet federal motor vehicle safety standard certification label requirements as specified in 49 CFR 567.
The vehicle is used exclusively in the conduct of agricultural operations.
A self-certification process for Ag CMVs was also created. The Ag CMV Self-Certification is not required. It is provided as a convenient reference for operators and law enforcement.
Pesticide sprayers, lime and fertilizer spreaders (not including manure) with extending tires, fenders and fender flares may not exceed 12 feet in width.
These width limits are not applicable on interstate highways. The width limit for Ag CMVs on interstate highways is 8 feet, 6 inches. An Ag CMV over 8.5 feet wide requires an oversize permit to operate on interstate highways.
Three vehicle combination - two towed units drawn by a power unit.
IoH/Ag CMV Maximum Weight Limits table applies: A maximum single axle weight of 23,000 pounds and a gross vehicle weight of 92,000 pounds dependent on number of axles and axle spacing.
These higher weight limits are not allowed on interstate highways. (s. 348.15(9)(c)2.(d), Wis. Stat.) However, I-41 travel is exempted from this provision.
Exempt from axle weight limits on local roads unless local government authority passes ordinance or resolution limiting Ag CMV axle weight.
A No Fee Permit may be required if gross vehicle weight or axle weight is greater than the IoH/Ag CMV weight table.
Ag CMVs traveling for delivery, service, repair by dealer or farmer within 75-mile radius of business or farm are exempt from gross vehicle weight and axle weight limits. Exemptions also pertain to Class B highways.
Operators are allowed to transport Ag CMVs on trailers or semi-trailers to and from farm-related destinations under the same weight limits that are allowable for the Ag CMV being operated on the highway. If the 2-vehicle combination transporting an Ag CMV exceeds the Ag CMV axle weight or gross weight limits, the operator may apply for a no-fee permit.
The weight exemption described above for Ag CMV - Category 1 also applies if it is transported by a 2 vehicle combination via trailer or semi-trailer to or from a farm-related destination.
Ag CMVs up to 12 feet wide are allowed to be trailered without a permit, except on interstate highways. On interstate highways, an oversize permit is required to trailer Ag CMVs over 8 feet, 6 inches wide. Review the safety webpage for lighting and marking requirements for trailered vehicles.
To trailer an Ag CMV that exceeds the above dimensions on a local road, contact the local maintaining authority for information.


text:
Kukoak (Quqoā'q)), jedno od nekoliko lokalnih skupina Songish Indijanaca, porodica Salishan, koji su živjeli uz još nekoliko grupa (Chikauach i Chkungen) na McNeill Bayu na južnoj obali kanadskog otoka Vancouver u provoncija Britanska Kolumbija,.

Izvori

Songish
Indijanci, Britanska Kolumbija
Indijanska plemena

redpajama_set_name: RedPajamaWikipedia


Martin was settled by Henry C. Black, the agent for the Atlanta & Charlotte Air Line Railroad when the station was established in 1877. It was incorporated in 1891 and named for early governor John Martin, a native of Rhode Island who was elected in 1782.
Your Extensive Photos of Georgia are extraordinary. I was in charge of most of the restoration of The Ritz Theater In Toccoa. It was named The Schaefer Center as contract requirement when the Schaefer family sold the Theater to the Arts Council at a significantly reduced price. Dr. Schaefer was a prominent surgeon who built the Ritz.The building was closed when purchased. Restoration funds were raised from community leaders. Several years later governmental entities and A Fox Foundation donation provided funds to install the present front and marquee.
Thanks, Alton. I hope to return to Toccoa this year and get more photographs. I love Stephens County.


Please note due to the high volume of e-mails it might take us up to seven days to respond to your e-mail and all e-mails are handled by Nina's office staff, no e-mails go through directly to Nina.
Alle deutschsprachigen Anfragen werden auf deutsch bearbeitet! Bitte haben Sie Verständnis ,dass keine emails direkt an Nina gehen.Unser Team bearbeitet alle Anfragen. Bitte haben Sie etwas Geduld bei der Terminvergabe/ Warteliste.
Enter comments here!Media request also!
Nina Andeson is not a medical doctor or associated with any branch of medicine. Nina works in Integrative Medicine. She offers her opinions based on her intuition, and her personal energy healing work, which is not a substitute for medical procedures or treatments. Always consult a physician or trained health care professional concerning any medical problem or condition before undertaking any diet, health related or lifestyle change programs. As in traditional medicine, there are no guarantees with medical intuition, intuitive anatomical medical diagnosis or energy medicine.
" Nina is the best naturally medicin "
Nina is a magnificent and gifted healer, and the best clairvoyant,there is no doubt about it!"
Content copyright ANDESONSUCCESS.COM. All rights reserved.


Distance from Logan International Airport to John Glenn Columbus International Airport is 640 miles / 1030 kilometers / 556 nautical miles.
Estimated flight time from Logan International Airport (BOS) to John Glenn Columbus International Airport (CMH) is 1 hour 42 minutes.
There is no time difference between Boston and Columbus.
Flight map from Boston to Columbus.
Boston (BOS) → Columbus (CMH).