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Captain Alastair Cook described the moment he became England's leading Test run-scorer as "special" and "humbling".
Alastair Cook's up-and-down England career makes his record-breaking feat all the more special, says Nasser Hussain.
Alastair Cook became England's leading Test run scorer and Adam Lyth posted his first international ton on a gripping day two at Headingley.
Pakistan will play their final one-day international against Zimbabwe despite a suicide bomb attack close to the game's venue.
Hashim Amla played a starring role as the Derbyshire Falcons stumbled to a five-wicket win over the Lancashire Lightning.
Luke Ronchi was delighted with his first day as a Test cricketer despite narrowly missing out on a century at Headingley.
James Anderson says it feels "a bit surreal" to have become the first England bowler to reach 400 Test wickets.
Azhar Ali played a captain's innings to lead Pakistan to a series-winning six-wicket victory over Zimbabwe in Lahore.
After watching James Anderson claim his 400th Test wicket, Sir Ian Botham has backed the England paceman to reach 500.
James Anderson became the first Englishman to secure 400 Test wickets before New Zealand closed day one of the second Test on 297-8.
Vernon Philander will leave Nottinghamshire earlier than anticipated after being recalled by Cricket South Africa.
We look at James Anderson's career highlights after he took his 400th Test-match wicket at Headingley.
Over-by-over commentary on day two of the second Test at Headingley - watch live on Sky Sports 2 HD.
Over-by-over commentary from the second Test at Headingley
England's limited-overs skipper Eoin Morgan has welcomed the appointment of Trevor Bayliss as the new national coach.
Kevin O'Brien and Grant Elliott produced a batting masterclass as Leicestershire eased to an eight-wicket win over Durham at Grace Road.
England captain Alastair Cook is fully over his batting ills as he closes on Graham Gooch's record, says Nasser Hussain.
Two of England's key players could set landmarks in the second Test between England and New Zealand at Headingley.
New Zealand captain Brendon McCullum believes England may not have seen the best of his side.
Sir Ian Botham thinks new England coach Trevor Bayliss and assistant Paul Farbrace can make a successful team.


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Rapid enzyme-linked immunosorbent assay for detection of the algal toxin domoic acid.
KEY WORDS: ASP, domoic acid poisoning, ELISA, mussels, scallops, razor clams, test kit
Domoic acid (DA) is a potent toxin produced by bloom-forming phytoplankton in the genus Pseudo-nitzschia (Fig. 1). It is a glutamate analog, which acts as a potent excitatory neurotransmitter and causes amnesic shellfish poisoning (ASP) in humans (Quilliam & Wright 1989, Quilliam et al. 1989b, Wright et al. 1989). Symptoms include vomiting, diarrhea, and in more severe cases confusion, loss of memory, disorientation, and even death. As a tricarboxylic acid, fully ionized at seawater pH, DA can behave as a potent trace metal ligand (Rue & Bruland 2001, Wells et al. 2005). DA can bioaccumulate and rapidly transvectors throughout the food chain via clams, mussels, crabs, filter feeding fish, and other organisms (Homer & Postel 1993, Scallet et al. 2005, Vigilant & Silver 2007). DA poisoning was first recognized after a lethal event on Prince Edward Island, Canada in 1987 (Wright et al. 1989). Since that time, a number of toxic events have occurred on the United States west coast where DA has been shown to commonly accumulate in the edible parts of razor clams (Siliqua patula), mussels (Mytilus californianus or edulis), and Dungeness crabs (Cancer magister) (Wekell et al. 1994, Horner et al. 1997). High levels of DA in razor clams in Oregon and Washington are responsible for beach closures that can last for more than a year. Losses of more than $20 million annually result from these closures caused by lost tourism and reduced recreational and commercial and tribal clam harvests (Adams et al. 2000). DA has also been implicated in the death and illness of brown pelicans (Pelecanus occidentalis) and Brandt's cormorants (Phalacrocorax penicillatus) (Fritz et al. 1992, Work et al. 1993), California sea lions (Zalophus californianus) (Scholin et al. 2000, Trainer et al. 2000, Brodie et al. 2006), sea otters (Enhydra lutris) (Kreuder et al. 2003), and possibly whales (Lefebvre et al. 2002).
The regulatory method for DA detection sanctioned by the Interstate Shellfish Sanitation Conference (Quilliam et al. 1989a, Quilliam et al. 1995) is a high performance liquid chromatography (HPLC) assay (Quilliam et al. 1991, Hatfield et al. 1994). Though accurate, these analyses are generally run by centralized state facilities with results typically not available for 3-14 days after the samples are collected. In more remote communities, many of which depend heavily on subsistence clam harvests, these long delays and the costs of sample analysis are causes for public health concern. The average cost of approximately $100 per sample limits the number of samples that can be analyzed (Harold Rourk, WA State Department of Health, pers. comm.). Resource managers in coastal communities have expressed their desire for a cost-effective method for rapid and accurate determination of DA concentrations in shellfish and phytoptankton samples. This paper describes the development and optimization of a robust monoclonal antibody based enzyme-linked immunosorbent assay (ELISA) test kit for DA that will meet management needs for rapid detection of DA in environmental samples.
Assay Kit Overview
The DA assay kit was developed jointly by NOAA's National Centers for Coastal Ocean Science, Nationa| Ocean Service, and the Northwest Fisheries Science Center, together with ah industry partner Mercury Science, Inc., Durham, NC (NOAA/MSI). It was designed as a sequential competitive enzyme linked immunosorbent assay (ELISA) utilizing a high avidity monoclonal antibody (mAb) to DA to ensure assay specificity and consistency across production lots. In the current format, a fixed number of anti-DA mAb binding sites are incubated with dissolved DA in the sample followed by the addition of a DA--horseradish peroxidase (HRP) conjugate. As these binding events occur, the anti-DA mAb molecules are simultaneously captured by antimouse antibodies affixed to the surface of the microtiter plate wells. Subsequent HRP derived color development, readable on standard microplate readers, was inversely proportional to the concentration of DA in the sample matrix. The assay reagents were titrated so that the amount of mAb and the DA-HRP conjugate added produced a maximal absorbance signal of 3 absorbance units when no DA was present. The implementation of this ELISA system required the development and validation of two essential reagents, a high avidity monoclonal antibody to DA and a stable DA-HRP conjugate recognized by the same mAb.
Production of the Anti-Domoic Acid Antibody
Domoic acid (Sigma-Aldrich, St. Louis, MO), was conjugated with bovine serum albumin (BSA) using dicyclohexyl carbodiimide and N-hydroxysuccinimide by a two-step synthetic pathway (Adamczyk et al. 1994). Ten mice were immunized with the DA-BSA immunogen. Serum titers were determined five days after each boost. A fusion was performed on the three mice that showed the greatest response. Hybridoma cell lines and monoclonal antibody production was performed according to the method of Fenderson et al. (1984). The 10 clones with highest affinity mAbs were selected for further growth and their affinity to DA was compared. The most sensitive clone was ultimately selected as the primary mAb for use in the assay development.
DA-HRP Conjugate
Domoic acid (Sigma) was cross-linked to horseradish peroxidase (HRP) using the procedure of Yoon et al. (1993). The reagent was tested for stability and was used to screen for high affinity mAbs after the fusion and for assay development.
Domoic Acid Standards
The DA standards used to calibrate the assay were purchased from the Certified Reference Materials Program at the National Research Council of Canada Institute for Marine Biosciences (Halifax, Nova Scotia, Canada).
Assay Calibration
A series of dose response curves using varying amounts of antibody and DA-HRP were performed to optimize the assay sensitivity. The optimal assay conditions were found to have an effective linear range from approximately 0.1-3.0 ppb. These conditions were used in all the subsequent phases of assay development. The antibody was also tested for cross-reactivity with varying concentrations of kainic acid, glutamine and glutamic acid. These compounds are structurally similar to various portions of DA molecule and have the potential to cross-react with anti-DA mAbs. Glutamine and glutamic acid, in particular, are common in animal tissues, including shellfish.
Calculation of the Parameters Needed to Construct an Internal Domoic Acid Standard for Each Well Strip
Using the optimized DA assay, multiple dose response curves were made using the NRC standards diluted to between 0 and 10 ppb (1-10 ng [mL.sup.-1]) in the assay reaction buffer. The average response derived from each of the individual response curves was calculated anda dose response curve was generated using a four parameter logit-log curve fitting analysis (Ritchie et al. 1981; Fig. 2). Four parameters were derived from this analysis. This first was [B.sub.o], the maximal signal, which occurred when no sample DA was present (Fig. 3A). The second was B, the signal produced by a known amount of sample DA. The third was the slope of the logistic transformed data [proportional to the linear portion of the sigmoidal curve describing the relationship between the In sample DA concentration versus signal (B)]. And the fourth was [ED.sub.50], the DA concentration at the mid point of the slope curve where half the available anti-DA mAbs in the well are bound to DA-HRP (Fig. 3A). Because the concentration ratio of anti-DA antibody and DA-HRP conjugates are standardized within reagent lots, the kinetics of the reaction were fixed between assay runs (assuming constant temperature), such that the slope and [ED.sub.50] values remain constant. This made it possible to calculate DA concentrations using the four parameter model.
DA concentration = [ED.sub.50][[([B.sub.o]/B) - 1].sup.-slope]
Because the slope and [ED.sub.50] are constants, all that was needed to calculate the DA concentrations was an accurate [B.sub.o] and the B estimates from individual samples. In the assay, the mean value for [B.sub.o] for each strip of wells was determined by adding sample dilution buffer lacking DA to the first two wells in that strip. Duplicate aliquots from each of three extracted samples diluted with sample buffer were then added to the six remaining wells to obtain the B values. Duplicates were run to ensure assay replicability. It should be noted that [B.sub.o] (the maximal value with no DA added) can have noticeable variation between assays depending on differences in temperature and development time as shown in Figure 2A. However, when the B values for each strip are divided by [B.sub.o], the kinetics of the curve become normalized (i.e., replicable between strips and between runs) (see Fig. 2B). In this way the average [B.sub.o] values serves as an internal standard that can be used in place of a standard curve provided the variation in the [B.sub.o] is not above or below certain limits, which are specified in the calculation software described later.
Domoic Acid ELISA Test Kit Procedure
The 96-well assay tray used in the assay contained 12 strips. Each strip of 8 wells could be removed and stored until it was needed. The first two wells of each strip were used as a control (no DA added). The remaining six wells were used to analyze three samples in duplicate. This format provided the flexibility of running anywhere from 3-36 duplicate samples at a time. For unknown sample analysis, extracts were diluted to a final concentration ranging from 0.3-3 to ppb using the sample buffer (phosphate salt solution, pH 7.8, containing casein). For clam tissues containing DA, sample dilutions of 1:50 and 1:1000 were typically used. Preliminary tests with razor clam extracts showed that a 25-fold dilution in sample dilution buffer eliminated matrix effects in ELISA analysis.
The assay was initiated by adding 50 [micro]L of the anti-DA antibody to each well using a multi channel pipettor. Next, 50 [micro]L of the control solution (sample buffer without DA) was added to the first two wells in each row. Duplicate 50 [micro]L aliquots from the diluted DA extracts were then added to the remaining wells in each strip and the plate incubated at room temperature for 30 min on an orbital shaker set to vigorously mix the solution in each well (PerkinElmer Waltham, MA 1296-004 DELFIA Plateshake set on high). Vigorous mixing is key to obtaining replicable results from one run to the next. In this step, the bulk of the native DA will bind to available mAbs in proportion to the DA concentration. At the end of the incubation, 50 [micro]L of DA HRP conjugate was added to each well and the plate incubated a second time for 30 min at room temperature on an orbital shaker. The DA-HRP will then bind to remaining available mAb sites. After the incubation, the plate was washed three times with wash solution [Tris-HCl buffered salt solution (pH 7.8) containing Tween 20 and sodium azide as a preservative] using a commercial plate washer, making certain the fluid was completely aspirated from all the wells. Alternatively, these washes can be done manually by adding wash solution to wells using a multichannel pipettor and then flicking all fluid from the wells. The manual method may result in slightly higher variability. Next, 100 [micro]L of K-Blue TMB substrate (5.5'-tetramethylbenzidine, Neogen Corporation, Lexington, KY) was added to each well. The plate was placed on an orbital shaker for no more than 5 min, or until adequate color development was observed. Color development was terminated by adding 100 [micro]L stop solution (1N hydrochloric acid) to each well. The absorbance in each well was measured at 450 nm using a Thermo Ascent MultiSkan plate reader (Thermo Scientific, Waltham, MA). The DA concentrations were determined using the sample (B) and control ([B.sub.o]) absorbances, the original tissue weights, and the volume of 20% or 50% methanol used to extract each sample. The actual calculations were made using a Microsoft Excel work sheet (Microsoft Corporation, Redmond, WA), which incorporates the constants for the four parameter model described above. This worksheet can be downloaded from Stewart (2008). Processing time for this assay was ~1.5 h.
Routine Tissue Extraction
In the case of razor clams and scallops, pooled samples of 10-12 individual shellfish were cleaned, and ground to a smooth and uniform homogenate in a commercial blender (Waring model HGBSS56, Torrington, CT). Clams were pooled because previous studies of DA in razor clams from the Washington coast indicated that the coefficient of variation for DA between clams in a population exceeded 100% (Wekell et al. 2002). If the homogenate appeared to be forming a gel caused by unusually high lipid content, an equal weight of water was added and the dilution noted. Approximately 2 g of homogenized tissue were added to a tared 50 mL conical tube and the weight recorded to the nearest 0.01 g. Next, 18 mL of 50% methanol were added and the samples mixed at high speed on a vortex mixer for 2 min. Once the extraction was completed the tubes were spun in a table top centrifuge for 20 min at 10,000 x g or until a tight pellet and clear supernatant were obtained. If the samples did not clear despite the spinning at high speed, the supernatant was poured into a syringe, then passed through a 0.45 [micro]m Millex HA syringe filter (Millipore, Billerica, MA) to remove proteins and other compounds that can form micelles, whereas soluble DA remained in the filtrate. At this point the homogenate was ready for analysis by ELISA and HPLC. If necessary, the sample was stored at 4[degrees]C for up to 24 h in an explosion proof refrigerator prior to analysis.
Phytoplankton Extraction
Approximately 0.1-1.0 L of cultured cells or sea water samples were filtered onto a GF/F filter, which was immediately frozen at -80[degrees]C until the filter could be processed. For processing, the filter was placed in a 5 mL conical BD Falcon Tube (Becton Dickinson, Franklin Lakes, NJ) and 3 mL of 20% methanol were added. The samples were then sonicated using a Thermo Fisher Scientific Model 100 Sonic Dismembrator with a 1/8 inch probe (model 15-338-80, Fisher Scientific, Waltham, MA) until the filter was completely homogenized. Care was taken to prevent the probe from rupturing the tube. The sonicator probe was cleaned very carefully with 20% methanol between samples to prevent cross-contamination. Next the homogenate was centrifuged at 3000 g for 10 min. The supernatant was then passed through a disposable Whatman GD/X 0.2 [micro]m syringe filter (Florham Park, NJ) into a 5 mL tube. At this point the sample was split for analysis using both the ELISA and HPLC assays.
HPLC Validation of DA concentration from Razor Clam Tissues
HPLC is the accepted standard method for measuring DA and is the basis of the current official method for regulatory action in the U.S. (AOAC Official Method 991.26). The lower detection level for the standard assay is ~0.5 ppm. This technique was used to validate the DA concentration in the razor clams in this study. Briefly, 10-15 mL of the clarified supernatant prepared as described above was transferred into a 25 mL disposable plastic syringe and filtered through 0.45 micron HA Millipore filter (Bedford, MA) into a labeled scintillation vial. Salt clean-up was done with solid phase extraction columns (Hatfield et al. 1994). Strong anion exchange (SAX) solid phase extraction (SPE) cartridges (Whatman, Florham Park, NJ) were conditioned by washing successively with 6 mL of methanol, 6 mL of deionized water, and 6 mL of 50% methanol. The SPE clean up also removes tryptophan, which is a major source of false positives in HPLC-UV detection of DA because it coclutes with DA. Each sample was then drawn through a conditioned SAX SPE cartridge at a rate of 1 drop per second using a vacuum manifold. Flow was stopped when the meniscus was just above the top of the column. The columns were washed with 5 mL of 0.1 M NaCl in 10% aqueous acetonitrile (10% acetonitrile: 90% deionized water). The columns were immediately moved to a new row in the vacuum manifold and the DA eluted from the SPE cartridge using 5 mL of 0.5 M NaCl in aqueous 10% acetonitrile (10:90, acetonitrile:deionized water) and collected in 5 mL graduated centrifuge tubes. Flow was stopped when eluant reached 4.9 mL in the graduated centrifuge tube. The graduated centrifuge tube was removed from the manifold and the actual volume recorded. The graduated centrifuge tubes were capped and the eluant immediately mixed by shaking the tube vigorously 5-10 rimes. Tissues from the other invertebrate species examined (Table 1) were processed similarly, except that the extracts were filtered through Nanospec MF GHP 0.45 pm centrifugal filters (Pall, Ann Arbor, MI) instead of SPE columns before HPLC analysis. Eluted samples were transferred to HPLC analysis vials. The HPLC conditions were as follows: Vydac TP210 column (Grace, Deerfield, IL), 2.1 by 250 mm, 40[degrees]C, elution of DA in 10% acetonitrile containing 0.1% trifluoroacetic acid (TFA). Twenty [micro]l of each sample were injected into the column and eluted isocratically at 0.3 mL per min. The retention time for the DA peak was about 6-8 min depending on the column. Canadian NRC DACS standards at concentration of 1 ppm in 10% acetonitrile solution were run simultaneously (Hardstaff et al. 1990).
HPLC Detection of Domoic Acid in Phytoplankton Using Fluorenylmethoxycarbonyl (FMOC) Derivatization
A more sensitive fluorescent fluorenylmethoxycarbonyl chloride (FMOC) derivatization method (Pocklington et al. 1990) was used to determine particulate DA concentrations in phytoplankton samples, which typically contained less DA than shellfish tissues. The samples were processed on a Hewlett-Packard 1090 HPLC using a Vydac 201TP, 5 [micro]m, 25 cm column, HP 1046A fluorescence detector, and column heater set to 40[degrees]C with the following modification. In our analysis, solvents A (HPLC Water with 0.1% v/v TFA) and B (acetonitrile with 0.1% v/v TFA) were pumped at 0.2 mL/min and the linear gradient elution was changed allowing for increased separation and resolution of the domoic acid peak. The initial gradient went from 70% A and 30% B at time of injection to 60% A and 40% B over 0-10 min, then held constant for 10 min; adjusted to 0% A and 100% B from 20-30 min, held isocratic for 2 min; adjusted from 0% A and 100% B to 70% A and 30% B over 2 min, and then held constant at these (initial) conditions until the end of the run at 45 min. Dihydrokainic acid was used as an internal standard, as described by Pocklington et al. (1990).
A subset of phytoplankton samples was validated to confirm the presence of DA (by mass) using liquid chromatography-mass spectrometry (LC-MS) on a ThermoFinnigan Quantum Discovery Max TSQ ESI Mass Spectrometer coupled to a HP 1100 series binary pump HPLC, following the general protocol of Quilliam et al. (1989a). Samples for LC-MS were prepared as for HPLC, but were then dried down under vacuum and redissolved in 100% methanol prior to injection. The HPLC conditions for the reverse phase were programmed for a linear gradient elution of 10:90% acetonitrile:deionized water (both containing 0.1% formic acid) up to 0:100% water:acetonitrile over 30 min.
Testing Cross-Reactivity of the ELISA Against Glutamine, Kainic Acid and Putative Isomers Epi-DA and Iso-DA
Domoic acid is structurally similar to glutamine, glutamic acid and kainic acid, all of which can potentially co-occur with DA in sample extracts (Fig. 1). To test for potential cross-reactivity with these compounds, the NOAA/MSI ELISA kit was run using concentrations of glutamine, glutamic acid and kainic acid ranging from 10 ppb to 5 ppm. The [ED.sub.50] for each compound was calculated and then divided by [ED.sub.50] for DA and multiplied by 100 to determine percent cross-reactivity (Table 2). A majority of DA in razor clams and phytoplankton is in the form shown at the top of Figure 1. However, samples sometimes contain a larger quantity of compounds closely eluting with DA on standard HPLC runs that have been identified as the DA conformers epi- and iso-DA (Wright et al. 1990, Kotaki et al. 2005). To determine if the mAb used in this assay could detect these DA isomers, and the extent of interference by such coeluting compounds present in crude extracts of intertidal barnacle, limpet, and snail samples, crude methanolic extracts of these tissues were assays using HPLC-UV and both the NOAA/MSI and Biosense (Biosense Laboratories, Bergen, Norway) ELISA methods. These intertidal invertebrate extracts exhibited high levels of the putative epi-DA and iso-DA isomers as called by comigration on HPLC chromatograms. These compounds are generally near detection limits in razor clams, crabs, and to a lesser extent in mussels, and therefore these extracts provided novel matrices for evaluating the accuracy of NOAA/MSI ELISA.
Data Analyses
Analytical results for DA concentrations determined from razor clams, mussels, scallops and phytoplankton cells determined by HPLC, FMOC-HPLC, LC-MS and the NOAA/MSI ELISA were compared using linear regression analysis (Sokal & Rohlf 1995). The performance of the NOAA/MSI and Biosense ELISA kits was also compared using a subset of the phytoplankton samples. This comparison involved simultaneously analyzing phytoplankton extracts using the two kits and comparing the results with those obtained using FMOC-HPLC. All samples were run within a 24 h period to prevent differential degradation of DA, which may occur in some samples. Data were compared using linear regression analysis.
The NOAA/MSI ELISA accurately measured NRC standard DA concentrations (Fig. 4) and gave equivalent results for razor clam (Fig. 5), mussel (Fig. 6), scallop (Fig. 7), and phytoplankton extracts (Fig. 8) as obtained when using HPLC, FMOC-HPLC, or LC-MS methods. When the variability in the NOAA/MSI ELISA and FMOC-HPLC method were compared using replicate phytoplankton extracts they were found to be comparable (Fig. 9). The primary advantage of the NOAA/ MSI ELISA over HPLC methods, besides a significantly lower cost per sample was much higher throughput. As many as 36 samples can be completed in <1.5 h after tissue extraction.
The NOAA/MSI formar was also flexible. An internal control was incorporated into each strip, which eliminated the necessity of running a standard curve each rime the assay was performed. Any unused strips could be removed and stored in a desiccator pouch at 4[degrees]C for at least six months without compromising assay performance. This allowed as few as 3 samples to be run in real time thereby avoiding the degradation of DA that can occur during storage, particularly once the samples have been extracted (Smith et al. 2006). For example, when phytoplankton samples were run within 24 h using the Biosense ELISA kit, which has been validated by an international collaborative study, and is officially approved by the AOAC International for regulatory detection of DA in shellfish, and the NOAA/MSI ELISA kit, equivalent results were obtained (Fig. 10, [r.sup.2] = 0.97). In contrast, when samples were run two weeks apart the correlation dropped to [r.sup.2] = 0.79, indicating DA degradation.
The ability to efficiently run a small number of samples in real time was not incorporated into other DA ELISA formats. For example, the Biosense DA ELISA kit includes reagents for only two standard curves (product insert), therefore, only two batches of samples can be run per kit. This means that when small numbers of samples are being collected, they may have to be stored until a sufficient number of samples have been accumulated to maximize the number of samples per kit. This could lead to sample degradation and a critical delay in reporting when samples surpass the regulatory limit of 20 ppm.
Another advantage of the NOAA/MSI assay is that it could be run in either a quantitative or screening mode when assaying shellfish tissues. For quantitative analysis, several dilutions were assayed simultaneously to obtain an accurate DA concentration. Alternatively, to rapidly screen for DA concentrations of concern, the sample extracts were diluted 1:1,000 before running the assay. Taking into account the 1:10 dilution that occurred during the extraction process, the 1:1,000 dilution reduced samples in the 20 ppm DA range to ~2 ppb in the diluted sample. This concentration was within the linear range of the assay (0.1-3 ppb). Tissue samples with 5-10 fold less DA, and far below levels of concern, would show no detectible DA at this dilution. Tissues containing initial DA concentrations >30 ppm would be off scale and indicate a significant DA concentration requiring action. Any samples from this rapid screening that were of concern could then be diluted and run again to obtain an accurate concentration. The NOAA/MSI ELISA test kit also comes with a simple Excel spreadsheet, which allowed the toxin concentrations to be quickly and easily calculated in either a quantitative or rapid screening mode. All that had to be entered was the [B.sub.o] (no DA added) and sample absorbance data from each strip, the weight of the extracted tissue samples, and the extraction volumes.
The NOAA/MSI and Biosense ELISA kits were tested against crude methanolic extracts of several intertidal invertebrates, which HPLC identified as containing >100 ppb levels of epi-DA and iso-DA. These compounds are reported to be less toxic DA congeners based on receptor binding assays (Sawant et al. 2007). Results from both ELISA kits revealed the presence of only trace amounts of DA equivalents in the extracts. The NOAA/MSI ELISA cross-reactivity with these compounds ranged from 0% to 5.3% and the Biosense ELISA cross-reactivity from 0.01% to 1.5% (Table 1) indicating that the ELISA assays are relatively insensitive to cogener interference. It should also be noted that the regulatory methods for assessing human safety are currently based on measuring DA alone, not the combination of DA, iso-DA and epi-DA. These results indicated that both the NOAA/MSI and Biosense ELISA methods provide DA values comparable to the HPLC values currently used as a basis for regulatory decisions.
Measuring low concentrations of DA in real rime is particularly important because the presence or absence of DA contamination is frequently patchy and associated with variable onshore transport of toxic phytoplankton blooms (e.g., Trainer et al. 2002). Depending on prevailing winds and currents, one harvest area can become highly contaminated over a short period whereas adjacent regions remain uncontaminated (Trainer et al. 2000). These differentially affected regions frequently include areas where significant commercial and recreational clam harvests occur. This variability complicates monitoring programs designed to protect human health. The current standard practice involves shipping shellfish samples to a centralized facility for HPLC analyses, introducing delays between 3-14 days from the date of sample collection to reporting results. This turnaround time is too slow to adequately protect subsistence shellfish harvesters who rely on clams consumed within a day or two of harvest. The cost of HPLC analysis is also relatively high per sample and requires a substantially higher capital investment compared with the NOAA/MSI ELISA method. Having an economical technique for better assessing the degree of contamination locally, and in real time, is of great value for local resource managers and public health officials.
The ability to detect DA in phytoplankton using the NOAA/ MSI kits would further benefit environmental monitoring programs designed to detect the early onset of toxic Pseudo-nitzschia blooms. It is known that increases in the Pseudo-nitzschia capable of producing DA often precedes the contamination of shellfish and other filter feeders by a week or two (Trainer & Suddleson 2005). A combination of cell counts and direct toxicity measurements should provide timely predictions for marine resource managers and public health officials. The kit is now commercially available with MSI authorized to market, manufacture and distribute the 96-well plate format test kits. We anticipate completing the necessary validation procedures to qualify the 96 well plate format for regulatory use by public health officials. We are also developing a field test kit that can be used to detect DA levels in shellfish tissues above or below 20 ppm within 10 min after extraction. The test will require no laboratory equipment other than a homogenizer and can be used directly in the field by non-technical personnel, including shellfish harvesters and members of citizen monitoring groups and local volunteers.
In summary, the NOAA/MSI ELISA test kit provides an accurate, flexible and cost effective method for measuring DA in clam, mussel and scallop tissues, as well as in phytoplankton samples. The assay yields concentrations for DA that are indistinguishable from those obtained by HPLC. With further validation, the NOAA/MSI ELISA kit is expected to be approved as a regulatory method for making decisions concerning public health. The short assay (1.5-h) processing time, and relatively low cost, compared with HPLC analysis, mean that the ELISA can be used in more remote locations by environmental managers and public health officials to provide near real-time monitoring capacities.
The authors thank Mitch Lesoing and Mel Moon of the Quileute Tribe whose initial request for development of a domoic test kit and constant encouragement were key to the success of the test kit. This work was supported by a competitive grant awarded by the MERHAB program specifically for assay development as well as a NOAA NCCOS Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) grant GAD# R83-1705) and two Monitoring and Event Response for Harmful Algal Blooms (MERHAB) grants: NA04NOS4780239-03 and NA05NOS4781228. J. Bastion and A. Odell's participation was funded by a surcharge to the Washington State shellfish license provided or ORHAB. Jonathan Deeds provided helpful edits and suggestions.
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Kotaki, Y., E. F. Furio, M. Satake, N. Lundholm, T. Katayama, K. Koike, V. P. Fulgueras, F. A. Bajarias, Y. Takata, K. Kobayashi, S. Sato, Y. Fukuyo & M. Kodama. 2005. Production of isodomoic acids A and B as major toxin components of a pennate diatom Nitzschia navis-varingica. Toxicon 46:946-953.
Kreuder, C., M. A. Miller, D. A. Jessup, L. J. Lowenstine, M. D. Harris, J. A. Ames, T. E. Carpenter, P. A. Conrad & J. A. K. Mazet. 2003. Patterns of mortality in the southern sea otter (Enhydra Lutris nereis) from 1998-2001. J. Wildl. Dis. 39:495-509.
Lefebvre, K. A., S. Bargu, T. Kieckhefer & M. W. Silver. 2002. From sanddabs to blue whales: the pervasiveness of domoic acid. Toxicon 40:971-977.
Pocklington, R., J. E. Milley, S. S. Bates, C. J. Bird, A. S. W. de Freiras & M. A. Quilliam. 1990. Trace determination of domoic acid in seawater and phytoplankton by high performance liquid chromatography of the fluorenylmethoxycarbonyl (FMOC) derivative. Int. J. Environ. Anal. Chem. 38:351-368.
Quilliam, M. A. & J. L. C. Wright. 1989. The amnesic shellfish poisoning mystery. Anal. Chem. 61:1053A-1060A.
Quilliam, M. A., M. Xie & W. R. Hardstaff. 1991. A rapid extraction q and clean-up procedure for the determination of domoic acid in tissue samples. Canadian National Research Council, Technical Report no. 64, NRCC 33001. Ottawa, Canada. 52 pp.
Quilliam, M. A., M. Xie & W. R. Hardstaff. 1995. Rapid extraction and cleanup for liquid chromatographic determination of domoic acid in unsalted seafood. J. AOAC Int. 78:543-554.
Quilliam, M. A., P. G. Sim, A. W. McCulloch & A. G. McInnes. 1989a. High-performance liquid chromatography of domoic acid, a marine neurotoxin, with application to shellfish and plankton. Int. J. Environ. Anal. Chem. 36:139-154. Int.
Quilliam, M. A., B. A. Thomson, G. J. Scott & K. W. Siu. 1989b. Ion-spray mass spectrometry of marine neurotoxins. Rapid Commun. Mass Spectrom. 3:145-150.
Ritchie, D. G., J. M. Nickerson & G. M. Fuler. 1981. Two simple programs for the analysis of data from enzyme-linked immunosorbent assays on a programmable desktop calculator. Anal. Biochem. 110:281-290.
Rue, E. & K. Bruland. 2001. Domoic acid binds iron and copper: A possible role for the toxin produced by the marine diatom Pseudo-nitzschia. Mar. Chem. 76:127-134.
Sawant, P. M., B. A. Wearea, P. T. Holland, A. I. Selwood, K. L. King, C. M. Mikulski, G. J. Doucette, D. O. Mountfort & D. S. Kerra. 2007. Isodomoic acids A and C exhibit low KA receptor affinity and reduced in vitro potency relative to domoic acid in region CAI of rat hippocampus. Toxicon 50:627-638.
Scallet, A. C., L. C. Schmued & J. N. Johannessen. 2005. Neurohistochemical biomarkers of the marine neurotoxicant, domoic acid. Neurotoxicol. Teratol. 27:745-752.
Scholin, C. A., F. Gulland, G. J. Doucette, S. Benson, M. Busman, F. P. Chavez, J. Cordaro, R. DeLong, A. DeVogelaere, J. Harvey, M. Haulena, K. Lefebvre, T. Lipscomb, S. Loscutoff, L. J. Lowenstine, R. Marin, III P. E. Miller, W. A. McLellan, P. D. R. Moeller, C. L. Powell, T. Rowles, P. Silvagni, M. Silver, T. Spraker, V. L. Trainer & F. M. Van Dolah. 2000. Mortality of sea lions along the central California coast linked to a toxic diatom bloom. Nature 403:80-84.
Smith, E. A., E. P. Papapanagiotou, N. A. Brown, L. A. Stobo, S. Gallacher & A. M. Shanks. 2006. Effect of storage on amnesic shellfish poisoning (ASP) toxins in king scallops (Pecten maximus). Harmful Algae 5:9-19.
Sokal, R. R. & F. J. Rohlf. 1995. Biometry: The principles and practice of statistics in biological research. 3rd edition. New York: W. H. Freeman and Co. 887 pp.
Stewart, T. N. 2008. Template for calculating DA concentrations using the NOAA/MSI ELISA. http://www.mercuryscience.com/DA.html. Last accessed 4/22/2008.
Trainer, V. L. & M. Suddleson. 2005. Monitoring approaches for early warning of domoic acid events in Washington State. Oceanography 18:228-237.
Trainer, V. L., B. M. Hickey & R. A. Horner. 2002. Biological and physical dynamics of domoic acid production off the Washington USA coast. Limnol. Oceanogr. 47:1438-1446.
Trainer, V. L., N. G. Adams, B. D. Bill, C. M. Stehr, J. C. Wekell, P. Moeller, M. Busman & D. Woodruff. 2000. Domoic acid production near california coastal upwelling zones, June 1998. Limnol. Oceanogr. 45:1818-1833.
Vigilant, V. L. & M. W. Silver. 2007. Domoic acid in benthic flatfish on the continental shelf of Monterey Bay, California, USA. Mar. Biol. 151:2053-2062.
Wekell, J. C., V. L. Trainer, D. Ayres & D. Simons. 2002. A study of spatial variability of domoic acid in razor clams: recommendations for resource management on the Washington coast. Harmful Algae 3:1-9.
Wekell, J. C., E. J. Gauglitz, Jr., H. J. Barnett, C. L. Hatfield, D. Simons & D. Ayres. 1994. Occurrence of domoic acid in Washington state razor clams (Siliqua patula) during 1991-1993. Nar. Toxins 2:197-205.
Wells, M. L., C. G. Trick, W. P. Cochlan, M. P. Hughes & V. L. Trainer. 2005. Domoic acid: The synergy of iron, copper, and the toxicity of diatoms. Limnol. Oceanogr. 50:1908-1917.
Work, T. M., B. Barr, A. M. Beale, L. Fritz, M. A. Quilliam & J. L. C. Wright. 1993. Epidemiology of DA poisoning in brown pelicans (Pelecanus occidentalis) and Brandt's cormorants (Phalacrocarax penicillatus) in California. J. Zoo Wildl. Med. 24:54-62.
Wright, J. L. C., M. Falk, A. G. McInnes & J. A. Walter. 1990. Identification of isodomoic acid D and two new geometrical isomers of domoic acid in toxic mussels. Can. J. Chem. 68:22-25.
Wright, J. L. C., R. K. Boyd, A. S. W. de Freitas, M. Falk, R. A. Foxall, W. D. Jamieson, M. V. Laycock, A. W. McCulloch, A. G. McInnes, P. Odense, V. P. Pathak, M. A. Quilliam, M. A. Ragan, P. G. Sim, P. Thibault, J. A. Walter, D. J. A. Gilgan, D. J. A. Richard & D. Dewar. 1989. Identification of domoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Can. J. Chem. 67:481-490.
Yoon, D. Y., M. J. Choi, I. S. Choe, T. W. Chung & S. M. Byun. 1993. Influence of the conjugation site on the specificity of monoclonal antibodies to progesterone and on the performance of direct enzyme immunoassay. Biochem. Mol. Biol. Int. 31:553-563.
(1) National Ocean Service, NOAA, 101 Pivers Island Road, Beaufort, North Carolina 28516; (2) Mercury Science Inc., 2801 Blue Ridge Road, Suite G-70, Raleigh, North Carolina 27607; (3) Northwest Fisheries Science Center, NOAA, 2725 Montlake Boulevard East, Seattle, Washington 98112; (4) Ocean Sciences & Institute for Marine Sciences, University of California Santa Cruz, 1156 High Street, Santa Cruz, California 95064; (5) Moss Landing Marine Laboratories, 8272 Moss Landing Rd, Moss Landing, California 95039; (6) Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, AHF 301, Los Angeles, California 90089; (7) Quinault Indian Nation, Division of Natural Resources, Department of Fisheries, 1214 Aalis Drive Bldg C, Taholah, Washington. 98587; (8) Olympic Region Harmful Algal Bloom Partnership, 14 North Shore Place, Hoquiam, Washington 98550; (9) IFREMER- Centre de Brest, DYNECO, Pointe du Diable, BP 70, 29280 Plouzane, France; (10) IFREMER, 13 Rue Kerose, 29900 Concarneau, France
* Corresponding author. E-mail: email@example.com
TABLE 1. Intertidal invertebrates sampled from several locations around Monterey Bay in November 2006. HPLC-LJV analysis detected significant levels of compounds comigrating with iso- and epi-domoic acid standards. These crude methanolic extracts were used to challenge the NOAH and Biosense ELISAs. The goal was to establish the extent to which the ELISA assays are confounded by the presence of coeluting compounds called as the domoic acid isomers epi- and iso-domoic acid by HPLC-UV assay. Nondetect samples are represented as 0 values. DA Combined epi Concentration %, Total DA and iso-DA by NORA Detected by Organism by HPLC (ppb) ELISA (ppb) NORA ELISA Chthamalus fissus/dalli 281.7 0.00 0.00 Chthamalus fissus/dalli 1,137.1 15.41 1.36 Littorina scutulata 198.7 10.57 5.32 Littorina scutulata 682.0 15.98 2.34 Littorina scutulata 119.5 0.00 0.00 Lottia digitalis 236.7 0.00 0.00 Lottia digitalis 477.9 13.91 2.91 Lottia digitalis 390.6 10.31 2.64 DA % Total DA Concentration Detected by by Biosennse Biosense Organism ELISA (ppb) ELISA Chthamalus fissus/dalli 0.02 0.01 Chthamalus fissus/dalli 1.53 0.13 Littorina scutulata 3.02 1.52 Littorina scutulata 1.02 0.15 Littorina scutulata 0.17 0.14 Lottia digitalis 0.10 0.04 Lottia digitalis 0.09 0.02 Lottia digitalis 0.78 0.20 TABLE 2. Cross-reactivity of the NOAA/NISI ELISA with kainic acid. glutamine, and glutamic acid. % Reactivity in the Domoic Analyte Acid Assay Domoic acid 100 Kainic acid 0.3 Glutamine <0.1 Glutamic acid <0.1
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Johnson Level Green Magnetic Sheave Alignment - 40-6210
The GreenBrite magnetic pulley (sheave) alignment laser tool projects a plane of laser light parallel with the faces of drive and driven sheaves (or pulleys). Adjustable targets attach to sheaves, intersect the laser beam and visually show any misalignment. The Retro-Reflective film on the center line of the target reflects the laser beam and "glows" for enhanced visibility and accuracy. The reflective green film shows the laser's alignment line in "Real Time". This is called "Positive Tracking".


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Meet this cutie - Roo!UPDATED 5:55 PM HST Dec 06, 2012
It's time for Pets on Set and meet Roo, a 3-month-old domestic short hair kitten from the Hawaiian Humane Society. She's ready for adoption now and needs a new home for the holidays.
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text: ARARAT, Va. — Mr. Bobby Gray Wright, 54, of Ararat, Va., passed away Monday, May 23, 2011. Mr. Wright was born in Surry County on July 9, 1956, to Robert Wright and the late Pauline Duncan Wright. Mr. Wright was a loving father, son and brother who will be dearly missed. Surviving are daughters and a son-in-law, Teresa Martinez and Jesus Juarez, Jennifer Wright and Bobby Nester all of Ararat; Brandi Mayes of Cana, Va.; grandchildren, Ben Juarez, Gaby Juarez, Jesus J. Juarez, Joseph Nester and Jacob Nester; sisters and brothers-in-law, Patty Martin, Peggy and Richard Pell, Bonnie and Mark Donithan, June and Barron King all of Mount Airy and Libby and Dwight Grimes of Low Gap; and a brother, Mark Wright of Mount Airy; special nephew, Corbett Hodge; special nieces, Tee, Katie and Brandy Wright and several other nieces and nephews; and special friends, Arnold Ray Shinault Jr.; Charity Taylor and Earl and Becky Niten. In addition to his mother, Mr. Wright was preceded in death by a sister, Vicky Wright. Funeral services will be held at 11 a.m., Friday, May 27, 2011, at Moody Funeral Home Chapel by Bro. Don Collins. The family will receive friends Thursday night from 6 to 8 p.m. at the funeral home. Online condolences may be made at www.moodyfuneralservices.com.


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Secretary Course and Class Descriptions
Courses are available for would-be general secretaries, as well as aspiring legal assistants or administrative medical assistants. These courses are generally taken as part of a full undergraduate program.
Essential Information
Secretarial courses are commonly part of diploma, certificate and associate's degree programs, and occasionally bachelor's degree programs. Secretarial studies programs are also called secretarial science, administrative assistant, general office assistant or executive assistant programs. Specialized programs include those for aspiring medical administrative assistants and legal administrative assistants. Hands-on training during classes is usually provided, and cooperative education is part of some programs.
Common secretary courses include those in keyboarding, word processing, business math, business English, records management, transcription and office operations. While general programs provide students with a variety of basic office skills, specialized programs include courses geared toward the medical or legal field. For example, medical secretary programs often cover medical transcription, medical terminology and medical coding, while legal assistant programs cover legal research and writing, litigation and contracts.
Overview of Courses
Descriptions of general courses that appear in a program geared toward secretaries are shown below.
Keyboarding and Word Processing Course
This required secretarial course introduces students to the alphanumeric keyboard, including function keys and 10-key numeric data entry, on a computer and sometimes a typewriter. Speed and accuracy is stressed. Students also become familiar with working from handwritten or electronic files.
Students in secretarial courses like this learn how to draft business letters, reports, statistical tables and business forms using Microsoft Office. Students in specialized medical or legal secretary programs may learn keyboarding for additional specialty software programs as required.
Mathematics for Business Course
Business math skills include the ability to perform arithmetic and understand basic financial transactions. Bookkeeping and accounting skills are also taught, along with how to perform inventory. Some programs may require this course.
English for Business Course
Strong communication skills are vital for secretarial professionals, including good grammar, punctuation, vocabulary and spelling. English courses generally include the development of reading comprehension skills and expository writing ability. This course is required in some programs.
Transcription Course
A transcription course is a core class in most programs. Transcription includes both operating the transcription or dictation equipment and creating a written record of spoken communication. Students learn to transcribe quickly and with a minimum of errors.
Records Management for Business Course
Secretarial skills include knowing the principles of creation, storage, maintenance and disposition of paper and electronic records and files. Filing and sorting systems are explored. Records management classes are required by some secretary courses.
Office Operations Course
Students are introduced to basic office work flow, proper e-mail, Internet and telephone protocols, preparing business documents like correspondence and reports, booking meetings, welcoming visitors and collaboration with colleagues. The use of business machines, including postage meters, telephone systems, fax machines and photocopy machines, is introduced. Students also learn business ethics in this required class.
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Property Details
Studio, 0 Bedrooms, 1 Baths, 3 Sleeps
Note that WEEKLY Rates Do Not Include Taxes & $50 Cleaning Fee.
$150 Fully REFUNDABLE Security Deposit is Required for Summer Rentals.
By the Month OFF Season Rates Available and Includes All Utilities (See Below)
Also, We Are a Non-Smoking Property both Inside the Unit and on the Common Grounds. Public Street is located just in front of the Bldg
Our "ALL-Antic" Condotel provides nothing but FUN-FUN-FUN in Beautiful Atlantic Beach, North Carolina quietly located on an Ocean Side dead-end street on the Bogue Banks (the southern point of the Outer Banks system in Coastal Carolina)!!!
Welcome Friends, Family, Beachgoers, Fisherman, Water Enthusiasts hard working Military, Students and the Like. An Oasis Awaits you while you Rest & Play!!
This All-In-One (Room) Efficiency Unit Sleeps up to 3 People, Provides a Equiped Kitchen, and a Flat Screen TV above The Bar! Two Twin Beds w/Bolsters Double as Couches. For Sleeping, the Twins can be easily joined to make a King Bed or pull-out and pop-up the Trundle for your #3. Unit Offers Wireless Internet, Cable TV and DVD Player for your inside pleasure.
Although Affordable, This Unit is Recently Remodeled, Clean and Comfortable Day and Night, Perfectly Located between Fort Macon State Park and the Oceana Pier (both walking distance). Enjoy the Ocean Breeze and View from your Shaded 2nd-Story Veranda right outside your Front Door (see pic#6)! Very Short Walk to a Full Amenities Beach / Ocean Access (pic#7), or the opposite way toward the sound side Triple S Marina, Store & Boat Ramp. Sit in the shade out back or take a Swim in the Gorgeous Pool, the choices are yours!
Please No Smoking Inside the Unit or on the Common Ground Areas!!!!
About the Owner
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Activity and Interests
See Ideas On the Right But... OUR FAVORITES other than the Great Big Clean Beautiful Beach less than a block down the street is are: Beaufort & Morehead Citys Waterfront (shops, restaurants, bars, boat & fish traffic galore!) . A Ferry ride either from Beaufort to Shackleford Banks, or Harkers Island to Cape Lookout Lighthouse or if your up for a very full day the Cedar Island Ferry to Ocracoke (make reservations!) are very fun too.
Check out crystalcoastnc events calendar on line for the many local events and celebrations of our terrific coastline and coordinate your trip accordingly. Our favorites are the Seafood Festival in Morehead (October), The Powerboat Races (around Sept), the the Beaufort Music Fest (May) but there are many many others!!!!!
Rental rates
- Nightly Rate: $100 - $100
Weekly Rate: $500 - $500
Monthly Rate: $550 - $850
Winter Rates Available!! $550 Per Month Plus $250 Security
Security Deposit Required*
Small/Med Clean Well-Behaved Pets Welcome
(Additional $10/per Night, $50/ per Week, $100 per Month or longer)
Responsible Military & Students Welcome
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Property Type
- studio
- Adventure
- Budget
- Family
- Romantic
Pool / Spa
- Outdoor Pool
- pets considered
Location Type
- Beach View
Sports & Adventure Activities
- golf
- 1 Bathroom
- Bathroom 1
- Studio, Sleeps 3
- Nice 2nd Story Veranda with Bench 2 Twins Beds make it great for both individuals or couples to share as 1 king Pop up Trundle accomodates third individual or child Fully Equiped Kitchen w/Full Size Refrigerator Flat Screen TV w/ Cable WIRELESS INTERNET NOW AVAILABLE Roomy Bathroom with Tile Shower Beautiful Pool / Sand Volley Ball Court / Private Shaded Grassy Yard Area great for Big BQ (pits available) Public Beach Access w/Full Restroom Facilities, Gazabo & Playground < 100 Feet Triple S Marina (one Block North) provides a Convenience Store, Boat Ramp, and potential dockage.* * (You must make other boat/trailer parking arrangements however)


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Here you will find a quick synopsis of all news from Microsoft's Cloud Platform organization as it is released with links to additional information.
The Official SBS BlogThe System Center Team BlogSQL Server Team BlogWindows Azure Team BlogVirtualization Team BlogWindows Server DivisionSQL Azure Team Blog
by STB Blogger
Windows Azure Mobile Services: New support for iOS apps and renewed alliance with Twilio
This week, Microsoft announced a number of new improvements to Windows Azure Mobile Services, including how developers can now use Mobile Services to develop native iOS apps. Additional updates include: Facebook, Twitter, and Google authentication support; blob, table, queue, and Service Bus support from within your Mobile Service; sending emails from your Mobile Service (in partnership with SendGrid); and the ability to deploy mobile services in the West US region in addition to the East US region.
In concert with the Mobile Services updates, Twilio announced a renewed alliance with Microsoft, which will allow for Twilio's voice and messaging APIs to easily be available to Windows Azure customers, including Windows Azure Mobile Services. Further information on these improvements and how you can get started, can be found on Scott Guthrie's blog.


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|Property type
|Minimum Stay
|2 - 4 nights
With spectacular views of mountains, you may find Antelope, Moose, Bear and Deer impeding your views from time to time. A large walk-out deck, charming decor and open floor plan make this great for a small family or couples retreat. Enjoy a new log cabin on several acres without neighbors crowding in. This getaway has a comfortable living space, in a peaceful mountain location, this wonderful cabin is sure to make your next vacation a hit! Inclusive of panoramic views, you are centered between the trail systems and several mountain ranges. Snowmobile access is easy and in every direction, and ATVs trails are just out your front door. Such a great value without compromise. The property was built as a future guest cabin to a main home to be built someday! Property is for sale at $119,500 and includes 1.74 acres with the cabin...
*Peak Property is the only company offering FREE trash service to its guests! Book with us to avoid hauling your trash to the dump!
Rating Details:
The property manager was very helpful.
The vacation rental was spotless.
I was very happy with the location.
I was more than satisfied with the condition of the vacation rental.
Overall, I recommend this vacation rental.
Reviewer Comments:
My wife and I had a very comfortable and fun stay. It was clean, well kept, and had all the appliances and comforts we needed. Ran the shower with straight hot water, and still never seemed warm enough for showers though. I imagine this is only a problem in the winter.
*Plus compulsory or optional charges based on actual occupancy or consumption. The payment has to be made in the currency offered by the property manager, before use of conversion tool. Payments in other currencies have to be negotiated beforehand with the property manager. The currency conversion tool is provided for your convenience only and is non-binding.


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text: The TouchPad uses the WebOS user interface -- the multitasking is even more compelling. Activity cards (representing data or apps) are grouped into card stacks (this is the same as the phone, but I imagine it would be so much better on a tablet). (Note: After the event we got a demo of this and it was very compelling.)


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Feb. 27, 2014
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Sunday, June 22nd:...
Chicago Witch: Witchy Living in the Windy City
A perspective of a modern, urban Witch who strives to balance faith with family and career.
Lori Dake
Lori Dake is a life-long native several generations back , is a mom to an adult son and married for over fifteen years. She is a leasing agent primarily serving the North Side and has also worked as a paralegal for several years. Sometimes, she's a hardcore fashionista, and sometimes a concert shirt and jeans are more her style. Hobbies include painting while listening to 80's metal, writing, participating in various forms of philanthropy and creating fabulous meals on a budget.


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Dump Truck
Stolen Dump Truck, Boat, ATVs Recovered in Jefferson County
Police say Douglas Buffer is charged with Theft over $500, but he's accused of taking items worth $115K
Reggie Wayne's 4 Most Ridiculous Colts Training Camp Entrances
Indianapolis Colts wide receiver Reggie Wayne has earned a reputation for his flashy training camp arrivals and this year was no different. In keeping with tradition, Wayne stepped up his training camp arrival game.
St. Louis Police Cruiser Collides with Dump Truck
A St. Louis police officer suffered a head injury after his cruiser collided with dump truck Tuesday.


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The Rutland County Humane Society (RCHS) is launching a campaign to help raise funds and build a walkway to your new best friend. Brick by brick our walkway will be built. Bricks are ideal gifts to commemorate a special event, honor a special person or pet, dedicate the memory of a loved one, celebrate a milestone, holidays and more. 4" x 8" bricks are $50 and 8" x 8" bricks are $200. To order your brick or for more information please contact RCHS at 483.9171 or visit www.rchsvt.org. Together we can build a pathway to a brighter future for the homeless animals in Rutland County.
3 year old. Spayed Female. Cocker Spaniel.
I am a pretty little girl who has nice leash manners and enjoys quiet attention from people. For a small dog, I do have a big reaction when strangers come to the door-barking and growling-so I am looking for a quiet adult-only home where I won't get stressed out. Adult only is also good for me because I get nervous when I am hugged and tugged.
7 month old. Spayed Female. Dachshund mix.
I am a sweet girl who likes people and will be able to live with other dogs if they are well-socialized and open to a canine companion to live with. I am a little timid around other dogs but should be able to bounce back. Originally, I was brought here as a puppy from the Spay the Mom program.
1 year old. Spayed Female. Domestic Long Hair Black and White.
I want to be Queen Bee so I may do well as an only kitty because of my bossiness. I am very affectionate with human contact and cannot seem to get enough loving from people that visit. I like to play with all sorts of cat toys so I would suggest having lots around.
2 year old. Neutered Male. Domestic Short Hair Gray Tiger.
Now, I am a cat with an attitude with no doubt. If you are looking for a good mouser and someone around to tell a story to I am the one for you. I really don't care much for being picked up or snuggled; I guess you could say I am a macho type of guy.
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Cheap ALC to Rockford flights
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Route information for flights from Alicante Intl. to Greater Rockford.
4,454.40 miles
ALC - Alicante Intl. Airlines
Browse the most popular airlines servicing the Alicante (ALC) airport. Visit their flights pages to learn more about the most popular routes these carriers fly.


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nwheeler89's Mom Answers Activity
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Biting is a big concern. But not all daycare has camera installed in the class room. So it totally depend on the teachers. Daycare is not...posted by GinaDL
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I'm 8 days late and all 4 tests have come back negative. I'm never late! What should I do?????
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It takes about two weeks after a missed period for a test to read positive. If it still comes back negative after you've waited and taken...posted by nwheeler89
Is it normal that my son isn't talking as much as others?
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thanks. i don't think we have an early childhood place around here. he goes to daycare, so I'm hoping that they help him out as much as t...posted by nwheeler89
How do I get my 13 month old to let me brush his teeth?
Most recent answer (11/16/2011):
Thank you. I will try that this time and see if it works.posted by nwheeler89


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Joan Voight | May 7, 2012 | Comments
The "broad" from New York straddled the traditional and Internet agency worlds.
Read the whole Digital Mad Men/Women series.
When ad giant Omnicom decided to invest in interactive startups back in 1996, it turned to Felice Kincannon. An elegant former agency chief who could have been the older sister of the young startup founders, she was charged by Omnicom CEO John Wren to cull a list of 25 prospective shops down to six, in which the holding company would buy significant minority stakes. From her Boston base, where she once managed ad shop Hill Holliday, the "broad from Omnicom" hopped around the country to size up - and woo - digital marketing's emerging leaders.
She was infinitely patient, except with those "stupid" traditionalists who warned her she was wasting her promising career. After the deals were finalized Wren promoted Kincannon to managing director. Her job was to bring work from existing Omnicom agency clients to the startup partners - an exercise in intricate diplomacy. Her stable of digital talent was impressive: Agency.com, Interactive Solutions, Razorfish, Red Sky, Think New Ideas and Organic. A few years of that was enough; in 1999 she left to start a marketing consultancy with a Harvard professor friend. These days Kincannon is still press shy as she chases the cutting edge, quietly advising major brands on how to thrive by being sustainable and socially responsible.
ClickZ: What was it like visiting all those fledgling Web agencies?
Felice Kincannon: I was the afternoon entertainment at their offices. Their attitude seemed to be, "Here's that broad from New York, what does she know?" But for me, it was a blast. I'd get the meetings because these firms wanted access to holding company clients. Once I was there I could win them over because I understood how important the technology was and I really respected what they could do.
Keep in mind I was well into my 40s, about 10-15 years older than these agency guys. I was dressed in stylish, conservative pants and jacket, not jeans and a T-shirt or all leather. I represented a different culture, at first. In all, I visited 13 firms.
CZ: Once the six Web shops were brought into the holding company fold, how did you introduce them to the main Omnicom agencies?
Kincannon: My job was to go to the standing agencies and explain how they could use these interactive shops. Often I'd ask the [traditional] agency to show me some online work they'd done. And it would take forever to load - we called it the World Wide Wait. I'd show them similar online work from one of the interactive shops and it would load really fast. The agency staff would ask, "How did they do that?" And I'd tell them, "It's because they understand the technology. And they'll share that with you."
I was incredibly enthusiastic about the Internet, but it helped that I had spent 15 years in the traditional ad business.
CZ: With your ad industry roots, did your colleagues ever question your career path?
Kincannon: Some friends told me that I was wasting my time with Internet marketing. And I'd think that if people were going to be that stupidly negative, I couldn't convince them otherwise. But I knew I was right and they were wrong. In the end we saw that I was right.
In that vein, you've got to give credit to Omnicom's senior folks. The more they understood what the interactive shops could do and got to know the founders, the more weight they gave the category.
CZ: Did you run across other women at the Web agencies you visited?
Kincannon: Generally, I was the only woman in the room. At that time, the lack of [senior-level] women was true of business in general as well as the interactive industry. When I met with standing Omnicom agencies, sometimes another woman would be present. It helps to keep in mind that my role was unique. I wasn't a secretary, an account executive or a developer.
CZ: Who inspired you during that topsy-turvy era?
Kincannon: It's not you'd think. It's my grandfather, who was a phenomenally creative person. He was the art editor at Collier's magazine, he started Glamour magazine, he worked on a radio show and he published comic books. He had the ability to roll with the times. But most importantly, he could understand the essence of any medium and how to leverage that essence. I wanted to cultivate that ability of his as we learned how to grasp the essence of interactive media.
CZ: What do you miss from the 1990s?
Kincannon:I loved the rapid change and the opportunity to look around the corner and see what was coming next. I don't think the marketing world has had that since then.
On the heels of a fantastic event in New York City, ClickZ Live is taking the fun and learning to Toronto, June 23-25. With over 15 years' experience delivering industry-leading events, ClickZ Live offers an action-packed, educationally-focused agenda covering all aspects of digital marketing. Early Bird Rates expire May 29. Register today and save!
Joan Voight is a Contributing Editor to ClickZ. Based in the San Francisco Bay Area, she has covered online and offline media, marketing and advertising since the mid-1990s for several business publications. She spent nine years at Adweek magazine, where she was San Francisco bureau chief, national senior writer and contributing reporter.
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This Magic Quadrant examines leading digital commerce platforms that enable organizations to build digital commerce sites. These commerce platforms facilitate purchasing transactions over the Web, and support the creation and continuing development of an online relationship with a consumer.
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Google reports that paid search ads are currently driving 40+ million calls per month. Cost per click is increasing, paid search budgets are growing, and mobile continues to dominate. It's time to revamp old search strategies, reimagine stale best practices, and add new layers data to your analytics.
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Those 13-year-olds in Gaza sure grow up fast. Maybe it's hormones in the milk. Or maybe they haven't been 13 year olds in this century.
Elder of Ziyon notes something rather odd about one 13-year-old boy slain by the brutal Zionist overlords of doom.
The Hamas Interior Ministry reported that 13-year old Ibrahim Jamal Kamal Naser was killed on July 19.
Here is the shahid poster of "13-year old" Ibrahim Nasser, killed on that same day in that same place, by the Abu Rish Battalions of the Al Aqsa Martyrs' Brigades
Maybe a really old 13. A 13-year old who has been left back more times than Arafat at the airport security checkpoint.
Elder has found a bunch of other "children" who could easily get a drink in any bar. Maybe they're young at heart.} add_action( 'genesis_after_post_content', 'single_sub' ); function single_sub() { ?>


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More and more Americans are consuming artificial sweeteners as an alternative to sugar, but whether this translates into better health has been heavily debated.
NPR's Allison Aubrey explains a new study in the journal "Trends in Endocrinology and Metabolism" that looks at the brain science behind drinking diet soda.
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Monty Python
Keeping milk and poultry prices high makes no senseCanada should get rid of its supply management system for agriculture
A new, older Doctor Who is back to scratch your itchDeft, occasionally dazzling, light of heart and lively in spirit, Doctor Who continues to be one of the small screen's strangest anomalies.
Fantasia International Film Festival comes of age(WITH VIDEO) Now in its 18th year, the celebration of genre cinema continues to promise weird and wonderful discoveries.
Just for Laughs: Mike Birbiglia is happy to lose the plotThe masterful monologist downplays the narrative structure of his previous shows in Thank God for Jokes.
Bill Brownstein: David Heti elevates his act by putting himself downThe Montreal comic takes self-deprecation to another level.
North Carolina twist: Church challenges ban on gay marriageHmm . . . I know there is a Monty Python reunion in the works, so I have to wonder if they have anything to do with the news that a church is suing the state of North Carolina for the right to perform same-same marriages.
The Big Bang spurt theory, Part 2Continuing from the preceding post , for the fun of it . . . One reader in my other blog brought up Monty Python -- does that surprise anybody? If you would like to comment or theorize, feel free to do so at my other blog at jillianpage.


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Hooking 110 power cord to shore power?
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Re: Hooking 110 power cord to shore power?
Got it now. This is what you are asking for.
Marinco Shore Power Pigtail Adapter
However, powering the boat this way for an extended period isn't a great idea. Stopping over for a few hours maybe okay. The household plug does not lock in well, despite the screw, and the weight of your shore power cord has to be relieved or it is constantly trying to pull it out. I use this adapter over the winter on the hard to power up onboard battery chargers.
The other concern you'll have to deal with is your boats power draw. If she has 30 amp cables, you may overwhelm a 15 or even 20 amp household circuit. Smokey the Bear disapproves. Not all breakers will pop.
Our panel has an electronic switch, which can be set to the shore power input so it won't draw more than able. When we charge over the winter, we only have the chargers running. Nothing else and I stay there to supervise until done.
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Just finished our season by winning the USGF 8U state championship!
Open tryouts Saturday, August 4th, 6pm at Dan J Buckner Park in Dickson.
Please call or email any questions.
Brian Ellis 615-406-3538
Tryouts will be held on Field 5 @ Buckner Park
If tryouts have to be canceled due to weather, it will be posted on here ASAP.
If you would like to receive a text if we have to cancel, please send a text to 615-406-3538.
Hope to see you soon!
Tryouts moved to field 4, but are still on as scheduled :)


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Improving Stable Boundary-Layer Height Estimation Using a Stability-Dependent Critical Bulk Richardson Number
- H. Richardson,
- S. Basu,
- A. A. M. Holtslag
- … show all 3 hide
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For many decades, attempts have been made to find the universal value of the critical bulk Richardson number ( \(Ri_{Bc}\) ; defined over the entire stable boundary layer). By analyzing an extensive large-eddy simulation database and various published wind-tunnel data, we show that \(Ri_{Bc}\) is not a constant, rather it strongly depends on bulk atmospheric stability. A (qualitatively) similar dependency, based on the well-known resistance laws, was reported by Melgarejo and Deardorff (J Atmos Sci 31:1324–1333, 1974) about forty years ago. To the best of our knowledge, this result has largely been ignored. Based on data analysis, we find that the stability-dependent \(Ri_{Bc}\) estimates boundary-layer height more accurately than the conventional constant \(Ri_{Bc}\) approach. Furthermore, our results indicate that the common practice of setting \(Ri_{Bc}\) as a constant in numerical modelling studies implicitly constrains the bulk stability of the simulated boundary layer. The proposed stability-dependent \(Ri_{Bc}\) does not suffer from such an inappropriate constraint.
- Anderson, WC, Basu, S, Letchford, CW (2007) Comparison of dynamic subgrid-scale models for simulations of neutrally buoyant shear-driven atmospheric boundary layer flows. Environ Fluid Mech 7: pp. 195-215 CrossRef
- Andreas, EL, Claffey, KJ, Makshtas, AP (2000) Low-level atmospheric jets and inversions over the western Weddell sea. Bound-Layer Meteorol 97: pp. 459-486 CrossRef
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- Arya, SP (1999) Air pollution meteorology and dispersion. Oxford University Press, New York
- Basu, S, Porté-Agel, F (2006) Large-eddy simulation of stably stratified atmospheric boundary layer turbulence: a scale-dependent dynamic modeling approach. J Atmos Sci 63: pp. 2074-2091 CrossRef
- Basu, S, Porté-Agel, F, Foufoula-Georgiou, E, Vinuesa, JF, Pahlow, M (2006) Revisiting the local scaling hypothesis in stably stratified atmospheric boundary layer turbulence: an integration of field and laboratory measurements with large-eddy simulations. Bound-Layer Meteorol 119: pp. 473-500 CrossRef
- Basu, S, Holtslag, AAM, Wiel, BJH, Moene, AF, Steeneveld, GJ (2008) An inconvenient "truth" about using sensible heat flux as a surface boundary condition in models under stably stratified regimes. Acta Geophys 56: pp. 88-99 CrossRef
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- Basu S, Holtslag AAM, Bosveld FC (2011) GABLS3 LES intercomparison study. In: ECMWF/GABLS workshop on "Diurnal cycles and the stable atmospheric boundary layer", ECMWF, pp 75–82
- Brost, RA, Wyngaard, JC (1978) A model study of the stably stratified planetary boundary layer. J Atmos Sci 35: pp. 1427-1440 CrossRef
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- Clarke RH, Dyer AJ, Brook RR, Reid DG, Troup AJ (1971) The Wangara experiment: boundary layer data. Technical paper no. 19. CSIRO, Division of Atmospheric Physics. Aspendale, Australia, p 362
- Esau, I, Zilitinkevich, S (2010) On the role of the planetary boundary layer depth in the climate system. Adv Sci Res 4: pp. 63-69 CrossRef
- Esau, IN, Zilitinkevich, SS (2006) Universal dependences between turbulent and mean flow parameters instably and neutrally stratified planetary boundary layers. Nonlinear Process Geophys 13: pp. 135-144 CrossRef
- García, JA, Cancillo, ML, Cano, JL (2002) A case study of the morning evolution of the convective boundary layer depth. J Appl Meteorol 41: pp. 1053-1059
- Garratt, JR (1992) The atmospheric boundary layer. Cambridge University Press, Cambridge
- Gryning, SE, Batchvarova, E (2003) Marine atmospheric boundary-layer height estimated from NWP model output. Int J Environ Pollut 20: pp. 147-153
- Hanna, SR (1969) The thickness of the planetary boundary layer. Atmos Environ 3: pp. 519-536 CrossRef
- Heinemann, G, Rose, L (1990) Surface energy balance, parameterizations of boundary-layer heights and the application of resistance laws near an Antarctic ice shelf front. Bound-Layer Meteorol 51: pp. 123-158 CrossRef
- Holtslag, AAM, Boville, BA (1993) Local versus nonlocal boundary-layer diffusion in a global climate model. J Clim 6: pp. 1825-1842 CrossRef
- Holtslag, AAM, Bruijn, EIF, Pan, HL (1990) A high resolution air mass transformation model for short-range weather forecasting. Mon Weather Rev 118: pp. 1561-1575 CrossRef
- Holtslag, AAM, Steeneveld, GJ, Wiel, BJH (2007) Role of land surface temperature feedback on model performance for stable boundary layers. Bound-Layer Meteorol 125: pp. 361-376 CrossRef
- Hong, SY (2010) A new stable boundary-layer mixing scheme and its impact on the simulated East Asian summer monsoon. Q J R Meteorol Soc 136: pp. 1481-1496 CrossRef
- Jeričević, A, Grisogono, B (2006) The critical bulk Richardson number in urban areas: verification and application in numerical weather prediction model. Tellus 58A: pp. 19-27
- Jiménez, MA, Cuxart, J (2005) Large-eddy simulations of the stable boundary layer using the standard Kolmogorov theory: range of applicability. Bound-Layer Meteorol 115: pp. 241-261 CrossRef
- Kiehl, J, Hack, J, Bonan, G, Boville, B, Williamson, D, Rasch, P (1998) The National Center for Atmospheric Research Community Climate Model: CCM3. J Clim 11: pp. 1131-1149 CrossRef
- Mahrt, L (1981) Modelling the depth of the stable boundary-layer. Bound-Layer Meteorol 21: pp. 3-19 CrossRef
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- Nieuwstadt, FTM (1984) The turbulent structure of the stable, nocturnal boundary layer. J Atmos Sci 41: pp. 2202-2216 CrossRef
- Nieuwstadt, FTM A model for the stationary, stable boundary layer. In: Hunt, JCR eds. (1985) Turbulence and diffusion in stable environments. Clarendon Press, Oxford, pp. 149-179
- Ohya, Y (2001) Wind-tunnel study of atmospheric stable boundary layers over a rough surface. Bound-Layer Meteorol 98: pp. 57-82 CrossRef
- Ohya, Y, Uchida, T (2003) Turbulence structure of stable boundary layers with a near-linear temperature profile. Bound-Layer Meteorol 108: pp. 19-38 CrossRef
- Ohya, Y, Neff, D, Meroney, R (1997) Turbulence structure in a stratified boundary layer under stable conditions. Bound-Layer Meteorol 83: pp. 139-161 CrossRef
- Pichugina, Y, Banta, R (2010) Stable boundary layer depth from high-resolution measurements of the mean wind profile. J Appl Meteorol Climatol 49: pp. 20-35 CrossRef
- Saiki, EM, Moeng, CH, Sullivan, PP (2000) Large-eddy simulation of the stably stratified planetary boundary layer. Bound-Layer Meteorol 95: pp. 1-30 CrossRef
- Seibert P, Beyrich F, Gryning SE, Joffre S, Rasmussen A, Tercier P (1998) Mixing height determination for dispersion modelling, COST action 710-final report, harmonisation of the pre-processing of meteorological data for atmospheric dispersion models, L-2985. European Commission, Luxembourg, EUR 18195 EN (ISBN 92-828-3302-X)
- Sørensen, JH, Rasmussen, A, Svensmark, H (1996) Forecast of atmospheric boundary-layer height utilised for ETEX real-time dispersion modelling. Phys Chem Earth 21: pp. 435-439 CrossRef
- Steeneveld, GJ, Wiel, BJH, Holtslag, AAM (2007) Diagnostic equations for the stable boundary layer height: evaluation and dimensional analysis. J Appl Meteorol Climatol 46: pp. 212-225 CrossRef
- Stoll, R, Porté-Agel, F (2008) Large-eddy simulation of the stable atmospheric boundary layer using dynamic models with different averaging schemes. Bound-Layer Meteorol 126: pp. 1-28 CrossRef
- Stull, RB (1988) An introduction to boundary layer meteorology. Kluwer Academic Publishers, Dordrecht CrossRef
- Troen, I, Mahrt, L (1986) A simple model of the atmospheric boundary layer; sensitivity to surface evaporation. Bound-Layer Meteorol 37: pp. 129-148 CrossRef
- Vickers D, Mahrt L (2004) Evaluating formulations of stable boundary layer height. J Appl Meteorol 43: 1736–1749
- Vinuesa, JF, Basu, S, Galmarini, S (2007) The diurnal evolution of $$^{222}$$ Rn and its progeny in the atmospheric boundary layer during the Wangara experiment. Atmos Chem Phys 7: pp. 5003-5019 CrossRef
- Vogelezang, DHP, Holtslag, AAM (1996) Evaluation and model impacts of alternative boundary-layer height formulations. Bound-Layer Meteorol 81: pp. 245-269 CrossRef
- Wiel, BJH, Moene, AF, Jonker, HJJ, Baas, P, Basu, S, Donda, JMM, Sun, J, Holtslag, AAM (2012) The minimum wind speed for sustainable turbulence in the nocturnal boundary layer. J Atmos Sci 69: pp. 3116-3127 CrossRef
- Yamada, T (1976) On the similarity functions A, B and C of the planetary boundary layer. J Atmos Sci 33: pp. 781-793 CrossRef
- Zilitinkevich S, Baklanov A (2002) Calculation of the height of the stable boundary layer in practical applications. Bound-Layer Meteorol 105:389–409
- Title
- Improving Stable Boundary-Layer Height Estimation Using a Stability-Dependent Critical Bulk Richardson Number
- Journal
Boundary-Layer Meteorology
Volume 148, Issue 1 , pp 93-109
- Cover Date
- 2013-07-01
- 10.1007/s10546-013-9812-3
- Print ISSN
- 0006-8314
- Online ISSN
- 1573-1472
- Publisher
- Springer Netherlands
- Additional Links
- Topics
- Keywords
- Boundary-layer height
- Large-eddy simulation
- Low-level jet
- Resistance laws
- Stable boundary layer
- Industry Sectors
- Authors
- H. Richardson (1)
- S. Basu (1)
- A. A. M. Holtslag (2)
- Author Affiliations
- 1. Department of Marine, Earth, and Atmospheric Sciences, North Carolina State University, Raleigh, NC, USA
- 2. Meteorology and Air Quality Section, Wageningen University, Wageningen, The Netherlands


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Tom Cruise is a Flying Mad Man!
Nov 3, 2014
Tom Cruise is famous for being brave enough, and sometimes reckless enough, to do his own stunts ... but this time he's gone overboard with the whole stunt thing! Tom is working on the upcoming Mission Impossible movie, which means ... Read More


text: Publications Search Results Users may wish to consult What's Included in the Bibliography. Results 1 - 1 Results per page: 25 50 100 Sort by: Pub Date (newest) Title Recent Additions Author Clear All Pub. Year Citation 1993 Greene, Ruth L., Jackson, James S., Neighbors, Harold W. Mental health and help-seeking behavior. Aging in Black America. Thousand Oaks, CA: Sage. Export Options: RIS EndNote XML [1]


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William Ampey, 89Published 10:08pm Thursday, July 26, 2012
William Ampey, 89
Born: Jan. 15, 1923
Died: July 25, 2012
William E. Ampey, 89, of Cassopolis, died Wednesday, July 25, 2012, at his residence. He was born Jan. 15, 1923, in Calvin Township to Carl and Mamie (Lawson) Ampey.
He was a member of Kalamazoo Lodge, F & AM and Penn Friends Community Church, Cassopolis.
He was preceded in death by his parents; one granddaughter, Emily Hunley; sisters, Martha Emily Wright and Jessie Powell; and brothers, Clarence Ampey and Oren Chavous.
William is survived by one daughter and son-in-law, Rebecca and David Hunley, of Cassopolis; one son, William O. Ampey, of Cassopolis; two granddaughters, Pamela Osborn and August Hunley; one grandson, Matthew Hunley; and sister-in-law, Ruth Ampey, of Clovis, N.M.
Family and friends will gather at 2 p.m. Saturday, July 28, for a graveside service in Calvin Community Cemetery, Calvin Township, with the Rev. Carl Wright, pastor of First Baptist Church, Decatur, officiating.
The family prefers memorial contributions Hospice at Home, Inc.-St. Joseph, 4025 Health Park Lane, St. Joseph, MI 49085.
Arrangements have been entrusted to Wagner Family Funerals, Cassopolis.
Online condolences may be posted at wagnercares.com.


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Nick & Sarah's Betrothal Party
August 10, 2008 Sacramento, California
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On March 8, 2013, U.S. Citizen and Immigration Services (USCIS) published a new two-page version of Form I-9, together with six pages of instructions. The new form will be required for use by all employers beginning May 7, 2013. Until May 7, employers may continue to use the versions dated 08/07/09 and 02/02/09. However, employers may also begin using the new form now.
Employers do not need to complete the new Form I-9 for current employees for whom there is already a properly completed Form I-9 on file unless reverification is necessary. While the new form is available in fillable Adobe format, to avoid allegations the employer (or someone else) completed Section 1, for now employers are encouraged to print the new form and have it completed it by hand. In some jurisdictions, Immigration and Customs Enforcement (ICE) may consider an Adobe-filled Form I-9 to be an "Electronic I-9," which would bring into play additional regulations concerning the creation and storage of Electronic I-9s.
Listed below are some of the notable features of the revised Form I-9.
In addition, the new Form I-9 contains two spaces for a "3-D Barcode." For now, this space is a place- holder for use in the future. USCIS has indicated it is developing an "enhanced Form I-9," presumably to be completed online, that will capture the information presented by the employee and the employer and create a barcode in these spaces. USCIS has also separately announced that changes to the E-Verify memorandum of understanding are being considered. There is no way at present to determine if there will be any ties between an enhanced Form I-9 and E-Verify, but it certainly seems a possibility.
The release of the new Form I-9 comes at a time when there is considerable discussion in Congress regarding a federal mandate to require use of E-Verify by more employers. Nearly half the states require at least some employers to use E-Verify, and CPB is considering converting to paperless I-9s. Overall, the changes to Form I-9 and its expanded instructions should be seen as an evolving process that is intended to help lessen some of the confusion that has arisen in the completion of the form, while suggesting additional changes may be on the way for employers.
DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
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text:
Assembly.ReflectionOnlyLoad Method (String)
Loads an assembly into the reflection-only context, given its display name.
Namespace: System.Reflection
Assembly: mscorlib (in mscorlib.dll)
'Declaration Public Shared Function ReflectionOnlyLoad ( _ assemblyString As String _ ) As Assembly
- assemblyString
- Type: System.String
The display name of the assembly, as returned by the AssemblyName.FullName property.
Return ValueType: System.Reflection.Assembly
The loaded assembly.
assemblyString is Nothing.
assemblyString is an empty string ("").
assemblyString is not found.
assemblyString is found, but cannot be loaded.
assemblyString is not a valid assembly.
Version 2.0 or later of the common language runtime is currently loaded and assemblyString was compiled with a later version.
Dependencies are not automatically loaded into the reflection-only context.
You cannot execute code from an assembly loaded into the reflection-only context. To execute code, the assembly must be loaded into the execution context as well, using the Load method.
Whether certain permissions are granted or not granted to an assembly is based on evidence. The rules for assembly and security evidence merging are as follows:
Reflecting on executable files compiled in C++ might throw a FileLoadException. This is most likely caused by the C++ compiler stripping the relocation addresses or the .reloc section from your executable file. To preserve the .reloc address, specify /fixed:no when you are linking.
The reflection-only context is no different from other contexts. Assemblies that are loaded into the context can be unloaded only by unloading the application domain.