Abe 's family moved from the Bahamas to Japan, so they had convert their money into Japanese yen. Their checking account  had 6359.0 yen, and he has spent 3485.0 yen since then. How many yen do they have left? Answer:

If a farmer buys chicken feed for £4 per tonne, pig feed for £3 per tonne and cattle feed for 40p per tonne. How much of each would he have if he had 100 tonnes for £100?
Answer: 8 tonnes of chicken feed, 12 tonnes of pig feed and 80 tonnes of cattle feed.

Joe’s code has 20 bugs. If Joe fixes 2 bugs per hour for 8 hours, how many bugs does Joe’s code have now? Answer: 27.

Right now, Dr. Johnson is three times older than his son.
When Dr. Johnson was 37 years old, his son was 3 years old.
How old are Dr. Johnson and his son now?
Answer: Dr. Johnson is 51 and his son is 17 years old.
Assume that Dr. Johnson is X years old and his son is Y years old.
Then 3Y = X, and X – Y = 34.
Solving for X and Y, we get X = 51 and Y = 17.

Subtraction is the method by which we know the remaining after taken away some quantity from a certain quantity.
Step 1: Arrange the digits of the numerals as per the place value chart.
Step 2: Now subtract ones from ones, tens from tens and so on.
Minuend: The number from which other number is subtracted.
Subtrahend: The number which is subtracted from the other number.
Difference: When a number is subtracted from other, the product is called difference.
Subtract 15 lakhs from 12 crores.
The sum of two numbers is 7894562. If one of the numbers is 564897 then find the other.
A water tank contains 9800365 litre water. 456876 litre water is replaced by 9864 cubic stones. Find the remaining amount of water in the tank?
There are two boxes, first box contains only red balls whereas second box contains blue and white balls. Number of red, blue and white balls are 456456, 745879, and 25463 respectively. Jack threw all the white balls out of box. Find the remaining number of balls in the second box.
Second box contains two types of coloured balls blue and white. Jack threw all the white coloured balls out of box.
A and B represents two different villages. Population of the village A is 758945 in which 548796 are educated. Population of the village B is 879456 in which 256456 are educated. Find the total number of uneducated people.
Therefore, total number of uneducated people = 210149 + 623000 = 833149 .
Addition is the operation to know the total quantity when two or more than two quantities are taken together.
The numbers which are added together called addend and the problems is called sum.
When two or more than two numbers are added, the product is called sum.
Subtraction is the method by which we know the remaining after taken away some quantity from a certain quantity.
The sum of a number and 0 is equal to the number itself.
The result of subtractions is called the difference between the numbers.
Plus and minus are Latin words. In Latin plus means more and minus means less.
In the USA, -7 is pronounced as negative seven instead of minus seven.
Choose correct option for the expression 12 crores - 12 thousands.
Cody goes 45607 km north and returns back then he goes 84659 km east and returns back. Find the total distance covered by him.
Total distance covered by Cody = 45607 km + 45607 km + 84659 km + 84659 km = 260532 km.
Difference of the two numbers is 5648 less than 908564. If one of the numbers is 789006, find the other number from the following.
P = 454658, Q = 400085, R = 408789. Which one of the following is the greatest?
A + B is greater than C + D. Choose the correct option.
(a) If C is greater than A, B must be greater than D.
(b) If A is greater than C, D must be greater than B.
(c) If A is greater than D, B must be greater than D.
(d) If D is greater than A, B must be equal to D.

What will be the unit digit of 3√15625 ?
9 is the cube root of __________ .
What will be the unit digit of the cube root of a number ends with 7?
How many digits will be there in the cube root of 46656 ?
If 8³=512 , then3√512 = __________ .

Question: There are several books on a bookshelf. If one book is the 4th from the left and 6th from the right, how many books are on the shelf?
Answer: 9. Try sketching the problem out to see it.
Haha. There could also be seventeen in that case.

What is the value of 8 in 894,610?
Write the following number in numerals.
Write 795,201 in expanded form.
Write 512,070 in word form.
The population of Baroda is five hundred forty-four thousand seven hundred sixty. Write this in numeral.
Write 452,920,178 in expanded form.
Round 892,402,942 to the nearest hundred million.

There are 12 dialysis providers in Decatur, Georgia, providing a total of 241 dialysis stations and an average QIP rating of 0 out of 100.
Of these providers, 12 offer Hemodialysis, 4 offer Peritoneal Dialysis, 3 offer Home Training, and 2 of them are open after 5pm. For profit dialysis providers account for 10 of the facilities, with the other 2 listed as Non-Profit. 10 facilities are part of a chain of dialysis providers.

Everyone knows that Hong Kong is the vibrant financial hub of Asia and that Taiwan leads the world with delicious rice noodles and the best cycling environment. But did you know that both destinations offer amazing scenery, trails, and golden beaches along with fascinating cultural events?
"Cycling is very popular in Taiwan," says Linda Lin, Director – San Francisco Office of the Taiwan Tourism Bureau, "with Sun Moon Lake elected by CNN Travel as one of the world's best cycling routes.
Island hopping is a popular tourist pursuit in Hong Kong. The outlying islands offer a sharp contrast from Hong Kong's trademark hectic pace.
"Take the island of Cheung Chau, for example," says Michael Lim, Director of Canada, Central and South America for the Hong Kong Tourism Board, "it has no cars, beautiful scenic walks, fresh seafood and the popular Bun Festival. It's a great place to relax and experience the culture and lifestyle of the fisher folk."
With nine national parks and a rich marine ecology, it's not difficult to see why the Portuguese used the name "Formosa," meaning beautiful island, to describe Taiwan when they arrived in the 16th century. In the 21st century, the customs and traditions of Taiwan's indigenous people are still celebrated through events such as the Harvest Festival (Smatto), the Worship of Hunting (Mabuasu), snake worship, totemism, and various other spiritual rituals.
More information on Taiwan's vibrant cultural and traditional festivals and activities can be found here.
"The best thing to do," Michael says, "is to come and walk through the various neighbourhoods in Hong Kong and Taiwan and enjoy the different experiences. See how people live and interact with each other. Enjoy the street markets of Hong Kong and make sure to visit the night food markets in Taiwan. Both destinations are about living the culture."
For the first time visitor, the Tourism Boards of Taiwan and Hong Kong in conjunction with China Airlines can recommend a wide variety of packaged trips or guided tours to experience the fascinating cultures, vibrant cities and dramatic outdoor experiences of both Hong Kong and Taiwan.
For more information on exploring the vibrant cities, peaceful bike trails, and golden beaches of Hong Kong and Taiwan, visit www.taiwanhongkong.com.

Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.
Clostridium chauvoei is an anaerobic, endospore-forming Gram-positive bacterium known to be the causative agent of blackleg, a severe disease with high mortality affecting young cattle and sheep of any age. While C. chauvoei infections are specifically found in ruminants, a case of human fulminant gas gangrene caused by C. chauvoei was recently described . Ingestion of C. chauvoei spores is probably the most common form of exposure and infected ruminants do not directly transmit the disease to other animals. The endospores of C. chauvoei can lie dormant in the soil for years and, after ingestion by the animal, they are assumed to cross over the gastro-intestinal tract, enter the bloodstream and finally migrate in various organs and muscles, where they remain dormant until stimulated to cause disease . These spores are only activated in a low oxygen environment, such as that of damaged or bruised tissue in cattle. In sheep, blackleg is frequently associated with wounding, such as at shearing cuts, tail docking and castration site . Once these stimulating events occur, the spores in the infected tissue germinate and multiply into the disease-causing C. chauvoei bacteria. Then, the disease progresses rapidly, with the infected animal dying in 12 to 36 h after the appearance of the first symptoms . The molecular mechanisms of blackleg are not clearly understood. Due to the peracute evolution with severe oedematous lesions restricted to the local area, fever, loss of appetite, lameness and depression observed in blackleg outbreaks, it is speculated that virulence of C. chauvoei is caused by rapid spread of the activated, vegetative form of the bacterium in the infected tissue, followed by the production of potent toxins [4, 5]. Although sialidases and toxins produced by C. chauvoei are believed to play a significant role in the spread of the pathogen and in the induction of lesions in affected tissues , they have not yet been characterized at the molecular and genetic level. It has to be mentioned here that, to date, only a few DNA sequences of C. chauvoei have been deposited in DDBJ/EMBL/GenBank databases including the 16S and the 23S rRNA genes, the 16S-23S rRNA spacer region, and the flagellin gene [6, 7].
Sialidases, or neuraminidases (EC 3.2.1.18), are enzymes that cleave N-acetylneuraminic acid from carbohydrate polymers, such as mucin, glycoproteins, gangliosides and other sialoglycoconjugates, located on many mammalian cell membranes. The mature sialidase is generally composed of three main domains (Figure 1). At its N-terminal end, it contains the F5/8 type C domain, alternatively designated CBM32 (carbohydrate binding module 32) or discoidin domain, that appears to be primarily involved in binding of terminal galacto-configured sugar residues and is also found in the C. perfringens sialidase NanJ . The central domain contains a CBM40 module (or lectin-like sialic acid binding domain) that was shown to be specific for binding sialic acid residues in NanJ of C. perfringens. The enzymatically active site of sialidases is in the C-terminal end that contains the usual catalytic residues that are common to all sialidases [9–11], including a catalytic nucleophile tyrosine, an arginine triad that interacts with the carboxylate group of sialic acid, a tryptophan residue that forms a weak H-bond with the glycerol side chain of sialic acid, and an aspartic acid that forms H-bonds with the four OH-groups of sialic acid. Furthermore, the enzymatically active domain contains four "Asp-boxes"  characterized by the motif Ser-X-Asp-X-Gly-X-Thr-Trp, and the first arginine of the catalytic triad of sialidases generally belongs to a RIP (Arg-Ile-Pro) motif .
Schematic representation of the NanA sialidase of C. chauvoei. Upper part: DNA sequence of the promoter region of nanA of strain JF4135 with -35 and -10 boxes and the ribosome binding site (RBS) underlined. The translational start codon (ATG) is indicated by upper case letters. Lower part: main domains of the NanA polypeptide. Numbers refer to amino acid positions and black bars represent the characteristic "Asp-boxes". Si is the signal sequence and RIP indicates the position of the first Arg residue of the catalytic triad with the characteristic Arg-Ile-Pro sequence. The horizontal lines indicate the part of NanA that was expressed as a recombinant 40 kDa polyhistidine-tailed peptide to produce monospecific polyclonal antibodies in rabbits (upper) and the part of NanA expressed as enzymatically active recombinant protein (lower).
Sialidases have been found in viruses, bacteria, protozoa, fungi and metazoans . In viruses, parasites and bacteria, sialidases are thought to be important for survival and pathogenicity of the organisms . It has been established that viral sialidases have a central role in the pathogenesis of influenza [14, 15]. The importance of sialidase in respiratory tract infections has been demonstrated for two bacterial species: Streptococcus pneumoniae and Pseudomonas aeruginosa. The neuraminidases (sialidases) NanA and NanB of S. pneumoniae have been shown to be essential for successful colonization of both upper and lower respiratory tract and for pneumococcal survival in the blood using an outbred MF1 mouse or chinchilla model [16, 17]. The sialidase produced by P. aeruginosa has been shown to be a critical factor for colonization of the upper respiratory tract. It has also been shown that viral neuraminidase inhibitors blocked biofilm production and the bacterial colonization by P. aeruginosa. Recently, Mally and colleagues  demonstrated the impact of sialidase in Capnocytophaga canimorsus, a commensal canine bacterium of the oral flora, that can cause severe septicaemia and meningitis in humans . Wild type C. canimorsus is dependent on a surface exposed sialidase for growth and persistence in a murine infection model, whereas a sialidase deficient C. canimorsus mutant was unable to grow and persist in mice . Sialidase may have an important role in blackleg by degrading tight junctions upon cleavage of sialic acids at a high rate, thus allowing the bacterium to spread through host tissue . A sialidase activity has been reported in C. chauvoei, but the sialidase enzyme has not yet been characterized in detail [22, 23]. Furthermore, the molecular and genetic basis of sialidase in C. chauvoei is still unclear.
This study reports the cloning of the nanA sialidase gene from C. chauvoei, its expression and the characterization of the sialidase NanA protein. Under native conditions in bacterial culture medium, C. chauvoei sialidase is found as a 150 kDa protein, in the form of a dimer of two NanA peptides with an apparent molecular mass of 72 kDa.
The seven C. chauvoei strains used in this study are listed in Table 1. The C. chauvoei strains were grown anaerobically on Tryptic Soy Agar (TSA) medium containing 5% sheep erythrocytes (bioMérieux, Geneva, Switzerland) or in TGY broth (3% tryptic soy broth (Becton Dickinson AG, Allschwil, Switzerland), 2% glucose (Sigma-Aldrich chemical, St. Louis, MO, USA), 1% yeast extract (Becton Dickinson AG), and 0.1% L-cysteine (Sigma-Aldrich chemical)) at 37°C for up to 72 h. Escherichia coli strains were grown in Luria-Bertani (LB) broth or on LB agar at 37°C. Antibiotics (ampicillin, 100 μg/mL) were added for selection of E. coli carrying recombinant plasmids.
DNA from C. chauvoei strains was extracted by the guanidium thiocyanate method . Ligation, subcloning, plasmid extraction of the DNA fragments and agarose gel electrophoresis (0.7%) were performed as described . Plasmid extraction from recombinant E. coli clones was done by alkaline lysis using a Miniprep kit (PeqLab Biotechnologie GmbH, Erlangen, Germany). Sequence analysis was performed using an ampli-Taq FS dye terminator kit (Applied Biosystems, Foster City, CA, USA) and the universal primers complementary to the T3 and T7 promoters flanking the multiple cloning site of pBK-CMV, or primers that were derived from obtained DNA sequences.
Comparisons of DNA sequences and their corresponding amino acid (aa) sequences with DDBJ/EMBL/GenBank databases were performed using BLAST analysis . The molecular mass and theoretical isoelectric points (pI) of the sialidase were calculated using ProtParam . Pattern and profile searches were carried out at the online servers Motif Scan  and InterProScan . The presence and location of signal peptide cleavage sites in the amino acid sequence were predicted with the program SignalP 3.0 . Prediction of transmembrane helices and topology of the sialidase was carried out with the programs HMMTOP 2.0  and Phobius . The neighbour joining tree of aa sequences from clostridial sialidases was constructed with multiple amino acid sequence alignment by ClustalW2 .
A culture (100 mL) of C. chauvoei strain JF1863 (Table 1; type strain ATCC10092) grown for 72 h in TGY medium was centrifuged at 9000 × g at 4°C for 20 min. Proteins in the supernatant were precipitated overnight at 4°C upon addition of 70% (w/v) ammonium sulphate. Precipitated proteins were sedimented by centrifugation for 10 min at 12000 × g. The pellet was suspended in 10 mL of dialysis buffer (0.1 M Na-bicarbonate, pH 8.3, 0.25 M NaCl) and then dialyzed overnight at 4°C with gentle stirring. Aliquots of 500 μL containing 200 μg of dialyzed proteins or 50 μg of purified recombinant protein were mixed with the same quantity of Gerbu adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany) for immunizing rabbits three times at intervals of two weeks. Rabbits were bled two weeks after the last immunization and all serum samples were then incubated for 30 min at 56°C to inactivate complement.
Genomic DNA of C. chauvoei strain JF4135 (other name: C6 O/D1126/04; Table 1), a strain that caused acute blackleg in Switzerland in 2004, was partially digested with Sau 3AI and fragments from 2 to 4 kb were selected to construct a genomic library, using Bam HI-digested λ-ZAP-express vector arms, which was packaged with the Gigapack-11 packaging system (Stratagene, La Jolla, CA, USA). The phage library was plated using standard protocols on the E. coli strain XL-1 Blue MRF'. Screening of transformed E. coli was performed with rabbit serum against secreted proteins of C. chauvoei, raised as described above, used at a dilution of 1:100. Positive clones were amplified and finally subjected to in vivo excision using the f1 helper phage in E. coli strain XLOLR to obtain phagemid clones in expression vector pBK-CMV. A clone that strongly reacted with the rabbit hyper-immune serum was revealed to contain a 1.7 kb fragment whose sequence showed similarity to the sialidase gene of Clostridium septicum, as determined by BLAST analysis. The remaining part of the sialidase gene, named nanA (neuraminidase A), was detected by screening the same genomic DNA library by DNA:DNA hybridization with a 512 bp digoxigenin-labelled probe amplified from the cloned 1.7 kDa fragment with primers digSiaCC_605F (TTACTAGTGGAAATGGTG) and digSiaCC_1117R (CTTTTGCTGTAGTTTCAC). Digoxigenin labelling of the probe was done according to the manufacturer's instructions (Roche Diagnostics, Rotkreuz, Switzerland). Upon analysis of few clones harbouring partial sequences of nanA by primer walking with an ABI Prism model 3100 genetic analyzer (Applied Biosystems), the complete DNA sequence of the cloned gene and the sequences upstream and downstream were determined. Sequence alignment and editing were performed using the software Sequencher 3.0 (GeneCodes, Ann Arbor, MI, USA). The full length nanA gene of strain JF3703 (other name: 595E; Table 1), a highly virulent strain isolated in New Zealand in 1959, was determined by PCR amplification and DNA sequence analysis using the same primers as for strain JF4135. The sequence data of the sialidase gene nanA have been deposited in the EMBL Nucleotide Sequence Database (European Bioinformatics Institute, Cambridge, UK) under accession numbers FM213081 for strain JF3703 and FM213082 for strain JF4135.
The presence of the nanA gene of C. chauvoei was demonstrated in all seven C. chauvoei strains examined (Table 1) by PCR using the primers SiaCC_1371F (ATCAGCAATAGATACATC) and SiaCC_1789R (TGACCTCTTCCTGGTCCTGT). PCR was carried out in 30 μL reaction mixtures containing 1 × reaction buffer B (supplied with FIREPol® DNA polymerase), 2.5 mM MgCl2, 0.4 mM of each primer, 1 mM dNTPs, and 2.5 U of FIREPol® polymerase (Solis BioDyne, Tartu, Estonia). Approximately 100 ng of genomic DNA was added as template. Cycling conditions on a 9800 Fast Thermal Cycler (Applied Biosystems) were 3 min denaturation at 94°C, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 60 s. A final extension step for 7 min at 72°C was included. Amplicons were purified with the High pure PCR product purification kit (Roche Diagnostics).
To generate polyclonal antibodies directed against the CBM40 sialic acid binding domain of NanA, the coding region from nt position 367 to 1389 of the nanA gene (corresponding to aa 123 to 463), which also includes the first Arg residue and the RIP (Arg-Ile-Pro) motif of the catalytic site of sialidases, was amplified from C. chauvoei strain JF4135 using the primers Sia40_NcoIF (ggtcccatgg GTATAAAAGAATACAAAATTTATGCG) and Sia40_HindIIIR (cccaagctt GATGTATCTATTGCTGATGATGCTCC) (nucleotides in lower case are extensions and underlined nucleotides are recognition sites for the restriction enzymes Nco I or Hin dIII). The resulting PCR product was cloned into the pGEM-T easy vector (Promega Corp., Madison, WI, USA) and transformed into E. coli strain DH5α. Recombinant plasmids with the correct sequence were then digested with Nco I and Hin dIII, and the DNA fragments were inserted into the expression vector pETHIS-1 . The cloned insert was sequenced to ensure correct fusion with the vector's poly-His codons before being transformed into E. coli BL21(DE3) cells  for expression. Induction and subsequent purification of the recombinant 40 kDa His-tagged protein was performed as described . Purified 40 kDa His-tagged protein was used to immunize rabbits as described above.
Pellets and supernatants of C. chauvoei strains (Table 1) incubated at 37°C under anaerobic conditions in TGY medium for 72 h were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting on nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated on 12% acrylamide gels by SDS-PAGE under denaturing or non-denaturing conditions . Non-denaturing gel electrophoresis was performed in the absence of reducing agents. In order to detect the sialidase, the immunoblot membranes were incubated with rabbit polyclonal anti-sialidase 40 kDa peptide serum (diluted 1:1000), followed by incubation with a phosphate- or peroxidase-conjugated goat anti-rabbit IgG heavy and light chains (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) diluted 1:2000.
To express enzymatically active recombinant NanA, we amplified the part of the nanA gene from nt position 364 to 2316 (corresponding to aa 122 to 772), encoding CBM40 (sialic acid binding module) and the enzymatically active domain but not CBM32 (Figure 1), from C. chauvoei strain JF4135 using the primers SiaCC_BssHIIF (ttggcgcgc AATGATAAAAGAATACAAAATTTATGCG) and SiaCC_HindIIIR (cccaagctt TATAAATTTCCATTTTCTGTTATTAAACC) (nucleotides in lower case are extensions containing recognition sites for the restriction enzymes Bss HII and Hin dIII, respectively, that are underlined). The resulting PCR product was cloned into the vector T-easy (Promega Corp.) and transformed into E. coli strain DH5α. Recombinant plasmids with the correct sequence were then digested with Bss HII and Hin dIII, and the DNA fragment was ligated into the multiple cloning site of vector pFastBac1 (Invitrogen, Carlsbad, CA, USA) digested with Bss HII and Hin dIII. Vector pFastBac1 was chosen in order to express active NanA in both prokaryotic and eukaryotic cells, such as insect cells, utilizing the baculovirus expression system. This plasmid harbours the polyhedrin promoter from the baculovirus, which was shown to also have an adequate ability for initiating expression of introduced heterologous genes in E. coli. E. coli DH5α was transformed with pFastBac1::nanA and with empty vector pFastBac1. Sialidase activity of recombinant E. coli was measured using the sialidase spot test described below.
The fluorogenic substrate 2'-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MU-Neu5Ac) (Sigma-Aldrich chemical) was used to assay sialidase activity in a spot test  and in a fluorimetric determination in culture fluids [41, 42].
For the spot test, a working solution of 15 μM MU-Neu5Ac in 0.17 M sodium acetate buffer (pH 6.5) was prepared. Either colonies of C. chauvoei, recombinant E. coli, or 10 μL of supernatant from liquid C. chauvoei cultures were spotted onto 1-mm Whatman filter paper that had been previously moistened with the MU-Neu5Ac working solution, and then incubated at 37°C for 15 min. The reactions were stopped (0.085 M glycine, 0.2 M sodium carbonate, pH 9.4), the filter papers air-dried and fluorescence observed under a UV light (360 nm excitation wavelength). Observable bright blue fluorescence was recorded as positive. Anti-sialidase 40 kDa peptide serum or pre-immune serum (both diluted 1:10) obtained from the same rabbit were spotted onto the filter paper, at some distance from the bacterial spots but still partially overlying them, to assess the neutralizing effect of the anti-sialidase serum against sialidase activity.
For the fluorimetric determination of culture fluid sialidase activity, strain JF4135 was grown anaerobically for 1 day at 37°C. After cell counting (OD600 nm), the culture was centrifuged at 2000 × g for 10 min and the supernatant was centrifuged again at 15000 × g for 15 min to obtain cell-free supernatant. The reaction was performed in a total volume of 100 μL containing 30 μL of 0.1 M sodium acetate buffer (pH 6.0), 10 μL of a 1 mM MU-Neu5Ac solution in water and 60 μL of sample (each sample consisted of 54 μL cell-free supernatant pre-incubated for 30 min at 37°C with 6 μL of anti-sialidase or pre-immune serum (at several dilutions) prior to fluorimetric quantification of sialidase activity). Samples were incubated at 37°C for 60 min and quantification of sialidase activity was carried out kinetically (at 30 sec intervals) in triplicate with an FLx800 fluorescence microplate reader (BioTek, Luzern, Switzerland). Liberated 4-methylumbelliferone (MU) was quantified at 360/40 nm excitation and 460/40 nm emission in relation to a calibration curve obtained with MU standards (serial 2-fold dilutions ranging from 0 to 100 μM).
The full length sialidase gene nanA of C. chauvoei strain JF4135 consists of 2319 bp. A strong ribosome binding site (AGGTGG) is found eight bases upstream of the putative start codon ATG and is preceded by a potential clostridial promoter sequence including a TATA box (TATAAAAT) and a -35 element (TTTACA) (Figure 1). The NanA protein derived from the nucleotide sequence consists of 772 aa, with a predicted molecular mass of 84.0 kDa and a pI of 4.89. The aa sequence derived from the DNA sequence reveals a leader sequence peptidase cleavage site between position 26 and 27 (Ile-Tyr-Ala↓Asp-Ile), which is characteristic for secreted proteins of Gram-positive bacteria. The putative mature sialidase consists of 746 aa with a calculated molecular mass of 81.1 kDa. At its N-terminal end, aa residues 42 to 186 (numbering always includes the aa residues of the precursor) form the CBM32 module (Figure 1). In the central portion of NanA, aa 190 to 370 form the CBM40 module. The enzymatically active site ranging from aa 384 to 770 contains the usual catalytic residues, common to all sialidases: Tyr-728 (catalytic nucleophile), Arg-400, Arg-613 and Arg-693 (forming the arginine triad, whereas Arg-400 is contained in the RIP motif), Trp-577 (that interacts with the glycerol side chain of sialic acid) and Asp-582 (that forms H-bonds with the four OH-groups of sialic acid). Moreover, the four "Asp-boxes" are found at aa positions 437-444, 570-577, 634-641 and 707-714 (Figure 1).
The aa sequence of NanA from strain JF3703 differs from that of JF4135 at 18 aa loci that do not affect catalytic domains or "Asp-boxes". Amino acid similarities to NanA were found with several clostridial sialidases (Figure 2). Most closely related is the sialidase from C. septicum with an aa homology of 79% and 82% identical nucleotides at the gene level.
Neighbour joining tree of clostridial sialidases. The tree was constructed by ClustalW2 with gap open penalty of 10 and gap extension penalty of 0.05. The bar (0.10) represents substitutions per site. The following sialidase amino acid sequences were aligned: C. chauvoei (strain JF4135; this study), C. tertium (GenBank: CAA69951), C. sordellii (GenBank: AAB38298), C. septicum (GenBank: CAA44916), C. perfringens NanH (GenBank: P10481), C. perfringens NanI (GenBank: NP_561641) and C. perfringens NanJ (GenBank: NP_561469).
All seven C. chauvoei strains used (Table 1) contained the nanA gene as revealed by PCR. Sequencing of the amplicons revealed minor nucleotide changes that do not affect however the size of expressed proteins. Monospecific polyclonal antibodies directed against the recombinant 40 kDa peptide representing the CBM40 domain of NanA (Figure 1) reacted on standard immunoblots containing supernatants of C. chauvoei cultures of all strains with a 150 kDa and to a lesser extent with a 72 kDa band (Figure 3 shows the results for strains JF3703 and JF4135). The latter band was somewhat smaller than the predicted molecular mass of 81 kDa for mature NanA calculated from the DNA sequence of the nanA gene. When non-denaturing gels were used for the immunoblots, only the 150 kDa band was detected (Figure 3), indicating that the C. chauvoei sialidase is a 150 kDa dimer of the 72 kDa NanA polypeptide.
Dimer form of NanA. Western blot with anti-sialidase 40 kDa peptide serum under denaturing conditions (panel A) showing the 72 kDa monomer, or under native conditions (panel B) showing the 150 kDa dimer. Lane 1, C. chauvoei strain JF3703; lane 2, C. chauvoei strain JF4135.
All strains were positive in the sialidase spot test, showing the same spot diameter. Sialidase activity was observed directly with bacterial colonies spotted onto the filter, as well as with supernatant of C. chauvoei cultures grown in TGY medium (Figure 4), showing that sialidase was secreted. Moreover, when part of the nanA gene encoding the lectin-like sialic acid binding module CBM40 and the enzymatically active domain, but not CBM32, was cloned into plasmid pFastBac1 and transformed into E. coli strain DH5α, sialidase activity was found in culture supernatant of the recombinant E. coli strain harbouring the plasmid pFastbac1::nanA but not in the E. coli control strain harbouring the empty vector pFastBac1 only (not shown). Sialidase activity secreted by all C. chauvoei strains used in this study and by recombinant E. coli was neutralized by monospecific polyclonal antiserum raised in rabbit against the recombinant 40 kDa peptide (Figure 4 shows the results for strain JF4135), showing the functional impact of the CBM40 module covered by this peptide.
Sialidase neutralization spot test. The solid rings show the diffusion zone of the culture supernatant of C. chauvoei strain JF4135. The dotted rings show the diffusion zone of: left, anti-sialidase 40 kDa peptide serum; right, pre-immune serum from the same rabbit.
Similarly, the anti-sialidase 40 kDa peptide serum had an inhibitory effect on the sialidase activity in culture supernatants of C. chauvoei as determined by a kinetic fluorimetric assay (Figure 5 shows the results for strain JF4135). The assay monitored the cleavage of the glycosidic linkage of 100 μM of substrate MU-Neu5Ac upon incubation at 37°C for 60 min with C. chauvoei culture fluids. While the culture fluid of 5 × 107 cells was able to release approximately 10 μM MU from the substrate, anti-sialidase serum could inhibit this activity by 60% when diluted 1:10 (Figure 5). Anti-sialidase serum diluted 1:100 and 1:1000 inhibited the activity by 40% and 20%, respectively. As expected, pre-immune serum from the same rabbit, used as a control at different dilutions, did not affect the C. chauvoei culture fluid sialidase activity.
Fluorimetric determination of culture fluid sialidase activity. Cell-free supernatant of C. chauvoei strain JF4135 was pre-incubated with different dilutions of anti-sialidase 40 kDa peptide serum or pre-immune serum from the same rabbit. Liberated 4-methylumbelliferone (MU) obtained upon cleavage of substrate MU-Neu5Ac with sialidase in culture fluids of 5 × 107 C. chauvoei cells was recorded at 30 sec intervals. Black circles, no pre-incubation with antibodies; white triangles, pre-incubation with pre-immune serum (diluted 1:10); squares, pre-incubation with anti-sialidase serum (white, diluted 1:1000; gray, diluted 1:100; black, diluted 1:10). Plots are the mean of three kinetic measurements of sialidase activity.
Due to the rapid progression and severity of blackleg, it is appropriate to assume that potent toxins are responsible for the rapid spread of C. chauvoei and for the severe lesions that are produced by the pathogen in the target tissues. It has previously been hypothesized that sialidase may have an important role in blackleg by aiding the spread of the bacterium through the tissue . Furthermore, sialidases from S. pneumoniae, P. aeruginosa and C. canimorsus have been demonstrated to be crucial for infection and colonization of the host [16–19]. From these data, it can be hypothesized that the sialidase of C. chauvoei might have an important role in the early stage of blackleg infection by degrading tight junctions upon cleavage of sialic acid from mucins and glycoproteins in the infected tissue. Cleavage of sialic acid results in decreased rigidity of the cell surface, thereby facilitating cell motility and thus rendering the target site vulnerable to massive attack by the pathogen that rapidly spreads in the infected tissue. Then, the production of toxins such as hyaluronidases (gamma-toxins), deoxyribonucleases (beta-toxins) and oxygen-labile hemolysins by these bacteria may give rise to necrosis of the infected muscles and alteration of vascular endothelium, both known as characteristic signs of blackleg .
Sialidase activity of C. chauvoei was reported previously  but the corresponding enzyme was characterized only biochemically and not genetically [22, 23]. Our genetic and biochemical data reveal a sialidase, named NanA, in C. chauvoei that is a 150 kDa homodimer composed of two identical polypeptides of an apparent molecular mass of 72 kDa. Differences between the predicted size calculated from the nucleotide sequence of nanA (81 kDa) and the apparent size observed by SDS-PAGE (72 kDa) have previously been reported for several bacterial sialidases, such as S. pneumoniae[43, 44], Clostridium tertium and C. septicum. Native sialidase enzymes from bacteria are described as multimers, mostly dimers, of polypeptides having molecular masses ranging between 50 and 80 kDa , which agrees with our data for the sialidase of C. chauvoei. Although the 300 kDa sialidase described by Heuermann et al.  might represent a multimer of the 72 kDa NanA protein, we did not detect such a protein on our immunoblots. The sialidase NanA of C. chauvoei is most closely related to the sialidase of C. septicum and to NanJ and NanI of C. perfringens (Figure 2), the structures of which have been studied in detail [8, 10]. In this respect, it is interesting to note that C. chauvoei and C. septicum are phylogenetically very closely related  and at one time were classified phenotypically as the same species .
The C. chauvoei sialidase NanA displays the same motifs as the C. perfringens sialidase NanJ . NanA has a CBM32, or F5/8 type C domain, located at position 42 to 186, which is absent in C. perfringens NanI [8, 10]. This domain does not seem to be essential for enzyme activity as demonstrated by the sialidase activity of the recombinant NanA without CBM32. This is in agreement with recent data that classify the CBM32 module as a special domain that is involved in binding complex sialic acid substrates containing galactose residues [9, 11]. The lectin-like sialic acid binding module CBM40 and the enzymatic active domains show strong similarities to NanI and NanJ of C. perfringens. The latter domain contains all the characteristic residues necessary for sialidase activity, as seen in all prokaryotic and eukaryotic sialidases , including the catalytic arginine triad with the RIP motif on the first of the three Arg residues, and the characteristic "Asp-boxes". Antibodies directed against the recombinant 40 kDa peptide that includes the entire CBM40 module and part of the enzymatically active domain of NanA, in particular the RIP motif (Figure 1), neutralized the secreted sialidase activity of C. chauvoei strains (Figures 4 and 5), confirming their importance in sialidase activity. Furthermore, this result suggests that NanA is the sole or predominant secreted sialidase of C. chauvoei. If other sialidases antigenically similar to NanA were present in C. chauvoei, supplementary protein bands would have been detected on immunoblots using the same anti-serum against the 40 kDa peptide, but no such additional sialidases were observed (Figure 3).
Since prevention of contact of ruminants with the endospores of C. chauvoei that are present in the contaminated soil is virtually impossible, vaccination is the only preventive measure to effectively control blackleg. Current vaccination strategy against this disease relies on the use of a suspension for injection containing formaldehyde-inactivated cultures of virulent strains of C. chauvoei and culture supernatant, generally presented in polyvalent formulations. These vaccines are efficient but the particular antigens necessary to induce protective immunity against blackleg are unknown, thus requiring expensive work-intensive animal models for potency testing of vaccine batches. Moreover, molecular quality control of current vaccines, design of novel vaccines and diagnostic tests are mainly hampered by the lack of basic knowledge on the molecular mechanisms of pathogenicity of C. chauvoei. Studies on the role of sialidases of Streptococcus pneumoniae in otitis media in children and of Propionibacterium acnes in acne vulgaris have shown that sialidases of these species induce antibodies that inhibit propagation of bacteria in the host tissues [17, 49, 50]. When challenged, chinchillas vaccinated with recombinant sialidase from S. pneumoniae, expressed in E. coli, showed significantly reduced colonization and middle ear invasion compared with non-vaccinated individuals. Taken together, these findings in animal studies and the data of the present work, whereby antibodies raised against the sialidase 40 kDa peptide of C. chauvoei effectively neutralized the activity of the infectious agent, indicate that sialidase of C. chauvoei may be a good vaccine candidate to protect ruminants against the pathogen and to prevent the occurrence of blackleg. Furthermore, we expect that a combination of this antigen with one or more polypeptides derived from the toxins produced by C. chauvoei may have a potentiated protective effect against blackleg.
Edy M Vilei, Anders Johansson contributed equally to this work.
This study was financially supported by a grant from Intervet/Schering-Plough Animal Health. The authors are grateful to Elisabeth Bagge (National Veterinary Institute, Uppsala, Sweden) for kindly providing isolates from Swedish outbreaks of blackleg.
EMV and AJ made equivalent contributions to the study. EMV carried out the fluorimetric determination of culture fluid sialidase activity, devised the cloning strategies and participated in the sequence analysis. He drafted the manuscript. AJ constructed and screened the genomic library. He carried out the molecular biological studies and was responsible for expressing the recombinant sialidases as well as testing the resulting polypeptides in immunoblotting and sialidase activity spot tests. YS participated in sequencing and in the immunoassays. KR participated in the design of the study. JF conceptualized the study and supervised all facets of the research. All authors read and approved the final manuscript.

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There are 14 dialysis providers in New Orleans, Louisiana, providing a total of 244 dialysis stations and an average QIP rating of 0 out of 100.
Of these providers, 14 offer Hemodialysis, 9 offer Peritoneal Dialysis, 3 offer Home Training, and 3 of them are open after 5pm. For profit dialysis providers account for 11 of the facilities, with the other 3 listed as Non-Profit. 11 facilities are part of a chain of dialysis providers.

If you need to find a file, and are on a git repo, use `git grep` instead of grep. Similar options, much, much faster.
LC_ALL="C" grep -r ... ?
Did you do another grep test after the git-grep one ?
When I grep large folders the first grep takes ages but the next ones are incredibly fast. There must be some kind of cache. Or it's just zsh doing it for me, who knows ?
Yup, I did several of them, they all looked the same.
The second run will be faster always because the *filesystem* keeps an in-memory cache of recently opened files.
I'll try it again tomorrow and get some more numbers and screenshots, I'm pretty certain the numbers didn't change between runs.
There is some variation between runs.
How do you get the time counter?
Ack was 10 times slower than grep, even with --css.
Couldn't run git grep properly with the time command, as the output gets piped to a pager and I have to type q to quit (adding my finger typing to the time result).
Typing q as fast as I can gets me maybe 0.020s faster than grep. Too little project maybe. Well, git grep is faster to type anyway, saving a lot of time.
This isn't apples to apples. The two commands are not equivalent, so of course, one is faster.
In this case, I have a huge untracked logs directory in the root of this directory, and git-grep ignores anything not tracked in the repo. grep is much much faster by ignoring that log directory and binary files.
But since the counts don't match, it is still not apples to apples.
Anyway, I wasn't aware of git-grep, so thank you for the tip. Definitely useful if you're not already using an aliased version of grep.
Maybe you're using a slow grep.
The time difference seems to be solely due to what files are searched. 'git grep' only greps through the files that are tracked, so skips all binary files, while 'grep' by default also searches through compiled executables, .o files, etc.
Been using `ag` for a while https://github.com/ggreer/the_silver_searcher. Please do give it a try and compare with these results.
Did you see the comments on HN?

The first is Under Pressure Derivatization, which is used mostly for qualitative purposes, but has been neglected in regards to its qualitative aspects.
Plate treated in full within 3 seconds.
Clean, safe and ease of use.
Safety of the working environment.
And the second, is the acquisition of the plate’s image, by scanning and saving it.
The image acquisition and saving are done through a scanner.&nbsp.
Two detection methods: Visible and 254 nm (the latter is used 90% of the time).
Images of the TLC plates are scanned and saved through a basic program in various formats including: jpg, eps, pdf, tif, etc.
AR2i’s Derivapress and Chromimage are tools which will enable you to produce results, quickly and efficiently. They were created in the optic that, in this fast pace world, we need to save time and maintain a good stride on the production floor. By limiting your error margin in derivatizing, scanning and safekeeping your TLC plates, you are able, not only to keep up, but take the lead of this venture.

365 LIVE @ MOD Palm Springs with Rory Snyder, The DO-Its and MORE!
Its the fourth annual MOD Palm Springs and we record the podcast LIVE from the event. Join us in our hotel suite where we set up our studio and invite guests from the event to participate. Cocktails, special guests are just part of the fun. The DO-Its drop by, Rory Snyder makes an appearance and hotelier Harry drops in on us. And it wouldn't be a LIVE recording without everyones favorite podcast game show, "Whats in Kalanis Bra"?

There are 16 dialysis providers in Charlotte, North Carolina, providing a total of 386 dialysis stations and an average QIP rating of 0 out of 100.
Of these providers, 16 offer Hemodialysis, 11 offer Peritoneal Dialysis, 6 offer Home Training, and 2 of them are open after 5pm. For profit dialysis providers account for 14 of the facilities, with the other 2 listed as Non-Profit. 14 facilities are part of a chain of dialysis providers.

When my friend John posted some tweets using pencils as an allegory for educational technology, he didn’t expect the #pencilchat hashtag he used to go viral. He’s suggested some reasons why the topic went viral, and he’s probably right. The one addition to his list that occured to me is that the idea of critiquing some of the arguments against educational technology is timely, given many teachers’ current struggles with the use of technology in their classrooms.
There are reasonable arguments against the use of technology in schools, or at least in being critical about our use. Unfortunately, these are not the arguments people use to attack the use of technology in schools.
#Pencilchat highlights the issues educators face in their use of technology. If you want to see what these issues are for yourself, you can download this archive of 10,000 of the tweets (which unfortunately, include some spam from when the discussion was trending) from the viral discussion.
It could make an interesting research project to go through the tweets and find all of the various arguments people outline for and against the use of educational technology. While some of the tweets are quite funny, and there’s a fair bit of repetition, there are also some insights into issues facing schools today.
I am currently taking Dr. Strange’s EDM 310 class. For one of the assignments I, along with my classmates, had to read John Spencer’s post, Don’t Let Them Take Pencils Home. Sadly, many of my classmates did not catch on that Mr. Spencer was not literally talking about pencils; it was a metaphor for technology. I believe that many people are just not ready to see technology in that light. Pencils are something we are comfortable with; technology is scary to those who have not used much of it. I hope this changes soon.
I thought Mr. Spencer’s post was such an interesting post though. I had no clue that it had caused such tag, #pencilchat, on Twitter!
Thanks for the other links to look at!
People sometimes miss subtle metaphors. It happens, and I’ve done it myself (many times). Your reasoning about people being comfortable with pencils is interesting. I hadn’t thought that could be a reason that people would get confused over the analogy, but you are probably right.


There are 5106.0 books in the Oak Grove school libraries.  They donated 1986.0 to the public ibrary. How many books are left in the school libraries/ Answer:

There are 50 forms in the 'Insurance' category, available on 6 pages. (You are viewing page 2.) Forms 10 through 18 are on this page, shown below. Click one of the forms below to discover more information.

I’ve just finished podding peas. I bought a whole box off a farmer who had a stall at the side of the road. All young peas. All fresh.
There were three hundred and seventy-two peapods in the box. I counted them first. And then as I podded each peapod I counted backwards.
Not only that, but each time I podded I counted the peas in each pod, and added the total up as I went.
Three hundred and seventy-two. Six.
Three hundred and seventy-one. Eleven.
Three hundred and seventy. Fourteen.
Three hundred and sixty-nine. Nineteen.
When I got to the end I had three peapods left.
So I had to count all the emptied peapods again. And indeed it came to three hundred and seventy-two. And then I had to count all the peas again and there were one thousand six hundred and seventy-four. By dividing the peapod number into the number of peas, I got the average number of peas in each pod: which was four and a half.
I’m going to freeze these peas. It will make eight freezer bags of peas, with two hundred and nine and a quarter in each bag. That means there’ll be 0.5732602739726027 of a pea a day for me to eat until the next pea season.
Goodness! That’s not enough! I’ll have to buy another box. I wonder if it averages out at four and a half peas per pod again. Is that enough? Perhaps I should get two boxes.

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Now the nights are drawing in I love getting cosy with the fire on, lighting a candle and making the home smell lovely. I always have a stock of Christmas candles as I always buy too many every year, however the autumnal scents seem to slip away as I never buy many of them. I thought I would share with you the few scents I am using to make my home smell nice and feel cosy this time of year.
I tend to find the autumnal scents are the smaller candles in my collection as I will burn a Christmas candle right from November if I can get away with it! I did however find this lovely candle when food shopping this past weekend, I loved the orange tone of the wax, it feels very autumnal and the candle itself is called Radiant Autumn with some beautiful orange roses on the front. The candle has a sweet vanilla scent to it, with it being described online as smelling like the 'bright sun on turning leaves and the vanilla notes of change.' Whatever this is officially scented as, the sweet element of the vanilla is lovely and I am so excited to have my home smelling of this as I burn it over October.
Onto the smaller votive samplers that I will be using over the Autumn and transitioning into the Christmas period I have the Cosy by the fire scent, which as the name suggests smells like you are sitting by the open fire. If there is one thing that gets me excited for autumn and cosy nights in it is the thoughts of an open fire, unfortunately, open fires are not only messy and a lot of work, as well as the fact we couldn't have one at our house even if we wanted one, so the candles are the next best thing! It also reminds me of our honeymoon as our log cabin had an open wood fire in and it was simply magical!
Cosying up under a blanket as the nights draw in has to be a typical sign of the changing weather and the autumn drawing in, so this candle caught my eye just by the name and the image of the cosy knitted blankets. Described as 'A touch of amber, a dash of petals and lots of all things soft and comforting' the scent reminds me of fabric softener that you put in the washer when doing laundry. It makes me think of burying my face in a scarf on the cold autumn mornings as I walk through the leaves, keeping my chin and nose warm as the nights get darker and it slowly turns to winter. I love the clean fresh scents, and this is slightly different to the fresh linen, but has a similar kind of feel to the fragrance, while having warm cosy notes.
Japanese Maple is a candle that screams autumn, from the beautiful muted pink shade to the crisp autumnal leaves adorned on the sticker. The candle is a more floral scent, having soft floral notes, however a subtle sweetness to the candle stops is feeling like an overly summery floral and gives it a depth and warmth that makes you feel cosy. The candle is reminiscent of summer days without feeling like you are burning a summer candle, and its nod to autumn makes the change of season feel exciting and makes you eager to burn it. I like that this is a little different for autumn as it isn't the scent range I would usually go for, however I am really enjoying the scent and cannot wait to make my house smell like this!
I am always looking for new scents to burn to keep my house smelling fresh and make it cosy and inviting for guests, so I am always hoping for new recommendations, so please do leave your favourite candles in the comments for me to explore and find some beautiful new ones to try and fall in love with!

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Black Friday, Small Business Saturday, and Cyber Monday are a great fit for every business — awesome sales and happy customers! Not only does sending a timely promotional email help your contacts take advantage of your limited deals, but it reminds them they can avoid trampling and deal-crazed fans by clicking your links and buying online.
I’ve researched the most successful campaigns (based on click-through rate) to give you some tips on how to maximize your promotional email marketing results this holiday season.
Looking at the subject lines of the most successful campaigns, one thing is clear: it’s not very clear. There doesn’t seem to be a common thread, but it does look like simply having “Black Friday,” “Cyber Monday,” or “Holiday Sale” helps. That being said, it’s always best to try and stand out from the standard, “Check out my Black Friday Deals,” so try to spice it up a little. Remember that your readers’ inboxes will be full of holiday promotional emails.
Use subject lines to stand out from the standard.
The first suggests an extra incentive for opening the mail, which is always a great attention grabber, and the second suggests taking time out for everyone’s favorite person: yourself.
Please note: Many senders used a lot of CAPS and exclamation marks in their subject lines. I don’t recommend this, as it can get your mail flagged as spam and filtered. Better: keep it short, sweet and relevant.
It’s always best to place your most important links near the top of your promotional email. One thing I noticed quite consistently is the drop-off in click rates as I scrolled down emails. Most readers will spend less than 20 seconds looking at your promotional email, so this makes sense. Put your best foot forward when you have their attention!
Keep it simple! Just adding a linked phrase like “Shop Now” can do the trick.
Make your links noticeable. I noticed that the most clicked links have slightly larger text or are in CAPS, with a different color than the rest of the text (I’m not talking flashing lights here, but just a tweak can really help). Try using red.
Using just one link will help draw attention to it and will keep your promotion simple and clutter-free. Since 50 percent of emails are now opened on phones, it’s important to bear simplicity in mind.
You could also try using a linked image. It immediately catches the eye and can be used to incite a call to action. The example below (an awesomely laid out email) features a clickable image that yielded the lion’s share of the clicks.
I particularly like this promotion as it has an eye-catching, rich (but uncluttered) banner and a humorous opening that reminds customers of the hassle they can avoid by snatching up her deals from the comfort of … wherever they happen to be. The clickable image includes a “shop now” call to action and the phrase “holiday sale.” It communicates the sale details with noiseless clarity and November colors.
A study by offers.com shows that 74 percent of frequent shoppers watch for coupon codes.
Many successful promotional emails include a coupon code (a.k.a. a promo code) to offer customers a discount.
It lends to the sense of exclusivity and makes your loyal customers feel appreciated. Some successful campaigns also use “VIP” in their subject line. It’s simple — your customers/fans are the reason for your company’s success. Let them know they’re special, and you’ll both benefit.
I also picked this one as it illustrates an awesome, festive banner and another example of a highly clicked image.
So … paint your banners, chain those links and make sure you’re battle hardened and fully prepared to make the most of this awesomely busy weekend ahead.
Macdara Bracken is a contributor to the former Mad Mimi blog, writing about email marketing for businesses and individuals. GoDaddy joined forces with Mad Mimi in 2014 to give small business owners, freelancers, moonlighters and more a simple, beautiful and powerful way to use email marketing to accomplish their goals. Want to email all your customers before your first cup of coffee? Check out GoDaddy Email Marketing.

The number of housing of Paris was estimated at 1 336 209 in 2007. These accommodation of Paris consist of 1 139 192 main homes, 80 931 secondary residences and 116 087 vacant accommodation.
Area of Paris is 105,40 km² for a population density of 20 806,75 habs/km² and for a housing density of 12 677,50 housing/km².
Here are below a set of graphic, curves and statistics of the housing of the city of Paris.
Must see: the population data of Paris, the road map of Paris, the photos of Paris, the map of Paris.
The number of housing of Paris was 1 336 209 in 2007, 1 322 540 in 1999, 1 304 428 in 1990, 1 279 730 in 1982, 1 238 732 in 1975 and 1 221 954 in 1968.
In 2007 the accommodations of Paris consisted of 1 139 192 main homes, 80 931 second homes and 116 087 vacant accommodations.
The following graphs show the history of the number and the type of housing of Paris from 1968 to 2007. The second graph is drawn in cubic interpolation. This allows to obtain more exactly the number of accommodation of the city of Paris years where no inventory was made.
The population of Paris was 2 193 031 inhabitants in 2007, 2 125 851 inhabitants in 1999, 2 152 423 inhabitants in 1990, 2 176 243 inhabitants in 1982, 2 299 830 inhabitants in 1975 and 2 590 771 inhabitants in 1968. Meanwhile, the number of housing of Paris was 1 336 209 in 2007, 1 322 540 in 1999, 1 304 428 in 1990, 1 279 730 in 1982, 1 238 732 in 1975 and 1 221 954 in 1968.
The number of inhabitants of Paris per housing was 1,64 in 2007.
The accommodation of Paris in 2007 consisted of 11 600 houses and 1 276 670 flats.
Here are below the graphs presenting the statistics of housing of Paris.
In 2007, 375 578 main homes of Paris were occupied by owners. Meanwhile, 1 139 192 households lived in the city of Paris. The following graphs present the statistics of these households of Paris.

According to the article “4 Tips for Managing Cash Flow in a Seasonal Business” by contributor Lisa Stevens on Entrepreneur’s website, many small businesses encounter at least some degree of seasonality; in fact, according to a recent Wells Fargo/Gallup survey, almost half of small businesses report that they have predictable times of the year that are busier or slower than others.
Know your best season. The first step when creating a cash flow forecast is to know your busiest and slowest seasons. Be realistic: Don’t overestimate peak season revenue and slow season expenses. If you’re an established business, take a look at your historical sales and determine the months with the highest and lowest revenues. For startups, you will probably have to rely on competitive research to project your cash flow.
List your recurring variable expenses. Fixed expenses, such as rent and utilities, are fairly easy to remember, but don’t forget such variable quarterly, biannual or annual expenses as taxes, insurance premiums and months with extra pay periods, if applicable. Determine what these variable expenses are and plan them out for a more accurate forecast.
Consider establishing a business line of credit. Even if you create a very detailed and realistic cash flow forecast, unexpected expenses may crop up during the year, such as when you need to make a large purchase, or your income may not be as high during a season as you anticipated. Such unforeseen costs can be particularly difficult to shoulder during a slow season, but having a business line of credit can help you stay afloat until times get better. With a line of credit, you can access the capital you need, often at a lower rate than with credit cards. Work with your banker to determine if this is a plausible option for you.
Keep your forecast updated. Create a rolling 12-month spreadsheet, and be sure to update it at the end of every month. When updating this document, add a new month to the end of the forecast so you can have a more complete picture of your business’ seasonal and overall health. With regular updates, you will be able to anticipate financial shortages or take advantage of periods where you have more cash on hand.

12/31 How many members does each band have?
Great! Here comes the next one.
Show the nickname of the leader of each band, together with the corresponding band_id and the number of members the band has.

We will learn how to solve step-by-step the word problems on addition and subtraction of whole numbers. We know, we need to do addition and subtraction in our daily life. Let us solve some word problem examples.
1. The population of a country in 1990 was 906450600 and next year it is increased by 9889700. What was the population of that country in the year of 1991?
Therefore, the population of that country in the year of 1991 is 916340300.
2. Aaron bought two houses for $1668000 and $2454000. How much did he spend in all?
Amount of money spent in all $4122000.
3. The sum of two numbers is 41482308. If one number is 3918695 then, find the other number.
Therefore, the other number is 37563613.
4. Mr. Jones deposited $278475 in a bank in his account. Later he withdrew $155755 from his account. How much money was left in the bank in his account?
Therefore, Mr. Jones has $ 122720in his bank account.
Note: We need to be careful while arranging the addends in columns.

56 episodes available. A new episode about every 19 days .
Start listening to Rick Paris on your phone right now with Player FM's free mobile app, the best podcasting experience on both iPhone and Android. Your subcriptions will sync with your account on this website too. Podcast smart and easy with the app that refuses to compromise.

Monster Truck Jam 2018 Schedule nster Jam® is the most action-packed live event on four wheels where world-class drivers compete in front of capacity crowds in both monster truck racing and freestyle competitions.
Monster Jam. Monster Jam shows are held throughout the whole year with more shows during the winter than the summer, and travel around the country in U.S. and Canada. Shows are also being held in other locations around the world, such as Europe, Australia, Mexico, Costa Rica, The Philippines, Saudi Arabia and recently Japan.
Shows Added To 2018 Schedule. An additional 28 dates have been announced as part of the 2018 Monster Jam season schedule. Preferred Customers can purchase presale tickets beginning at 10 a.m. local time.
Discount Monster Truck Jam Tickets. Promo Code for Monster Truck Jam Tickets: DISCOUNT Monster Truck Jam Tickets: All 2018-2019 Events! QueenBeeTickets.com is a family-owned and operated ticket exchange offering authentic tickets and legitimate savings on today's most popular events.
Monster Jam Tickets 2019 2020 Schedule & Tour Dates. We have tickets to meet every budget for the Monster Jam schedule. Front Row Tickets.com also provides event schedules, concert tour news, concert tour dates, and Monster Jam box office information. Thank you for choosing Front Row Tickets.com to be your Monster Jam live entertainment website.

actualy I'm a new person in this high-tech component..so it had a problem in my computer. my invisible toolbar...how i'm going to do? pls give some suggeustion? pls send me the answer asap ok .
I had to run my computer in safe mode to fix some problems I had been experiencing. Since the reboot my color on my backgrounds and exploring the internet is hazzy and distorted. Does anyone know how to fix this problem??
Whenever I try to put an image as the wallpaper on my desktop the screen freezes. I pres Ctrl+Alt+delete to end task, but sometimes I have to restart the computer. How can I stop it from freezing everytime I want to change the wallpaper?
I have the same problem. I am trying to figure out how to fix the error. For now I ctrl-alt-delete scrool down to wtools end task gets rid of it for a while. -----Original Message----- Wtoolsa window will not close. I can only move it out of my way, but how do I get rid of it? .
-----Original Message----- error message Wtoolsa caused an invalid page fault in module KERNEL32.DLL How do i fix this? . Reply, I have the same problem and am investigating. Did you get any answers yet?
help please, my monitor flickers when i scroll up and down, its giving me a headache.
I have tried to transfer photos from my digital camera to my computer, but keep getting a fatal exception message. It reads ' A fatal exception OE has occurred at 0028:FF054203 in VXD USBSTOR(01) + 0000163'. Does anybody have any ideas on how to rectify this?
Hello Group... This is my first post. Here my problem: Some users on my Work use a DOS Accounting Program and sometimes when they change the screen mode to fullscreen, the Operating System freeze or crash. Then the famous "blue screen", and I need to restart. Why? This occurs randonly.

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A 10% fuzzy setting means there is a 10% deviation, or a 90% match.
A 20% fuzzy setting means there is a 20% deviation, or an 80% match.
A 30% fuzzy setting means there is a 30% deviation, or a 70% match.
First – to extract only first data from the value zone matching the value pattern specified.
Last – to extract only last data from the value zone matching the value pattern specified.
All – to extract only all data from the value zone matching the value pattern specified.
ALL – Extraction will be performed on all pages of the document.
FIRST – Extraction will be performed on first page of the document.
LAST – Extraction will be performed on last page of the document.
Weight: Configured value is multiplied with confidence score value to calculate new confidence score.
In case of multipage file the page number and image rendered while Editing KV Rule is same as page on which KV Rule was established. Eg: If the user has established KV Rule on Page Number: 3 then while editing the KV Rule overlays will be drawn on Page 3.

At some of the world's most idyllic beaches, mysterious surges in shark attacks wreak havoc for vacationers and locals alike. Many of these attacks occur suddenly, in quick succession, and at surprising locations, threatening to tear communities apart. Desperate for answers, scientists search for clues as to what is causing this terrifying upsurge.

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Unhappy With Your Career? Manage Up!
Career Management: How to Re-Energise Yourself with a Demanding.
Career Management: Are YOU Lazy?
Career Management: Are YOU Lazy?
When Should Career Management Start?
30 Career Management Tips – Do something proactive every day.
Is Attending College a Mandatory Career Management Strategy?
When Should Career Management Start?
So when exactly is a good time to start laying the groundwork for one’s career? In thinking about it, I realized there are opportunities that middle school students can capitalize on to help them with their future careers.
How would you use social media channels such as LinkedIn, Twitter, Facebook and Pinterest to manage your career? We’ll provide five tips about how social channels are powerful conduits to identifying and managing your digital profile and voice.
However, doing so now can set you up for a career management crisis. This means that hiring managers and recruiters are still being inundated with candidates. The rules for job seeking and career management have changed significantly.
There are three things in this life that you absolutely have to manage, or at some point they will catch up with you. The last essential, your career, is what we’re here to help you with. While there are still a few jobs out there with this arrangement, the vast majority of companies no longer expect their employees to stay in one place for their entire career. It means that those who don’t actively manage their careers are going to get left behind.
Career Management: Are YOU Lazy?
Career Management: Are YOU Lazy? Related: 6 Career Management Hacks That Will Get You Ahead. Career Management Help. Try some career management. The post Career Management: Are YOU Lazy? Work It Daily. Warning!
Career management is boring. Seriously, for as much written about career management, people don’t act on the writing. You might read about career management, but when was the last time you did anything more than think about your career?
Every manager wants a more engaged and productive workforce. To achieve it we need to create workplace cultures that empower and engage employees by offering career management processes and initiatives. Productivity and Priorities – Real Career Management.
It is here to stay, necessary and fundamental when it comes to managing your career. At Work Issues Careers Advice Networking Social Media and Branding Social media is impacting the world of work.
Not only are these seasoned athletes some of the most focused players around, they also know a thing or two about climbing the ladder to career success. Here’s what the Olympics can teach us about managing our careers and going for the gold: Building & Maintaing a Personal Brand.
Throughout my career in HR, I have seen more people leave their current job for the wrong reasons rather than stay for the right ones. Related: 6 Career Management Hacks That Will Get You Ahead. Here are six key steps of effective career management: 1.
Career management refers to the planning, supervising, controlling, handling, coping and administrating one’s professional life. Overall, managing your career will help maintain and develop your professional growth, development, and direction.
Career management is boring. Seriously, for as much written about career management, people don’t act on the writing. You might read about career management, but when was the last time you did anything more than think about your career?
Social media is impacting the world of work and it can have a profound impact on your career if you understand how to use it. Social is here to stay, necessary and fundamental when it comes to managing your career.
Here are just a few of his quotes that get you thinking about how you approach the management of your career: About attitude. So why do we oftentimes let ourselves get pulled into negative thinking that doesn’t benefit what we are trying to accomplish in our careers?
Career Management: Are YOU Lazy?
Career Management Help. Try some career management. The post Career Management: Are YOU Lazy? appeared first on CAREEREALISM. Workplace careerWarning! I’m about to speak in code.
In reality, career management is a very significant and specific process that, when done properly, helps to ensure long-term career success. In order to get the most out of your career, you have to put some effort into caring for it.
Having a career is nothing like having a job. When you find your career, you know it, and you should be spending some level of energy managing it. . Related: Career Management: Top 10 Career Limiting Moves. 5 Simple Career Management Tips You Should Follow.
Career management tends to be something that many people are guilty of neglecting. No thought about the direction or how to manage your career until a problem arises. Career Management Dorothy Tannahill-Moran SEO1We go to school, launch a job and the next thing we know, we’re on autopilot. You perform your job, go home and that’s pretty much it. If [.].
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. For career management purposes, you’ll need to go to those. The management challenge.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. Aspiring Cubicle Warriors can learn a lot from how consultants practice career management.
Is Attending College a Mandatory Career Management Strategy?
The term “ career management ” is a bit abstract for some people. In reality, career management is a very significant and specific process that, when done properly, helps to ensure long-term career success. And, before you know it, your career will be managing you.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. 30 Career Management Tips: Manage your meetings Written by Scot Herrick on September 24, 2007 in Time Management 5 Comments - Leave a comment! This month, I’m providing a career management tip-a-day (along with other posts) to help you trigger your own career management activities.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. 30 Career Management Tips — Manage E-mail Written by Scot Herrick on September 19, 2007 in Time Management 6 Comments - Leave a comment! This month, I’m providing a career management tip-a-day (along with other posts) to help you trigger your own career management activities.
Unhappy With Your Career? Manage Up!
Why not manage up ? According to personal and career coach, Beverly Flaxington , trying to manage up within your company is sometimes the better alternative than looking for opportunities elsewhere. “It’s Photo Credit: Shutterstock The post Unhappy With Your Career?
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. 30 Career Management Tips — Owning your mistakes Written by Scot Herrick on September 5, 2007 in Personal Branding 1 Comment - Leave a comment! This month, I’m providing a career management tip-a-day (along with other posts) to help you trigger your own career management activities.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. 30 Career Management Tips — Provide Personal Communication Written by Scot Herrick on September 7, 2007 in business networking 1 Comment - Leave a comment! This month, I’m providing a career management tip-a-day (along with other posts) to help you trigger your own career management activities.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. 30 Career Management Tips — Brilliant Information Filtering Written by Scot Herrick on September 9, 2007 in Time Management 6 Comments - Leave a comment! This month, I’m providing a career management tip-a-day (along with other posts) to help you trigger your own career management activities.
30 Career Management Tips – Do something proactive every day.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. Doing so will help your career — you fix problems before they occur. The management challenge.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. And it cost them their job, their career, and totally changed their personal brand. The management challenge.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. The management challenge.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. 30 Career Management tips — Give Credit to Others Written by Scot Herrick on September 5, 2007 in Personal Branding 1 Comment - Leave a comment! This month, I’m providing a career management tip-a-day (along with other posts) to help you trigger your own career management activities.
Home Blog Cube Rules Products Dream Job Coaching Media Contact Scot Cube Rules provides job advice and support for career-minded individuals working in corporate cubicles. 30 Career Management tips — Review your interview with a friend [.] The management challenge.
Home About Us How It Works FAQs Contact Resources Request a Proposal Monday, January 25th, 2010 Career Management: It Takes a Village Last week I attended the first of many college planning meetings at my daughter’s school.
Career Management: How to Re-Energise Yourself with a Demanding.

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