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http://petapixel.com/2016/03/24/googles … -100-free/
"Photo enthusiasts all over the world use the Nik Collection to get the best out of their images every day," Google writes. "As we continue to focus our long-term investments in building incredible photo editing tools for mobile, including Google Photos and Snapseed, we've decided to make the Nik Collection desktop suite available for free, so that now anyone can use it."
The Nik Collection has 7 desktop plug-ins that provide a wide range of photo editing features, including mimicking the look of old cameras and films, retouching and correcting photos, darkroom retouching, adjusting color and tonality, HDR, image sharpening, and noise reduction.
Free is good...I may try it; anyone else know about it?
by craftybegonia 7 years ago
What is the best free photo editing software available online?
by John Crozier 6 months ago
What is your favourite piece of photo editing software?
by Linnit 9 years ago
What is the best free photo editing tool to use on a mac?This question is different because it is about free mac photo editing tools. I need something that does more than iPhoto but isn't a huge suite like Photoshop
by metropolitanphoto 9 years ago
I am looking for people who are interested in photo editing.It would be great to share some knowledge with you and work together on different projects.If you are interested please send me a email and some editing examplesee you Carsten
by Melanie Palen 7 years ago
It's a photo editing software ad and I've been seeing it for a few weeks now. Anyway, this ad portrays what I think is a pretty girl and then does this "fabulous" editing to make her look airbrushed and fake. This fakey airbrush thing is the work of this great software... supposedly...
by Anusha Jain 5 years ago
If you had free video editing software and knowledge to use it, what kind of videos would u create?With improving cameras in mobile, Video Shooting is pretty common. But many a times, you would want to re-organize your clips in more presentable manner for yourself and others. So if you'd a free...
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subset: commoncrawl


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When you have invested the time and effort in creating an internal talent sourcing team it is important that you apply appropriate processes and methodologies and move the skills of the team forward. Too often the processes can vary with the individuals, so by establishing a talent sourcing methodology you can ensure that the overall skills of the department will transcend those of the individual consultant or manager.
It is important to define a standardised process with all the forms and all the steps to ensure consistency and quality in your approach. In this case standardised does not mean mundane or dumbed down.
These processes should grow and adapt over time but under a controlled method of change and not subject to the whim of individuals. This investment in time gives a thoroughly professional and resilient approach to the entire process.
The most effective companies value the concept of employee development and progression. So, when they are looking for talent to fill a particular position their initial reaction is not to look to the market to see what they can find. On the contrary, they look inside their business to see if they have skills which they can bring on.
The difficulty is that interviewing internal talent is different to interviewing someone who is external to the company and so the internal recruiters will require different skills and knowledge.
Interviewing someone who is internal means that there are elements of the normal interview process which can be truncated: such as spending time explaining the company structure and internal processes.
On the other hand there are aspects of working with the internal candidate which require some sophisticated skills to bring out the best from them at the interview.
You have to be able to get them to identify their worth to the business, to be confident about their achievements and not be concerned that they are in jeopardy of risking their current position by looking at a new role.
Making the internal development process work successfully requires the buy in, commitment and support of the business. However if it can be made part of the culture then it will deliver tremendous rewards in terms of employee satisfaction and retention. Some companies have excellent internal referral schemes and some don't. Developing internal referral schemes can be very effective. It's along the lines of "it's who you know" because your top talent will tend to gravitate towards their equivalent peers in other organisations … top talent knows top talent!
So creating a scheme that rewards your existing staff for opening up their network to you and getting qualified referrals can save many man hours of searching and can effectively expedite the recruitment process.
There are methods for gaining referrals in an acceptable and ethical way but so many companies are very bad at doing it. Tony teaches the techniques for achieving this both through reward based schemes, but also through the use of a strong networks who can deliver names and summaries of potential candidates.


text: Not sure if it's been posted already, but earlier this month PP announced several policy/pricing changes. One of them has a potential to really hurt if you sell (on eBay, etc) and issue refunds. In the past and currently, most of the PP fee could be reversed in case of refund. Not anymore. More on PP's Web site, DoC and lots of other places. Bad news.


text:
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One of them had two closets and in the wall separating them there was a small hole. Her duties commenced at 7:00 am and mostly continued until 7:00 pm. Andrews halted us.
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The Prescott Kiwanis Student of the Week for Jan. 23 is Prescott High School senior Kyra Miller, who was nominated by Prescott High School family and consumer education/health teacher Angie Boles.
"Kyra has been an asset to the Early Childhood courses," Boles said. "She is enthusiastic about the subject and does a great job."
Parents: Ben and Melissa Miller
Community service / outside clubs / activities: I work at New Adventures Learning Center.
Hobbies / interests: I love my job working with young children. I also like to spend time with my family and friends.
School activities: I played softball freshman and sophomore year.
Future Plans: Go to Dakota County Technical College for an associate's degree in child development.


text: What to do today: Mon. May 19 Chet Faker and the Ashbridges Bay fireworks by Kate Robertson May 19, 2014 9:08 AM Back to Search Results × Expand × Events I'm a little torn on this because fireworks are generally awful for the environment. But the lights, sounds and warm community feeling the 15-minute display at Ashbridges Bay Park bring sure is a fun way to end the weekend and kick off summer. You can see the fireworks from different spots in the east end, but people tend to meet in the park, and along Lakeshore between Carlaw and Woodbine. Keep in mind that Queen East is a complete nightmare on account of the streetcar construction. Starts at 9:45 pm. Music Ever since Australian Chet Faker wrote a hit, he's been trying to prove he's not all pop. You'll find him at the Hoxton tonight. More here. Comedy Tim Gilbert has a tumblr of horrible tattoos designed by him, and he's probably one of the funniest guys in town. Tonight and tomorrow, he's recording a live comedy album at the Ossington. Find out more here.


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I Want to Apply for Citizenship
If you would like to become an Australian Citizen you can either visit www.homeaffairs.gov.au to download the relevant forms or telephone 131 880.
After completing the relevant paper work, you will be interviewed and then all information will be assessed by the Office of Home Affairs.
Once your application has been assessed and approved, your Certificate of Australian Citizenship is forwarded to the Council. Council's Citizenship Ceremonies are held on the second Tuesday of even numbered months at 11.00a.m (except for December, Australia Day and February). New citizens are invited to bring along family and friends to participate in the ceremony.
Our Australia Day ceremony is held at the Lennox Community Centre at 9am.
So, in short, the steps to follow to become an Australian Citizen are:
- Visit www.homeaffairs.gov.au or phone 131 880 to complete and lodge your application for citizenship grant
- Attend an interview
- Await confirmation advice from the Office of Home Affairs that your application has been successful
- Await advice from the Council confirming that relevant paperwork has been received and advice of the date of the next citizenship ceremony
- Confirm your attendance at the ceremony and advise number of guests you would like to bring with you to your Citizenship Ceremony. Council is obliged to return your Certificate to the Office of Home Affairs if you cannot attend the Ceremony on the date allocated
- Enjoy your ceremony with family, friends and other new Australian Citizens.
Citizenship Ceremony Dates and Locations for 2019
- Saturday 26 January 2019 - 9am - Lennox Community Centre
- Tuesday 9 April 2019 - 11am - Council Chambers, Ballina
- Tuesday 11 June 2019 - 11am - Council Chambers, Ballina
- Tuesday 13 August 2019 - 11am - Council Chambers, Ballina
- Tuesday 17 September 2019 - 2pm - Council Chambers, Ballina (Australian Citizenship Day)
- Tuesday 8 October 2019 - 11am - Council Chambers, Ballina


text:
Scholastic Book ClubScholastic Book Club is a great way to help increase your student's home library for little money! I will be sending home Scholastic catalogs periodically throughout the year; however, please feel free to shop and purchase them online. It makes it easier for me and you! :)You can place your orders two ways:
Fill out the order form on the back of the catalog and send a check (NO CASH!!!) back to school with your student
Place your orders online yourself through the Scholastic website :
Everything you order through our class helps to earn points towards free books for our classroom library!


text:
We previously reported that mast cell α-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. α-Chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1's α-chymase-mediated cleavage products reveals hydrolysis at Phe12-Cys13 and Phe23-Val24 in loop 1 and Phe101-Val102 and Trp105-Asn106 in loop 3 of the NH2-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to α-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, α-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with α-chymase increases gel B activity 2- to 5-fold, suggesting that α-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of α-chymase blocks degranulation-induced TIMP-1 processing (absent in α-chymase-deficient HMC-1 cells). Purified α-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell α-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.
Activated mast cells respond to allergen, tumor invasion, and fibrogenic injury by releasing tryptase and α-chymase, which are granule-associated tryptic and chymotryptic serine proteases, respectively. Upon degranulation, both proteases participate in proteolytic and nonproteolytic pathways regulating matrix protein degradation, receptor activation, peptide inactivation, and fibroblast, smooth muscle, or epithelial cell mitogenesis (1). Tryptase and α-chymase may also act in a broader range of homeostatic and pathologic tissue remodeling processes via interactions with a family of Ca2+- and Zn2+-dependent matrix metalloproteinases (MMPs)3 (2, 3, 4, 5, 6, 7). Secreted or cell surface-associated membrane-type MMPs participate in physiologic pathways such as embryonic development, organ morphogenesis, angiogenesis, and wound healing, and also contribute to the pathogenesis of arthritis, cancer, cardiovascular disease, and lung fibrosis (8). Posttranslational regulation of MMP activity depends upon conversion of proenzymes to mature active forms and their inhibition by tissue inhibitors of metalloproteinases (TIMPs). Proteolytic activation of pro-MMPs involves one or more activator proteases which remove the propeptide domain containing a critical Cys residue, thus disrupting the cysteine switch which confers active site latency (9). TIMPs inhibit activity of active MMP species and block activation of pro-MMPs by forming TIMP-MMP complexes in a 1:1 molar ratio. NH2-terminal TIMP domains occupy the MMP active site and COOH-terminal domains confer binding specificity through interactions with COOH-terminal MMP domains (10).
Mast cells may regulate local MMP activity by contributing zymogen-activating serine proteases and by secreting pro-MMPs. Our previous work demonstrates that mast cells secrete progelatinase B (progel B; MMP-9), which is activated extracellularly by degranulated α-chymase upon cleavage of the catalytic domain at two sites (2, 3, 4). α-Chymase-dependent activation of progel B in vivo is hypothesized to switch nonangiogeneic tissues to an angiogenic phenotype in premalignant lesions in a murine model of epithelial carcinogenesis (11). In addition to progel B, α-chymase also directly cleaves and activates procollagenase (MMP-1) and prostromelysin (MMP-3) (6, 7). Mast cells also express these two MMPs plus progelatinase A (MMP-2), which shares in vitro substrate specificity with gelatinase B (gel B) (4, 12, 13).
Since cells secrete pro-MMPs bound to TIMPs in a complex, zymogen activation likely requires prior TIMP processing which precedes proteolytic activation of the MMP moiety. TIMP-1 binds to progel B, which is secreted as a monomer or dimer, or in disulfide-mediated linkage with neutrophil gelatinase-associated lipocalin (NGAL) to form a ternary complex of progel B monomer, TIMP-1 and NGAL (progel B-TIMP-1-NGAL) (14, 15). Unbound TIMP-1 may be inactivated in vitro by cleavage, degradation, or chemical modification via proteolytic and nonproteolytic mechanisms involving serine or thiol proteases and reactive oxygen species, respectively (16, 17, 18, 19). However, mechanisms which regulate processing of MMP-bound TIMP-1 remain unclear. In addition to its ability to block pro-MMP activation and inhibit activity of mature MMPs, TIMP-1 also demonstrates erythroid-potentiating activity, stimulates steroidogenesis, regulates mitogenesis, and controls apoptosis via mechanisms which are independent of its MMP inhibitory activity (10). Thus, processing of TIMP-1 in the inflammatory milieu may not only alter the protease:antiprotease balance to favor proteolysis, but also attenuate or abolish its non-MMP inhibitory effects on specific cell populations. We report here that mast cell α-chymase cleaves free TIMP-1 and processes the progel B-TIMP-1 complex by inactivating bound TIMP-1 and activating progel B, without involvement of other secreted proteases, and that degranulation of α-chymase regulates processing of TIMP-1 secreted by mast cells.
Materials and Methods
Cell culture
BR dog mastocytoma cells or HMC-1 cells (obtained from J. Butterfield, Mayo Clinic, Rochester, MN) were maintained in continuous suspension culture in DME-H16 medium supplemented with 2% bovine calf serum (2) or IMDM with 2% FCS (20), respectively, as previously described. Murine bone marrow-derived mast cells (MBMMC) obtained from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were cultured in RPMI 1640 medium supplemented with 10% bovine calf serum and 50% medium conditioned by WEHI-3B cells, as previously described (21). Cells were maintained at a final concentration of 1 × 106 cells/ml and incubated at 37°C in humidified 5% CO2 and 95% air. Cells were harvested by centrifuging at 500 × g for 5 min, washing three times in Ca2+-and Mg2+-free PBS, and resuspending in serum-free culture medium to a final concentration of 10–15 × 106 cells/ml.
RNA blotting
Poly(A)+ RNA was isolated from BR mastocytoma cells incubated alone or in the presence of 100 ng/ml recombinant canine KL (kindly provided by Keith Langley, Amgen, Thousand Oaks, CA) using the Poly(A)+3) cDNA to nylon membranes, posthybridization washings, and autoradiography were performed as previously described (4). Densitometric data were obtained by analysis of autoradiographic signals generated by hybridizing the blot with labeled probe. To account for possible variations in signal intensity due to differing concentrations of mRNA present in each lane, the blot was also hybridized with a labeled probe for γ-actin. Densitometric data were then compared with control values obtained with the γ-actin probe.32P]ATP-labeled 0.79-kb dog TIMP-1 cDNA (gift from Philip Davies, Merck Research Laboratories, Rahway, NJ) or 2.3-kb dog gel B (
Aliquots of medium conditioned by mast cells were concentrated by Microcon-10 filters (Amicon, Beverly, MA), subjected to electrophoresis, and blotted onto polyvinylidene difluoride membrane (Polyscreen; NEN Life Science Products, Boston, MA). Membranes were washed in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.3% Tween 20 and incubated with polyclonal rabbit anti-TIMP-1 (Chemicon, Temecula, CA; rabbit polyclonal Ab is specific for TIMP-1 and does not cross-react with other TIMPs in the manufacturer's quality control assays) at 22°C for 1 h. After incubation of membranes with HRP-linked anti-rabbit Ig for 1 h and luminol and peroxidase reagents (Phototope-HRP Western Blot Detection kit; New England Biolabs, Beverly, MA) according to the manufacturer's protocols, signals for immunoreactive proteins were visualized on Hyperfilm ECL (Amersham Pharmacia, Piscataway, NJ).
Substrate cleavage
Purified recombinant human (rh) TIMP-1 (Chemicon) or the progel B-TIMP-1-NGAL complex (Calbiochem, La Jolla, CA) was incubated either alone or in combination with different concentrations of activator proteases including: purified rhα-chymase (22), purified rhβ-tryptase (Promega, Madison, WI), human neutrophil elastase (HNE; Sigma, St. Louis, MO), l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma), and purified rh stromelysin (MMP-3) catalytic domain (22 kDa; Calbiochem). Reactions were performed in 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 10 mM CaCl2, and 0.02% NaN3
TIMP-1 activity
MMP inhibitory activity of rhTIMP-1 was determined by incubating intact or α-chymase-processed inhibitor with active gel B monomer (Calbiochem). rhTIMP-1 was incubated alone or in the presence of various concentrations of α-chymase in reaction buffer at 37°C for different time periods. Reactions were stopped by incubation with Ala-Ala-Pro-Phe-chloromethyl ketone (AAPF-CMK; Enzyme Systems Products, Livermore, CA) at 22°C for 10 min at a final concentration of 25 μM. Active gel B monomer was incubated alone or with aliquots of each reaction mix in a 1:1 molar ratio at 22°C for 10 min. The gel B activity in solution was determined in a final volume of 200 μl of reaction buffer containing 25 μM AAPF-CMK in 96-well plates (Becton Dickinson, Franklin Lakes, NJ) with 10 μM DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH2 (Cha = b-cyclohexylalanyl; Abz = 2-aminobenzoyl(anthraniloyl)] (DNP-PGCHAK; Calbiochem) (23). Fluorescent products released by cleavage of substrate were detected at different times at excitation and emission wavelengths of 360 nm and 460 nm, respectively, using a CytoFluor 2350 fluorometer (Millipore, Danbury, CT) and a sensitivity parameter of 6.
NH2-terminal sequencing
Amino acid sequence was determined by the Biomolecular Resource Center at the University of California San Francisco. The reaction mixture containing the TIMP-1 products of α-chymase cleavage was electrophoresed onto a 4–20% Tris-glycine polyacrylamide gel and blotted onto sequencing grade polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid buffer containing 10% methanol. Protein bands identified by Coomassie blue staining of the membrane were excised and subjected to Edman degradation using a Procise 491 Protein Sequencer (PE Biosystems, Foster City, CA). Protein sequence alignments were performed using MacVector software (Oxford Molecular Group, Hunt Valley, MD).
Gelatin zymography
The gel B activity resulting from α-chymase cleavage of the progel B-TIMP-1-NGAL complex was detected by gelatin zymography as previously described (2). Briefly, reaction products were electrophoresed through 10% polyacrylamide gels copolymerized with gelatin (1 mg/ml). After electrophoresis, gels were washed twice for 30 min in 2.5% Triton X-100 and incubated at 37°C for 18 h in 40 mM Tris buffer (pH 7.5) containing 200 mM NaCl and 10 mM CaCl2. Gels were stained with Coomassie blue for 10 min and destained in 10% acetic acid containing 50% methanol. Clear zones of lysis against a blue background indicated gelatinase activity.
Enzyme assays
Soluble MMP activity was determined by detection of products released upon cleavage of fluorescently labeled proteins or synthetic peptides by active proteases. The gel B monomer or the progel B-TIMP-1-NGAL complex was incubated alone or with activator proteases at 37°C for different time periods in 200 μl of reaction buffer in 96-well plates with various concentrations of DNP-PGCHAK (23) or DQ gelatin (fluorescein conjugate from pig skin) (Molecular Probes, Eugene, OR) to measure gel B activity or FITC-conjugated casein (Sigma) to assay stromelysin activity. Specific activity of active gel B monomer or active stromelysin catalytic domain using DQ gelatin or FITC casein was 186 fluorescent units/μg/min or 280 fluorescent units/μg/min, respectively. Background levels of substrate cleavage by α-chymase, HNE, or trypsin were subtracted from individual determinations of soluble activity.
Degranulation studies
Harvested cells were resuspended in serum-free culture medium to a final concentration of 10–15 × 106 cells/ml. Cells were incubated at 37°C for different time periods either alone, with 2 μM calcium ionophore A23187 (Sigma), or with ionophore in combination with individual inhibitors (Sigma) to identify protease classes involved in the processing of endogenous TIMP-1. Various concentrations of PMSF, aprotinin, or AAPF-CMK were used to block serine protease activity, while E64 or phosphoramidon and BB94 (gift from Marc Navre) were used to inhibit activity of cysteine proteases or metalloproteases (Affymay Research Institute, Santa Clara, CA), respectively. Aliquots removed at specified intervals were centrifuged to pellet cells. To identify the electrophoretic profile of products resulting from processing of native dog TIMP-1, various concentrations of purified dog α-chymase (24) were incubated with control BR cell supernatants for different periods of time. Cell supernatants and cleavage reactions were stored at −20°C before analysis by immunoblotting.
Statistical analysis
Differences with a p < 0.05 using Student's two-tailed t test were considered to be significant.
Mastocytoma TIMP-1 expression
Our previous studies established that mast cells express progel B, which could be purified in zymogen form free of TIMP-1 from dog BR mastocytoma cells, which phenotypically resemble human mast cells in their protease complement (2, 4, 25). Since cells usually secrete pro-MMPs complexed to TIMPs in a 1:1 stoichiometric ratio, expression of progel B by mastocytoma cells predicted coexpression of TIMP-1, which binds the zymogen and active forms of progel B (10, 26). As seen in Fig. 1⇓, A and B, autoradiography reveals a single TIMP-1 mRNA signal whose steady-state level remains unchanged following KL stimulation of BR cells; by contrast, KL increases gel B mRNA levels by ∼5-fold, as previously described (4). As seen in Fig. 1⇓C, analysis of media conditioned by cells incubated for 18 h reveals immunoreactive bands at ∼12 and 14 kDa (bands b and c), an unexpected electrophoretic profile since native dog TIMP-1 migrates at 35 kDa, a size which differs from that of human TIMP-1 (28.5 kDa) due to the presence of an additional N-linked glycosylation site (27, 28, 29). Since serine proteases process TIMP-1 in vitro (30), cells were incubated in the presence of increasing concentrations of PMSF to determine whether proteolysis obscures the identification of native TIMP-1 bands. Incubation with PMSF yields additional ∼8-, 20-, and 27-kDa immunoreactive bands, while the maximum concentration yields an ∼35-kDa band (band a). Thus, serine protease inhibition reveals the presence of native dog TIMP-1 protein and its intermediate cleavage products. These data demonstrate that mast cells express TIMP-1 mRNA and protein, which may be processed extracellularly by serine proteases released from secretory granules.
α-Chymase cleaves and inactivates TIMP-1
As shown in Fig. 2⇓A, incubation of rhTIMP-1 with rhα-chymase at a 1:1 molar ratio for 2 h generates two principle products with molecular sizes of ∼18 and 10 kDa. By contrast, the tryptic mast cell serine protease, tryptase, has no effect in agreement with previously published data (5). The electrophoretic profile of α-chymase-processed TIMP-1 products differs from that of HNE which yields bands at 17 and 16 kDa (30, 31) and additional products of 8–10 kDa. Thus, these data suggest that α-chymase cleavage of unbound TIMP-1 involves limited hydrolysis to generate products that differ from those generated by HNE. As seen in Fig. 2⇓B, incubation of α-chymase with TIMP-1 at a 0.5:1 molar ratio yields a faint ∼18-kDa band. Increasing the molar ratio yields a more intense ∼18-kDa band and an additional band at ∼10 kDa. The time course shown in Fig. 2⇓C illustrates that at a ratio of 1:1, α-chymase generates the ∼18-kDa band by 5 min with the ∼10-kDa band visible by 15 min. The later appearance of the 10-kDa band suggests that 1) it derives from a separate cleavage site on the TIMP-1 parent protein that is less avidly cleaved or 2) that it derives from the 18-kDa band via cleavage at a site that is accessible only after generation of the 18-kDa band. The effect of α-chymase processing on the MMP inhibitory activity of TIMP-1 was explored by determining its effect on the ability of TIMP-1 to inhibit soluble activity of active gel B. As shown in Fig. 2⇓D, α-chymase processing of TIMP-1 decreases its ability to inhibit active gel B monomer by >80% compared with that of unprocessed TIMP-1. Thus, cleavage by α-chymase attenuates the ability of TIMP-1 to inhibit gel B activity in solution.
α-Chymase cleavage sites of TIMP-1
To clarify the mechanism of α-chymase processing of TIMP-1, we characterized the electrophoretic profile of the cleavage products and identified sites of hydrolysis. As seen in Fig. 3⇓A, analysis of cleavage products by SDS-PAGE yields two bands at ∼18 and 10 kDa under reducing conditions. NH2-terminal sequencing of the ∼18-kDa product yields two sequences (X1NX2DLVI and VGTPEV) in a molar ratio of ∼1:3, while that of the ∼10-kDa product yields two sequences (VAPWN and NSLSLAQ) in a molar ratio of ∼1:4, as shown in Fig. 3⇓B. Alignment of these sequences with the primary sequence of human TIMP-1 (29) reveals that α-chymase cleaves the Phe12-Cys13 and Phe23-Val24 bonds in loop 1 and the Phe101-Val102 and Trp105-Asn106 bonds in loop 3 of the NH2-terminal domain (residues 1–124 (32)). Whereas residue X1 is indeterminate due to the inability of Edman degradation to detect Cys residues, no residue was assigned to position X2 due to insufficient discrimination of chromatographic peak amplitudes in successive cycles. Thus, the P1 residue at each of the identified cleavage sites is aromatic (Phe or Trp) as expected of hydrolysis by a chymotryptic enzyme. These aromatic residues are completely conserved in all mammalian TIMP-1. By contrast, the residue on the other (P1′) side of the scissile bond varies in mammalian TIMPs. Met or Leu substitutes for Val24 or Val102 in rodent sequences, respectively, while His, Arg, and Ser substitute for Asn106 in the rat, mouse, and canine sequences, respectively (Ref. 33 and GenBank Accession no. AAD10632). As seen in Fig. 4⇓, HNE cleaves TIMP-1 at a single site at the Val69-Cys70 bond which is situated in an area of intermolecular contact with stromelysin-1 (MMP-3) (32, 34). By contrast, α-chymase inactivates free TIMP-1 by hydrolyzing at four sites in loops 1 and 3, which are distant from the active site and located in the five-stranded β-barrel of the NH2-terminal domain, yielding two identifiable cleavage products.
α-Chymase processing of the progel B-TIMP-1-NGAL complex
To determine whether α-chymase also cleaves bound TIMP-1, increasing concentrations of the enzyme were incubated with a ternary molecular complex comprised of progel B, TIMP-1, and NGAL (progel B-TIMP-1-NGAL). As shown in Fig. 5⇓A, incubation of α-chymase with the progel B-TIMP-1-NGAL complex at a 0.5:1 molar ratio of α-chymase to the TIMP-1 component results in the appearance of an ∼18-kDa band. Increasing the molar concentration of α-chymase yields an additional band at ∼10 kDa, while a 10-fold molar excess decreases the 28.5-kDa band of intact TIMP-1. Thus, the electrophoretic profile of cleavage products resulting from α-chymase processing of bound TIMP-1 is similar to that generated upon cleavage of free TIMP-1. These data suggest that bound TIMP-1 is susceptible to hydrolysis by α-chymase.
As seen in Fig. 5⇑B, immunoblot analysis of cleavage products using polyclonal anti-gel B Ab detects bands at 82–84, 42, and 20–35 kDa. Increasing the molar ratio of α-chymase to progel B in the complex from 0.5 to 2.5 decreases the level of detectable 92-kDa proenzyme, whereas molar equivalents >2.5 generate a 42-kDa product. As shown in Fig. 5⇑C, gelatin zymography (performed under nonreducing conditions) detects the disulfide-linked complex of progel B (92 kDa) and NGAL (25 kDa) at ∼117 kDa following electrophoretic dissociation of TIMP-1, as seen in lane C. The band at >203 kDa likely represents progel B homodimer (15). Incubation of the progel B-TIMP-1-NGAL complex in the presence of increasing molar concentrations of α-chymase generates a lower band differing in size by ∼10–20 kDa. These data suggest that α-chymase processes TIMP-1-bound progel B via a mechanism similar to its activation of progel B monomer, whereby removal of an ∼10-kDa portion of the propeptide domain converts the zymogen to active species between ∼68 and 82 kDa (2, 3).
α-Chymase activation of complexed progel B
To determine the effect of α-chymase processing of TIMP-1-bound progel B, the gel B activity of the progel B-TIMP-1-NGAL complex in the presence of activator serine proteases was assayed in solution over 2 h using the fluorogenic substrate DNP-PGCHAK. As seen in Fig. 6⇓, incubation of α-chymase with the complex at a 0.1:1 molar ratio increases activity 2-fold. Activity of the complex alone likely occurs due to contaminating amounts of gel B monomer (35). Coincubation of the complex with α-chymase at ratios of 0.5:1 or 1:1 increases activity by 3.5- or 4.5-fold, respectively. Incubation of molar excesses of α-chymase with the complex does not further increase activity (data not shown). Similar molar ratios of trypsin, an activator of progel B monomer (35) (and additional log ratios between 10−10 and 10−1; data not shown), do not increase activity to the magnitude induced by α-chymase processing. These data demonstrate that the observed increases in the complex's soluble activity following α-chymase processing do not result from its activation of contaminating progel B monomer. Incubation of the complex with a molar equivalent or less (data not shown) of HNE does not increase gelatinolytic activity of the complex. Therefore, α-chymase processing of the progel B-TIMP-1-NGAL complex activates the zymogen, increasing the gelatinase activity of the complex in solution.
Activity of the progel B-TIMP-1-NGAL complex
As seen in Fig. 7⇓, the activity of progel B-TIMP-1-NGAL in solution after processing by activator proteases was compared with that of a molar equivalent of active gel B monomer. Whereas α-chymase increases the activity of the progel B-TIMP-1-NGAL complex, neither HNE alone nor a 4-h pretreatment of the complex with HNE before addition of 0.2 or 0.5 molar equivalents of active MMP-3 (a mechanism of sequential proteolytic processing previously shown to activate the progel B-TIMP-1 complex (30)) has any effect. Maximal soluble activity of α-chymase-processed complex represents ∼80% of the maximal activity of a molar equivalent of an active 82-kDa TIMP-free gel B monomer (relative to the 92-kDa progel B moiety in the complex). Thus, these data suggest that processing of the progel B-TIMP-1-NGAL complex by α-chymase overcomes TIMP-1 inhibition and results in recovery of the soluble activity of the gel B moiety following α-chymase-dependent zymogen activation.
Degranulation-dependent mast cell TIMP-1 processing
To determine the effect of degranulation on mast cell TIMP-1 processing, cells were incubated alone or in the presence of 2 μM calcium ionophore A23187 for 5 min. As seen in Fig. 8⇓A, supernatant of control dog BR mastocytoma cells contains an immunoreactive band at 35 kDa (band a), suggesting constitutive secretion of native TIMP-1; an additional band at 14 kDa (band b) suggests synchronous release of a related TIMP-1 product. Supernatant of either HMC-1 cells, a human mast cell leukemia line (20), or MBMMC reveals a similar pattern with bands migrating at 30 or 31 kDa, the expected sizes of human (with glycosylation-dependent variation from 28.5 to 34 kDa) or murine TIMP-1, respectively (29, 36, 37)). Supernatant of HMC-1 or MBMMC cells also contains the 14-kDa immunoreactive TIMP-1 band. These data suggest that mast cells constitutively release native TIMP-1 and that TIMP-1 secretion occurs in cells with either a normal or malignant phenotype. Upon degranulation, the electrophoretic profile of the supernatants of BR or MBMMC cells changes with the appearance of more bands between ∼17 and 29 kDa, in addition to the native and 14-kDa TIMP-1 bands present in control supernatants. By contrast, the profile of TIMP-1 bands present in HMC-1 degranulation supernatants remains identical to that of the control. HMC-1 cells are similar to BR or MBMMC cells in their expression of tryptase, which does not cleave TIMP-1 (as seen in Fig. 1⇑), but differ in their lack of expression of α-chymase (38). Therefore, these data suggest that degranulated mast cell α-chymase cleaves TIMP-1 extracellularly.
To identify the protease(s) involved in degranulation-associated TIMP-1 processing, BR cells were incubated with calcium ionophore A23187 alone or in combination with individual class-specific inhibitors. As seen in Fig. 8⇑B, incubation of cells with PMSF blocks the appearance of band c while aprotinin has no effect, thus implicating nontryptic serine proteases in processing of endogenous TIMP-1. By contrast, E64, phosphoramidon, or BB94 does not alter the profile, suggesting that cysteine proteases and metalloproteinases are not involved in extracellular TIMP-1 processing. By contrast, the appearance of the 14-kDa band remains unchanged, suggesting that its presence in supernatants is not due to extracellular proteolytic processing. Furthermore, the band appears not only in the supernatants of BR and HMC-1 cells (which are derived from a malignant phenotype), but also in that of MBMMC cells, which suggests that normal mast cells also release a 14-kDa form of TIMP-1 in a constitutive fashion. As seen in Fig. 8⇑C, incubation of cells with ionophore and increasing concentrations of AAPF-CMK (a serine protease inhibitor which inhibits α-chymase, but not tryptase) (2), diminishes the intensity of band c and blocks the appearance of the 17- to 29-kDa bands induced by degranulation. As shown in Fig. 8⇑D, incubation of purified dog α-chymase with control BR cell supernatants generates ∼8-, 12 (band c)-, 17-, and 27-kDa bands which share an electrophoretic profile similar to that of media conditioned by BR cells for 18 h (as seen in Fig. 1⇑); differences in the molecular weight of TIMP-1 cleavage products may result from species-dependent variations in glycosylation. Thus, addition of α-chymase alone results in processing of TIMP-1 present in BR cell supernatants. These data identify α-chymase as the degranulated mast cell serine protease responsible for extracellular cleavage of TIMP-1.
TIMPs act in diverse biological processes via mechanisms which may be either dependent on or independent of their ability to inhibit MMPs. In part, their myriad effects may be due to interactions involving structurally distinct NH2- and COOH-terminal domains, which block MMP-active sites and bind to MMP COOH domains, respectively (8). However, regulation of these TIMP domains is unclear. A unique feature of the gelatinase class of MMPs is binding of the zymogens, progelatinase A (MMP-2) and progel B, to TIMP-2 and TIMP-1, respectively (39, 40). Thus, TIMP-1 not only inhibits activity of mature gel B, but also blocks activation of the proenzyme. We previously reported that α-chymase, a chymotryptic serine protease unique to mast cells, activates progel B by cleaving the catalytic domain (3). The work here demonstrates that α-chymase also cleaves and inactivates TIMP-1 whether it exists free or bound in a complex with progel B. By cleaving TIMP-1 at exposed surface sites, α-chymase overcomes its inhibitory effects and converts complexed progel B to mature species with gelatinolytic activity in solution.
Whereas previous work demonstrated that mast cells express MMPs (2, 4, 12, 13, 25), the current data provide evidence that HMC-1 cells, dog BR mastocytoma cells, and MBMMC also secrete TIMP-1. Our previous data showed that ligation of mast cell kit receptor by KL up-regulates progel B mRNA expression ∼5-fold. By contrast, TIMP-1 mRNA levels remain unchanged following KL stimulation, possibly due to distinct mechanisms which regulate TIMP-1 expression via enhancement of mRNA stability (41). Thus, KL-kit interactions may alter local progel B to TIMP-1 ratios, favoring release of TIMP-1-free progel B, which would be more susceptible to activation. Mast cells not only release the native form of TIMP-1, but also an ∼14-kDa form whose presence in conditioned medium is explained neither by extracellular proteolytic processing nor by translation of alternatively spliced mRNA. Whether posttranslational intracellular processing or nonproteolytic extracellular mechanisms generate the 14-kDa form remains a subject of investigation. Both the native and ∼14-kDa forms of TIMP-1 also appear to be products of normal MBMMC, thus suggesting that the secreted TIMP-1 products of cultured canine and human mast cells do not only represent a malignant phenotype.
α-Chymase inactivates TIMP-1 by hydrolyzing bonds in the NH2-terminal domain. Whereas cleavage by HNE, trypsin, or chymotrypsin destroys the inhibitory activity of unbound TIMP-1 (31), only HNE has previously been shown to degrade TIMP-1 in the progel B-TIMP-1 complex (30). Like HNE, α-chymase cleaves TIMP-1, whether the inhibitor exists free or in a complex with progel B. However, α-chymase generates products that differ in size from those generated by HNE, suggesting that hydrolysis by α-chymase occurs at different sites. The four scissile bonds identified all contain aromatic P1 residues (Phe or Trp), which are favored by α-chymase and highly conserved in mammalian species, as seen in Fig. 4⇑; peptide sequencing did not identify cleavage at sites containing other α-chymase-preferred P1 residues (including Tyr and a hydrophobic residue, Leu), which localize to the surface of TIMP-1 (34, 42). This suggests that cleavage at these sites may not be favorable due to steric hindrance or that the cleavage products may either be transient or insufficient in quantity for detection and sequencing. Therefore, although α-chymase and HNE are similar in their inactivation of free or bound TIMP-1 via NH2-terminal domain processing, they differ in the number as well as general location of TIMP-1 bonds cleaved.
Conversion of complexed progel B to active forms requires processing which overcomes zymogen latency conferred by the cysteine switch (9) and inhibition of active species by bound TIMP-1. Cells secrete pro-MMPs noncovalently bound to TIMPs with 1:1 stoichiometry, suggesting that physiologic regulation of MMP activity depends on one or more activators, acting alone or in concert, to activate secreted proenzymes. Proteases which hydrolyze either free TIMP-1 or uncomplexed progel B monomer in vitro cannot cleave both components when complexed to each other (17, 30, 31). By contrast, at catalytic or stoichiometric molar ratios, α-chymase processing of the progel B-TIMP-1-NGAL complex results not only in cleavage of both TIMP-1 and progel B, but also in conversion of the zymogen to mature forms with up to a 5-fold increase in soluble activity. Activation of the zymogen in the progel B-TIMP-1-NGAL complex also suggests that NGAL (bound via disulfide linkage to progel B without altering the protease's activity (15, 43, 44)) does not hinder access of α-chymase to either TIMP-1 or progel B. Processing by catalytic amounts of α-chymase alone compares favorably with other proteolytic mechanisms which activate the complex via 1) molar excesses of either active stromelysin or matrilysin (MMP-7), which increase gelatinolytic activity by competitively binding TIMP-1 (without cleavage) and activating progel B (45, 46); or 2) sequential involvement of HNE, which cleaves and inactivates TIMP-1, and mature stromelysin, which activates progel B (30). In the present work, combined HNE and stromelysin processing of the complex did not increase its gel B activity, possibly due to differences in substrates and reaction conditions. Thus, α-chymase processing of the progel B-TIMP-1-NGAL complex not only cleaves and inactivates bound TIMP-1, whose hydrolysis appears to be rate limiting, but also converts progel B from zymogen to active forms.
Access to the NH2-terminal domain of complexed TIMP-1 is critical for cleavage and attenuation of its inhibition of gel B. Inactivation of TIMP-1 by HNE depends on cleavage of the Val69-Cys70 bond in the "C-connector loop" which forms the long edge that occupies the MMP-active site cleft (34). This region remains exposed and accessible to HNE when TIMP-1 binds to progel B. Other serine proteases such as trypsin and chymotrypsin can cleave free TIMP-1, but cannot process bound TIMP-1, suggesting that their preferred scissile bonds are obscured in the complex (30). By contrast, TIMP-1 regions cleaved by α-chymase are distant from the long edge and are thus likely to be accessible whether the inhibitor is free or bound, as seen in Fig. 4⇑. α-Chymase also cleaves TIMP-1 at multiple sites in loops 1 and 3, which may separate the NH2- and COOH-terminal domains. This contrasts with the single cleavage of TIMP-1 by HNE in loop 1, which leaves the domains linked via the disulfide bonds. Perhaps the liberated, inactivated NH2-terminal domain arising from α-chymase cleavage dissociates from the complex, facilitating subsequent activation of progel B. Whether the TIMP-1 COOH domain retains its non-MMP inhibitory functions following α-chymase processing remains unknown.
Hyperplasia and degranulation are key features of the mast cell response in inflamed tissues. Degranulating mast cells release α-chymase in active form from secretory granules where it is stored in high concentrations, second only in abundance to tryptase (1, 47). The current data demonstrate that secreted mast cell TIMP-1 remains in its native form until ionophore-induced degranulation releases α-chymase, which cleaves the inhibitor to generate products similar to those following incubation of purified α-chymase with mast cell supernatants. Lack of TIMP-1 processing by degranulating HMC-1 cells, which do not express α-chymase, further substantiates the dependence of mast cell TIMP-1 processing on exocytosed α-chymase. Whether nonproteolytic mechanisms may also process secreted TIMP-1 remains unknown. Mast cells express α-chymase at levels of ∼5 pg/cell (48), yielding a cellular concentration of ∼300 μM (which underestimates the concentration in secretory granules due to differences in granule and cytosolic volumes) that predicts even higher local tissue concentrations upon degranulation. In human tissues, α-chymase is found at a concentration of as much as ∼45 μg/g of tissue (48, 49), compared with a TIMP-1 concentration of ∼12.4 μg/g (50). Immunolocalization studies in a murine model of squamous epithelial carcinogenesis demonstrate limited diffusability of chymase; thus, it remains in the vicinity of degranulating mast cells situated near basement membranes, which are sites of extracellular matrix remodeling (11). The abundance of stored α-chymase and its limited diffusability suggest that in vivo mast cell degranulation may generate transient and localized molar excesses of α-chymase relative to its substrates such as TIMP-1. We speculate that such a quantum proteolytic event (51) may be an important mechanism whereby exocytosed mast cell α-chymase regulates both the MMP inhibitory and noninhibitory functions of TIMP-1 in remodeling tissues. Evidence of ongoing and gradual (or piecemeal) degranulation of mast cells in fibrotic tissues (52) suggests that such a mechanism may not only occur transiently, but also persist indefinitely. Therefore, the quiescence or activation of mast cells in lung, skin, heart, or gut tissues may determine not only the protease to antiprotease balance of MMPs to TIMPs which is critical in matrix remodeling and fibrogenesis (8, 52, 53), but also the ability of TIMP-1 to regulate apoptosis and mitogenesis via pathways which are independent of its MMP inhibitory functions (11, 54).
The current data suggest a role for mast cell α-chymase in physiologic pathways which regulate MMP activity. Unlike other proteases which require cascading activation of multiple proenzymes before activation of the target pro-MMP (2, 3, 5, 8, 17, 35, 55, 56, 57), α-chymase activates zymogen monomers of progel B, procollagenase, and stromelysin, without a requirement for other proteases or cofactors (2, 3, 6, 7). Data also show that infiltration of premalignant lesions by mast cells activates progel B via α-chymase processing, coincident with the activation of angiogenesis, thus demonstrating an in vivo role for α-chymase-dependent pro-MMP activation in tissue remodeling (11). In summary, our data show that cultured mast cells, such as HMC-1 or BR cells and MBMMC, secrete TIMP-1, which is processed by exocytosed α-chymase. Uniquely, α-chymase processes the progel B-TIMP-1-NGAL ternary complex by inactivating bound TIMP-1 and converting progel B to mature active forms. α-Chymase hydrolysis of TIMP-1 occurs at four scissile bonds containing exposed aromatic residues distant from regions which interact with the MMP active site. Cleavage attenuates the inhibitory activity of TIMP-1 and may separate its NH2- and COOH-terminal domains. Therefore, release of α-chymase may provide a novel mechanism for recovering and augmenting activity of progel B complexed to TIMP-1 during extracellular matrix remodeling in mast cell-rich environments.
We thank Ralph Reid and Wilfred W. Raymond for helpful discussions and Paul J. Wolters for providing murine mast cells. We also acknowledge the excellent technical assistance of Afshin Bidgol, John Blount, and Diego Muilenburg.
↵1 This work was supported by Grants HL-03345 and HL-24136 from the National Institutes of Health. K.C.F. is the recipient of a Mentored Clinical Scientist Development Award from the National Institutes of Health.
↵2 Address correspondence and reprint requests to Dr. Kenneth C. Fang, Box 0911, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0911. E-mail address: kfang{at}itsa.ucsf.edu
↵3 Abbreviations used in this paper: MMP, matrix metalloproteinase; AAPF-CMK, Ala-Ala-Pro-Phe chloromethyl ketone; DNP-PGCHAK, DNP-Pro-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH2; gel B, gelatinase B; HNE, human neutrophil elastase; KL, recombinant canine kit ligand; MBMMC, murine bone marrow-derived mast cells; NGAL, neutrophil gelatinase-associated lipocalin; progel B, progelatinase B; TIMP, tissue inhibitor of metalloproteinase; rh, recombinant human.
- Received October 4, 2000.
- Accepted December 7, 2000.
- Copyright © 2001 by The American Association of Immunologists


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By SCOOTER MACMILLAN
Staff Reporter
While several Vermont school systems are still struggling with school consolidation, not only has Charlotte and other towns in the Champlain Valley School District already dealt with merging, it wasn't their first trip to the school merger rodeo.
In 1869, Charlotte had at least 14 school districts, each with its own one-room school house. (If you have looked at the Beers Map of Charlotte dated 1869, you might be confused because you'll see District 15, but if you look more closely you will see there's no District 13. Local historian and president of the Charlotte Historical Society Dan Cole said his best guess is triskaidekaphobia, or fear of the number 13).
The number of schools went up or down as neighborhoods dwindled or grew until 1949 when all the Charlotte schools were merged into the brand-new Charlotte Central School.
One teacher taught all the subjects and all of the students, who ranged from 4 to 18 years old. Sometimes the students were older than the teacher, according to the book "Look Around Hinesburg and Charlotte, Vermont," published by the Chittenden County Historical Society in 1973.
In 1900, a teacher in the Charlotte schools earned $185 to $200 a year and they had to pay for their boarding.
"Originally teachers were boarded free, for families with school-age children were expected to take turns boarding the teacher for a month," says "Look Around Hinesburg and Charlotte, Vermont."
Some of the old one-room schoolhouses still stand, including the Quinlan School, the Emerson School and the Lakeview Seminary.
The Quinlan School
The Quinlan School is the old school that most people know about it since it's behind the Charlotte Library. It was disassembled and moved to its present location in 1996.
The Quinlan School is known as the Quinlan School because it was built by John Quinlan on his farm in 1855. It served elementary school students from Charlotte's District No. 7.
Coming to Charlotte in 1844, Quinlan cut firewood to make his living and went into farming. The first teacher was his daughter, Mary Quinlan, who taught there for more than 60 years until she died from influenza in 1918.
Local historian Dan Cole said that other teachers who served at the Quinlan School for many years were Susan Preston and Florence Horsford. When the Quinlan School reopened at the Charlotte Library, people donated old desks, so it is furnished much like a school in the 1800s.
The library has a history of the school, "The Quinlan School, A One Room Schoolhouse" by Jerrie Vane with more details about the school and a typical school day.
The Emerson School
The Emerson School is the oldest school in Charlotte, built in the 1791, according to "Look Around Hinesburg and Charlotte, Vermont."
And appropriately, it served school District No. 1.
This old school is located at the northwest corner of Thompson's Point Road and Lake Road. Bruce and Linda Williamson bought the old schoolhouse and renovated it into their home in 1986.
When they bought the building, it had been used as storage and one of the things that was stored in it was old school desks. They also found old report cards, said Bruce Williamson.
Although they've added onto it and raised the roof a bit, the building hasn't changed much.
The Lakeview Seminary
The Charlotte Female Seminary was built in 1836. When this building burned in 1880, a new school building went up that became the Lakeview Seminary.
The first principal of the Lakeview Seminary was the famous Burlington philosopher, psychologist, democratic socialist and educational reformer John Dewey.
He promoted "teaching by doing" and "hands on experience" in education, said Cole.
Dewey lasted at Lakeview Seminary for just one year, during the 1881-82 term. He said afterward that classroom teaching was not for him.
Dewey's and his wife's graves are the only ones on the University of Vermont's campus, next to the Ira Allen Chapel.
This school building on the east side of Greenbush Road just south of the intersection with Ferry Road became the school for District No. 5 as part of the Charlotte school system in 1895.
It was a public school from then until 1949, and Robert Larson went here for first grade in 1948 before moving to the new Charlotte Central School.
He said that there were two teachers, one taught first through third grade downstairs and the other had fourth through sixth graders upstairs.
"Our teacher would teach us then go on and teach the second graders and then we could go out and play," said Larson. "Or if we were smart enough could stay and learn with the second graders. And after she taught them, if could keep up to third graders, you could stay and learn with them."
Mark and Laurie Moser renovated the Lakeview Seminary building into their home in "1980 or 81," Mark says. It had been a home when they bought it, but before that it had been a post office.
Consolidation controversy is nothing new
The issue of combining schools and whether it makes schools better or less expensive isn't new. In an essay from 1899 (reprinted in "Around the Mountains"), William Wallace Higbee wrote:
"Close by stands the cozy little schoolhouse that used to be known as District No. 11 in the days before the legislature blotted the time-honored district schools from the map of the state. Were the question submitted to the people without prejudice from anybody, it is doubtful if they would pronounce our schools one whit better than under the old system. While so far as concerns expense, it costs the state thousands of dollars more than formerly."


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8 Quick Fixes to Cozy Up Your Sleep Space
by Talie Jane· October 22, 2015· in Bathrooms, DIY, Furniture & Fixtures, Home Decor, Tips and Trends· 0 comments · Decorating, Fall
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An Insider's Guide to Creating the Perfect Gallery Wall
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Home for the Holidays: Start Your Home Renovations Now!
by Talie Jane· October 08, 2015· in Home Decor, Tips and Trends· 0 comments · Holidays, Interior Design, Remodel
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10 Steps to Creating a Tailored Interior, Courtesy of Greg Natale
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Author's posts
Nov 28
Sheet metal deburring machine
Every owner in the industrial finishing and cutting business has come across problems caused by burring. Burrs are caused by general wear of smooth machinery parts and are causing tiny areas of damage. Over time these burrs are having a negative effect on your production line and the quality of your products. The metallic burrs …
Oct 13
Open a company in Netherlands
If you have plans to open a company in Netherlands, there are some strategic choices you can make to expand your business massively. When you're planning to start a company in the Netherlands, there are a few company structures you can consider; – Public companies (NV) – Private limited liability company (BV) – Foundations – …
Feb 10
IG Markets
IG Markets is the world's largest platforms for forex trading on normal shares without leverages and for CFD trading on shares, commodities and indices. The company is a worldwide market leader with offices in 17 countries. IG is an award-winning company and a reliable broker, regulated by the Financial Conduct Authority (FCA). Why trade with …


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The RCMP have laid terrorism charges in B.C. following an investigation named Project Souvenir.
John Stewart Nuttall and Amanda Marie Korody have been charged in relation to a plot to attack Canada Day celebrations at the B.C. legislature in Victoria.
Reaction on Twitter was swift.
Anthony Ho was quick to congratulate the RCMP on their work.
Following news that the devices found were similar to those used in the Boston bombings, the National Post's Jesse Kline poured scorn on the alleged attackers' lack of imagination.
For more reaction, see the Twitter gallery below:


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Last week, pharmaceutical giant Eli Lilly (NYSE:LLY) announced that its cancer drug candidate enzastaurin failed its late-stage clinical trial. Despite this news, shares remained practically flat following the announcement. Was this drug central to Lilly's pipeline, or was it one of many oncology drugs that the company is testing? Should investors instead be worried about Lilly's upcoming patent expirations? What about the ever-present competition from diabetes powerhouse Novo Nordisk (NYSE:NVO)? Health-care analyst Max Macaluso discusses these topics in the following video.
What Does This Failure Mean for Eli Lilly Investors?
Eli Lilly announced that it will stop developing cancer drug candidate enzastaurin. What does this mean for its business?
Max Macaluso
Max is the Technology, Biopharma & Health Care Bureau Chief at Fool.com. Prior to joining the Fool, he completed a PhD in chemistry at the University of Cambridge and an MBA at the College des Ingenieurs. Follow @TMFMassimo
Article Info
- May 13, 2013 at 6:30PM
- Health Care


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Everything tagged
Stories tagged: nfl
Thu., June 23, 2011
In brief: Vanderbilt eliminates Tar Heels from College World Series
Taylor Hill and Corey Williams combined on four-hitter, Connor Harrell and Curt Casali homered and Vanderbilt eliminated North Carolina from the College World Series with a 5-1 victory Wednesday night …
Wed., June 22, 2011
California stays alive at College World Series
College baseball: California coach David Esquer called on all hands to save his baseball program, and he's doing the same to keep his team playing in the College World Series …
Tue., June 21, 2011
NFL, team reps will meet
CHICAGO – Today in Chicago, two representatives from each NFL team are scheduled to attend what should be the most significant league meeting since the lockout began. But the meeting …
Sun., June 19, 2011
Saints' backup QBs grilling on a budget
NEW ORLEANS – With the NFL lockout putting the squeeze on both his wallet and pursuit of a pro football career, former Connecticut quarterback Tyler Lorenzen has created some of …
Sat., June 18, 2011
NFL players facing investigation surrounding casino
DOTHAN, Ala. – The NFL is investigating the reported investment by at least 25 NFL players in an Alabama casino that has been shut down, a business venture that might …
Thu., June 16, 2011
NFL fan code of conduct at center of court case
SAN DIEGO – City Attorney Jan Goldsmith believes the NFL has a right to enforce its code of conduct, including ejecting fans for making obscene gestures and using foul language. …
Sun., June 12, 2011
Expect owners to have their way in NFL, NBA negotiations
The NFL lockout drags on, with no indication it will be resolved before training camps are scheduled to open next month. NBA owners are preparing for a lockout of their …
Fri., June 10, 2011
Locker works out with Titans' starting job on line
NASHVILLE, Tenn. – The Tennessee Titans haven't seen much of rookie quarterback Jake Locker. That's not stopping his new teammates from thinking the eighth pick overall could be their starter …
Thu., June 9, 2011
In brief: NFL owners, players meet again in New York
NFL: Whether they are meeting in secret locations or in the middle of Times Square at rush hour, talks between NFL owners and players are a good sign. Commissioner Roger …
Wed., June 8, 2011
NFL, players gather for confidential meeting
NFL: The NFL and its players held talks Tuesday in New York, seeking a resolution to the labor impasse. Three people familiar with the meetings confirmed to The Associated Press …
Tue., June 7, 2011
Albert to broadcast NFL for CBS
Marv Albert is returning to the National Football League, whenever the lockout ends. The longtime play-by-play announcer is joining the NFL on CBS team, CBS Sports chairman Sean McManus announced …
Mon., June 6, 2011
Packers hit diamond
The Super Bowl champion Green Bay Packers finally got back together on the field Sunday – to play softball. Roughly three dozen Packers players gathered for wide receiver Donald Driver's …
Sat., June 4, 2011
NFL players urge appellate court to lift league's lockout
ST. LOUIS – NFL players urged a federal appeals court Friday to declare the lockout illegal, saying the league had no right to impose a work stoppage that is approaching …
Fri., June 3, 2011
In brief: McIlroy ties for first-round lead at PGA's Memorial
Golf: Rory McIlroy only gets to play three regular PGA Tour events this year. He showed Thursday why the Memorial tournament was one of them. On a Muirfield Village course …
Sat., May 28, 2011
Bruins advance to Stanley Cup finals to meet Vancouver
NHL: Nathan Horton scored with 7:33 left in Game 7, sending the Boston Bruins to the Stanley Cup finals for the first time in more than two decades with a …
Thu., May 26, 2011
In brief: Lightning force Game 7 with Bruins
Hockey: Martin St. Louis and Teddy Purcell each scored twice, resilient goalie Dwayne Roloson weathered a hat trick by Boston's David Krejci, and the Tampa Bay Lightning stayed alive in …
Wed., May 25, 2011
Chiefs go to work to aid tornado victims
KANSAS CITY, Mo. – Hundreds of cars lined up all day Tuesday in the Arrowhead Stadium parking lot as the Kansas City Chiefs filled two semitrailer trucks with bottled water …
Tue., May 24, 2011
Slouch starts to get gloomy thinking of holes in his life
This is as gloomy as Couch Slouch can ever recall: The possibility of no NFL season in the fall.
Sat., May 21, 2011
Sharks break through against Canucks in Western final
NHL playoffs: Patrick Marleau scored twice in the first period and Antti Niemi made the early lead stand up with 27 saves to get the San Jose Sharks back into …
Thu., May 19, 2011
In brief: Spokane Shock add two quarterbacks to roster
Arena football: The Shock added quarterbacks Steven Sheffield (in photo) and Casey Hansen to their roster Wednesday after the team had two quarterbacks injured in last week's win over Dallas. …
Wed., May 11, 2011
Bengals receiver Ochocinco takes on bull riding
NFL: Chad Ochocinco now is going to try bull riding. The Cincinnati Bengals receiver who earlier this year had a tryout with Sporting Kansas City of Major League Soccer, has …
Sat., May 7, 2011
Area will host 47th East/West game
Area will host East/West game Prep football: The 47th annual East/West All-Star Football game will return to the Inland Northwest on July 1.
Thu., May 5, 2011
In brief: Mavericks head to Dallas with 2-0 edge on Lakers
NBA: Dirk Nowitzki scored 24 points, Shawn Marion added 14 and the Dallas Mavericks stunned the erratic Los Angeles Lakers 93-81 in Game 2 on Wednesday night, taking a 2-0 …
Tue., May 3, 2011
Former Eagles star Jones happy he was drafted by Raiders
As far as his experience with the NFL Draft was concerned, Taiwan Jones liked the where much better than the when. The former Eastern Washington University All-American running back, who …
Tue., May 3, 2011
Heat ask fans to sing 'The Star-Spangled Banner'
Basketball: For the Miami Heat, one national-anthem performer will not be enough tonight. They want thousands.
Sun., May 1, 2011
Seahawks draft choice Carpenter lets game do talking
RENTON, Wash. – James Carpenter was called the quietest guy on Alabama's team by coach Nick Saban. Easy to understand why after Carpenter introduced himself to Seattle with one big …
Fri., April 29, 2011
Seahawks take Alabama offensive lineman James Carpenter at No. 25 overall
RENTON, Wash. – In his first draft in charge with Seattle, general manager John Schneider got his left tackle of the future. Schneider now hopes he's found his bookend on …
Thu., April 28, 2011
Spurs avoid elimination with late 3-pointer
NBA: Gary Neal caught an inbounds pass with 1.7 seconds left and forced overtime with a 3-point heave as time expired, and the San Antonio Spurs staved off elimination by …
Thu., April 28, 2011
Locker's draft status appears to have grown
Jake Locker's offseason journey to this week's NFL draft has like the joke about the two campers who come upon a bear. When one of the campers begins tying his …
Wed., April 27, 2011
Vancouver beats Chicago in Game 7 overtime thriller
NHL: Alex Burrows scored his second goal 5:22 into overtime, Roberto Luongo made 31 saves and the Vancouver Canucks avoided an historic playoff collapse by knocking the defending Stanley Cup …


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Bike Seat Dagger
The "dagger" consists of a 10-inch piece of solid steel, machined down to a spear-like point and then welded to the end of the bicycle seat post. It is easily concealed when the seat post is clamped in place in the bike's vertical seat-post tube.
Remember when dealing with cycling subjects, separate them from their vehicle to reduce the risk of them quickly accessing a hidden weapon.
Thanks to: Ofcr. Pete Beckwith for showing this weapon at the Street Survival Seminar
This concealed weapon threat was first published by Calibre Press.


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The head of a local nonprofit appeared before the Mayor and Board of Aldermen on Aug. 17 seeking help with an effort to receive a federal grant.
James Dunn, representing the Tunica County Community Development Coalition, said his organization recently applied for a grant from the Department of Planning and Safety.
"In order to make the application, we need an organization to agree to sponsor it," Dunn said.
The grant would cover costs for a Youth Entrepreneurship program that exposes kids to writing a business plan and running a business, Dunn said. The program features guest speakers and hands on experience. It operates on a part-time basis.
{paypalaccess}Read more{/paypalaccess}
Dunn said it was his wish to grow it into a 12 month program. The town could act as a clearinghouse for the grant.
Ann Johnson, who has helped oversee the program for Tunica County since 2002, said the program was a credible and valuable resource.
Dunn said the grant requires a 10 percent match by the sponsor.
Dunn said his organization could provide the match or find a sponsor.
Johnson said the grant could include five percent unaccounted-for funding, which would mean the town would only have to provide five percent.
Chuck Cariker asked if the program's personnel would be considered town employees.
Johnson said the staff would be specific to the program, and the town would only be responsible for the accounting.
Town clerk Celia Boren pointed out that federal funds require a singular audit.
Alderman Eloise Carson asked who was preparing the application.
Dunn said the nonprofit TCCDC, Inc. had initially submitted the application and were advised to get a sponsoring agency.
Alderman Edward Hunsucker asked where the classes were held. Dunn said the students meet at the Tunica Vo-Tech Center.
Hunsucker asked about the program's success rate.
Dunn said it has only operated on a part-time basis, but in the first year, 24 kids enrolled and 16 kids completed the program. He noted that it is designed to steer kids on the verge of becoming adolescent offenders on another path, but serves non at risk youth as well.
"It's really designed to get kids in the mindset of owning their own business or becoming the head of a household," Dunn said.
Hunsucker asked if it cover all aspects of entrepreneurship, including management and financing.
Dunn said the program uses a 96 page curriculum and students feed off into another program when this one is completed.
Hunsucker asked about the instructors.
Dunn said one staff member has a business background, including a business degree. The other is a retired educator.
Cariker asked how the bills for the program would be paid.
Johnson said the town would pay the bills and be reimbursed for them from the grant funds.
Hunsucker said he was concerned that it would require a lot of the town clerk's time.
Johnson said it wouldn't require any more time than the town's regular accounting duties.
Knabb asked if employees would be given a purchase order number.
Johnson said whatever the town's normal process was would be fine.
Boren said if the program's personnel were concerned town employees, they would be required to have benefits.
Dunn said the staff members were already TCCDC employees so they could be considered "contract labor" for the town.
Johnson said if the town agreed to sponsor the program, they would be wise to spend all the grant funding.
Following a few more questions from the board, Cariker suggested that the board take the request under advisement.
"Let's check with the state and decide by the next meeting," Cariker said.
Alderman Charley Knabb asked if there was a deadline.
Dunn said he was hoping to start the program after Labor Day.
Johnson said it was a little amount of money that can effect many lives.
Hunsucker asked about how students apply for the program.
"If you have room for 10 students and 20 apply, do you go to the ones who are borderline delinquent or try to mix it up?" Hunsucker asked.
"We intend to serve the kids who are doing well and the ones who are not doing so well," Dunn said.
Johnson said anyone who is put on the waiting list for the entrepreneurship program was cycled into another program until a spot open ups.
"We don't just leave kids hanging," Johnson said.


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Suburban Square Concert Series featuring Flying Ivories
A Dueling Pianos performance will turn the evening into something unforgettable. They will bring their show to the venue complete with lighting, sound and two custom-built black pianos. Next, they belt out an all-request, rock and roll singalong while the guests dance, hoot, holler and even join us on stage for a show that's part music, part comedy and all fun. From Billy Joel to Aretha Franklin, from Sinatra to Metallica, from Garth Brooks to Madonna, they have got it covered. The Attendees will control the set list, so no two shows are ever the same.


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The Yankee Plague: Escaped Union Prisoners and the Collapse of the Confederacy By Lorien Foote
Main Article Content
In our polarized society, even historians are playing along. Two distinct camps appear to contain most military historians of the Civil War: traditional militarists and the nontraditional militarists. Lorien Foote, who stands firmly in the latter camp, shows the merits of viewing society's impact upon the military rather than taking the more traditional top-down approach. In The Yankee Plague, Foote makes a compelling argument that during the latter months of the Civil War, waves of escaping Union prisoners of war hastened the collapse of the Confederacy.
Article Details
How to Cite
McKnight, B. (2019). The Yankee Plague: Escaped Union Prisoners and the Collapse of the Confederacy By Lorien Foote. Indiana Magazine of History, 113(2), 165-166. Retrieved from https://scholarworks.iu.edu/journals/index.php/imh/article/view/27600


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Current Projects
Desire Den (PC, VR, Mobile)
Desire Den is an adult fantasy game set in a swingers club full of sultry anthro characters for you to interact with and get to know on a very intimate level.
Wild World - Nomad No Mercy (PC, Mobile)
Wild World - Nomad No Mercy Is an open-world RPG set on an abandoned secret military testing island full of secrets to uncover, interesting allies (or enemies) and plenty of old junk to scrap into new weapons, tools, fortification and more!
Frosted Brain Shop


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Learn to connect position-time and velocity-time graphs. Explore velocity using an animated car icon connected to either a position-time or a velocity-time graph, or both. Then investigate other motion graphs. Describing Velocity is the fourth of five SmartGraphs activities designed for a typical physical science unit of study on the motion of objects.
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Bymark belongs in the Financial District. Where else but Bay Street can you charge these kind of prices for lunch. Fat cats and expense accounts flock here for their infamous $36.95 Bymark Burger.
"What is so appealing about Lynch Bages is that it has long been a classic Pauillac that, while not the most tannic and backward wine of the appellation, clearly possesses what most wine insiders love about Pauillac – lots of robust, rich, full-bodied crème de cassis fruit, 25-30 years of aging potential in top vintages, and an enormously satisfying, complex style."
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Specifications include, but are not limited to: It is the intent of the State of South Carolina, South Carolina Department of Public Safety (SC DPS) to solicit offers to provide two (2) motorized carts.
to solicitation details & future business opportunities.
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House Sitting
Drop-In Visits
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My dog gets super excited when Makaila comes to get him for his walk. His little tail can't swing fast enough. Makaila is such a loving and caring person. She walks my tootsie three times a week when I have to work late. She's dependable and I trust her with my dogs life. I know tootsie is in good hands. I will continue with Makaila to walk him.
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They'll love how to interactive I am with their pet , I love learning dogs personalities. I treat my dog just like he's my son. I treat other people's pet the way I would want my dog to be treated. I have a 7 year old shitzu mix. I had him since he was 12 weeks old . I did a pretty good training him and I know I can do the same for other ...
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I would like to know the breed. I don't want to handle a dog too large.


text: 4TH ANNUAL CHALDEAN FESTIVAL IN EL CAJON SEPT. 14 & 15 El CajonChaldean FestivalCentennial PlazaFestivals of the Month September 10, 2013 (El Cajon) – The 4th Annual Chaldean Festival well be held at Centennial Plaza in El Cajon on September 14 & 15 from 5 – 10 p.m. This free event features music, dance, food, games and vendor booths. There will also be a raffle for a Sports Car. All are welcome to this event sponsored by the Chaldean American Institute and St. Michael's Aid Society. Read more about 4TH ANNUAL CHALDEAN FESTIVAL IN EL CAJON SEPT. 14 & 15Log in or register to post comments


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The 16th Annual Bob Dylan Birthday Bash: Free Show presented by Kenn Kweder, John Train & many more TBA
feat. Kenny Aaronson (former Dylan bass player)
May 24
, 2018
Doors: 6:00 PM
Show: 7:00 PM
21 and over
Free (GA) / $15 Seated
The Ardmore Music Hall
23 East Lancaster Ave
Ardmore, PA
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This question already has an answer here:
- What is a transposing instrument? 1 answer
I recently was looking at trumpet models, and I saw something that baffled me. The trumpets had a note associated with them, an example being: "Standard Bb Trumpet". What do these notes (the Bb in the example) mean?


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Ethiopia, Deri Kochoha
Ethiopia, Deri Kochoha
Deri Kochoha is a classic fruit forward, natural process coffee from the Guji Zone with chocolate notes as it cools. Growing elevation is between 1800-2000 meters above sea level, providing exceptional sweetness.
With natural process, or dry process coffees, the ripe coffee cherries are picked and sorted for quality before being laid out to dry. The flesh of the cherry is then mechanically removed from the seed when dry. The extended contact time the flesh has with the seeds imparts additional sweetness and fruit attributes to the green coffee seed.
This is a good option for espresso as well as brewed coffee.
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