text:
Donver Stable's Tesseron, who last year was second
in the Grey Stakes (G3) at Woodbine and fourth in the Kentucky Jockey
Club Stakes (G2) at Churchill Downs, worked Wednesday morning in
preparation for a possible start in the $750,000 Toyota Blue Grass
Stakes (G1) on April 13. Ridden by Luis Contreras, the Tapit colt
covered six furlongs in 1:16.20.
"It was a good solid work, just what we needed," trainer Josie Carroll said.
Tesseron worked in company with Drenched, a maiden winner ridden by John Maguire who broke off several lengths in front.
"He likes to have a target to chase down," Carroll said about
Tesseron. "That's how he likes to run. He likes to sit and have
something to stalk."
Clockers caught Tesseron in fractions of :13.20, :25.60, :37.40, :49.60 and 1:02.
Carroll said the work would be the last for Tesseron before the
Toyota Blue Grass, a race the colt's connections chose because of its
"It's probably the last chance for Derby points, and it seemed like
the best spot for him," Carroll said, referring to the points system to
determine starters for the Kentucky Derby Presented by Yum! Brands (G1).
"He's a (April 23) foal. He's just starting to grow up. I didn't want
to overdo him coming in. We got one race in at Gulfstream. We'll take a
shot in the Blue Grass and hope he's good enough. And he likes this
In September 2012, Tesseron won his career debut at Woodbine. In his lone start of this year, he was fourth behind Black Onyx
in an allowance optional claiming race on February 23 at Gulfstream.
Black Onyx returned to win the Horseshoe Casino Cincinnati Racing Spiral
Stakes (G3) at Turfway Park on March 23.
Donver Stable represents Donna and Verne Dubinsky of Alberta, Canada.
For Donver, Carroll also trains 2011 Queen's Plate Stakes winner Inglorious,
a Canadian champion who has earned more than $1.4 million. Carroll said
the 5-year-old daughter of Hennessy, who is among her 12 horses here,
is scheduled to make her next start in the Hilliard Lyons Doubledogdare
Stakes (G3) on April 19.
In other Toyota Blue Grass news
Trainer Ken McPeek said Charles Fipke's homebred Java's War,
second in the Tampa Bay Derby (G2) on March 9 in his most recent race,
is scheduled to work at Gulfstream Park over the coming weekend then
ship to Keeneland for the Toyota Blue Grass.
Reynaldo Abreu, assistant to trainer John Terranova, said that Sam F. Davis (G3) winner Falling Sky is
at Churchill Downs, where he is scheduled to work Sunday toward a start
in the Toyota Blue Grass or the Arkansas Derby (G1) on April 13.
Another Terranova runner, West Hills Giant, worked a
half-mile at Belmont Park in :49.10 and is a possibility for the Toyota
Blue Grass or Coolmore Lexington Stakes (G3) on April 20. West Hills
Giant finished second in the Gotham Stakes (G3) on March 2 in his most
recent start.
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The American Triple Crown season has come and gone...
Trainer Josie Carroll has won Queen's Plates with ...
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text:
Submission + - Giant Rats Attack New York City (nydailynews.com) 1
KernelMuncher writes: It's not just the hurricanes New Yorkers have to fear. Giant rats have taken over an NYC housing project (with pic), terrifying small children and old people.
"Trust me. I know what I'm doing." -- Sledge Hammer

subset: commoncrawl


text:
BitFenix Ghost Mid-Tower Chassis Review
Accessories and Documentation
Part of the included hardware contains the SATA cover for the S4 bay to keep them clean and dust free. You also get grommets for the oval holes in the motherboard tray along with four rubber support pads.
I have to assume the risers are to replace the stud type on the tray, and you also get four long fan screws to add a second fan in the front. Then you see the seven expansion slot screws to hold cards in place. The bottom row has 12 screws to secure the motherboard and PSU, then six screws for the 5.25" bays, and a whole mess of tiny screws to use for adding 2.5" drives to the 3.5" trays.
These plastic trays were stuck in the 3.5" bays during its trip, but out of the bags you can see there are four for the 3.5" drives that will accept 2.5" drives, as well as a set of three specifically for the 2.5" drives.
I was also given two 30cm Alchemy LED strips, both are blue, and they are sent along to put under the chassis giving the chassis a glow that almost makes the case look like it is floating.
The quick start guide is plain black and white throughout to cut costs on the stuff that isn't used all the time to allow for all the features and goodies we just saw to come at this price.
Opening it to the middle I found a front fan installation guide insert added to the manual that covers everything else from start to finish with these top notch illustrations and basic text descriptions.
With a bit of super glue and the time it took me to get this far in the images, the base of the chassis is now one solid piece so I went ahead and added the Alchemy strips to it before I screwed it back onto the case. I also had to add pair of screws to the front to attach the bottom of the bezel to keep it from falling off the front since the pins were all sheared off.
PRICING: You can find products similar to this one for sale below.
United States: Find other tech and computer products like this over at Amazon's website.
United Kingdom: Find other tech and computer products like this over at Amazon UK's website.
Canada: Find other tech and computer products like this over at Amazon Canada's website.
- Page 1 [Introduction]
- Page 2 [Specifications, Availability and Pricing]
- Page 3 [Packaging]
- Page 4 [BitFenix Ghost Chassis]
- Page 5 [Inside the Ghost]
- Page 6 [Inside the Ghost Continued]
- Page 7 [Accessories and Documentation]
- Page 8 [The Build and Finished Product]
- Page 9 [Final Thoughts]
Recommended for You
- We at TweakTown openly invite the companies who provide us with review samples / who are mentioned or discussed to express their opinion of our content. If any company representative wishes to respond, we will publish the response here.
- Response:
- It's a shame that our Ghost sample arrived in such poor condition for the review. We want to make sure our fans are thrilled with their BitFenix purchase, so if anyone out there experiences something similar, please do not hesitate to write to our support department at support [at] bitfenix [dot] com, and we will make it right. A big shout out to the folks at TweakTown for taking the time to check out our latest creation!
The BitFenix Crew
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text:
hmmmmmmm asks
Am I overreacting? Is there a way to deal with his drinking and running away from arguments?
Dating | Closed
My boyfriend and I have been together for 5 years and are both 24. We moved out together about a year ago which only lasted 6 months because of his constant drinking and deserting me whenever we had a fight. When we would have 'these' fights he would storm out, not answer my phone calls and not come home until the next day sometimes still drunk to the point where he would come home and throw up. I've told him that's it's over many times but he always comes back about a week later saying that he will change which lasts for about a week. I don't want to end the relationship because I think we do care about each other, but just that he doesn't know how to deal with emotional issues. Does anyone have any experience with this?
She's Acting Like I Chose To Inconvenience Her!
92% vs. 7%Marriage
Bachelor Party Guy Trip Suspicions!
54% vs. 45%Marriage
Why Can't My Wife Work Out At Home?
55% vs. 44%Marriage
- salt780 asked the question "How to get alone time with a lover"
- madewell231 voted for OKracer (side 1) on the story "My Dad Won't Let Me Date A Black Guy"
- krismiss7 voted for ForSale (side 1) on the story "Making Fun Of Me For Pooping My Pants"
- krismiss7 voted for grapewoman (side 1) on the story "Ever Been Told You're An Idiot?"


text:
Native Plants
Q. Who is Mr. Smarty Plants?
A: There are those who suspect Wildflower Center volunteers are the culpable and capable culprits. Yet, others think staff members play some, albeit small, role. You can torture us with your plant questions, but we will never reveal the Green Guru's secret identity.
Did you know you can access the Native Plant Information Network with your web-enabled smartphone?
Ask Mr. Smarty Plants is a free service provided by the staff and volunteers at the Lady Bird Johnson Wildflower Center.
rate this answer
Tuesday - July 05, 2011
From: fairfax, VA
Region: Mid-Atlantic
Topic: Pests, Herbs/Forbs, Wildflowers
Title: Mosquito-deterring plants for shady hillside
Answered by: Guy Thompson
QUESTION:We have a part to full shaded hill side/ native woodland area that was once covered with english ivy..we managed to get rid of all the ivy but now we are overtaken with violets..maybe they are even native..but they are everywhere and they are drowning out all other plants and are harboring tons of mosquitos.is there another choice of ground cover for the area? we have cinamon ferns/maiden hair/ service berry/ fringed bleeding hearts/ cardinal flower/ sea oats all doing very well..
Mr. Smarty Plants assumes that you have pinpointed violets as the plants that harbor mosquitos. They do have broad leaves growing close to the ground, where they will retain moisture. Mosquitos will also hide in other humid locations, such as a dense cluster of ferns. I suggest that you replace the violets with plants that send their stems higher and/or have few or small leaves near ground level. For example, Aquilegia canadensis (Eastern red columbine), Packera aurea (Golden ragwort), Lobelia cardinalis (Cardinal flower) and Symphoricarpos orbiculatus (Coralberry). There are many other plants to choose from, depending upon your conditions, e.g. full shade, part shade, moist, dry, need for low-growing or taller species. Grasses generally do not do well in shade, but you could use a sedge, such as Carex pensylvanica (Pennsylvania sedge). This would look nice interspersed with other short species like Mitchella repens (Partridgeberry), . Ferns should do well. If taller plants are desirable, consider Hypericum prolificum (Shrubby st. johnswort), Maianthemum racemosum ssp. racemosum (Feathery false lily of the valley), Maianthemum stellatum (Starry false lily of the valley), and Lobelia siphilitica (Great blue lobelia). Symphyotrichum novae-angliae (New england aster) should thrive in partly sunny areas.
Certain plant species have been shown to have insect-deterrent activity. Most of those species I found are not native to your area. The sages generally occur naturally further west, but one species, Salvia azurea (Pitcher sage), is native to Virginia, and it may have unreported deterrent properties.
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text:
Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability
- First Online:
- Received:
- Accepted:
DOI: 10.1007/s00262-008-0518-1
- Cite this article as:
- Schmidt, M.M., Thurber, G.M. & Wittrup, K.D. Cancer Immunol Immunother (2008) 57: 1879. doi:10.1007/s00262-008-0518-1
- Citations
- 359 Views
Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids.
KeywordsTumor targetingCEAEndocytosisAntibody fragmentsAffinity
Antibody drugs have begun to show clinical promise for the treatment of a variety of cancers—there are currently nine FDA approved antibodies for cancer treatment with dozens more in clinical trials [3]. By specifically targeting antigens overexpressed on tumor cells, these drugs have potential to improve the efficacy of cancer treatment while limiting toxic exposure to healthy cells. Despite this progress, there are still significant barriers that must be overcome in order for these drugs to reach their full potential. In particular, efficacy has been limited in the treatment of solid tumors [6]. The reasons for incomplete cell killing in solid tumors are complex, but one significant factor is that antibodies often fail to fully penetrate the tumor tissue leaving regions of untargeted, viable cancer cells [6].
The transport of antibodies into solid tumors is a complex process involving circulation and clearance from the plasma, extravasation across capillary walls, binding and diffusion in the tumor interstitium, and internalization and catabolism in tumor cells. Solid tumors typically exhibit abnormal physiology characterized by high interstitial fluid pressure, insufficient vascularization, and dense extracellular matrix that limit antibody movement [21]. After entering the tumor, high affinity antibodies rapidly bind to free antigen depleting the pool of free antibody and further hindering penetration [2, 16, 18]. Recent theoretical analyses have suggested that antibody internalization and catabolism in tumor cells may also contribute to poor antibody transport [37]. Specifically, the time scale model derived by Thurber et al. predicts that incomplete antibody penetration will occur if the rate of antibody catabolism is faster than the rate of antibody extravasation and diffusion. The antibody internalization rate has also been predicted to influence the length of antibody retention in the tumor, surface accessibility of antibody for ADCC or pretargeted radioimmunotherapy, and the cytotoxicity of immunotoxins and antibody–drug conjugates [5, 35, 39]. In order to test these predictions, it is imperative to measure accurate rate constants for antibody uptake in tumor cells, which can vary widely for different antigen targets [1, 4]. Additionally, it is important to understand how antibody properties such as affinity, valency, and stability influence these rates, in order to select agents with suitable properties for a given targeting application.
Carcinoembryonic antigen (CEA) is a 180 kDa GPI-linked cell-surface glycoprotein normally expressed in the fetal gut and on the lumenal surface of the adult colon [19]. During colorectal carcinoma oncogenesis, CEA loses its polarity and becomes overexpressed throughout the tumor tissue. High levels of CEA expression have also been observed in epithelial tumors in the lung, breast, thyroid, and ovaries [19]. Due to this selective tumor overexpression, antibodies against CEA have been investigated as targeting agents for a number of imaging and therapeutic approaches including SPECT and PET imaging, pretargeted radioimmunotherapy, and ADEPT [9, 22, 32, 38].
Although CEA is often referred to as a shed or non-internalizing antigen [7, 8, 25], there have been sporadic reports that antibodies and immunoconjugates against CEA are in fact slowly internalized by CEA expressing tumor cells [14, 33, 34]. However, these studies have lacked: (1) quantitative measurement of the bound antibody internalization rate constant (ke) and (2) systematic study of how antibody properties such as affinity, stability, and valency influence this uptake. These limitations are addressed in the current study in which the internalization rates of a series of anti-CEA antibodies and antibody fragments with differences in affinity, stability to protease degradation, valency, and target epitope have been quantitatively measured in CEA expressing cell lines.
Materials and methods
Materials and cell lines
Anti-CEA IgGs M85151a and M111147 were purchased from Fitzgerald (Concord, MA). LS174T cells were obtained from ATCC (Rockville, MD). LIM1215 and SW-1222 cells were the kind gift of the Ludwig Institute. Cells were grown in minimum essential media (MEM) supplemented with 10% FBS and 1% penicillin–streptomyocin. CEA expressing HT-1080 cells (HT-1080-CEA) were created by transfecting HT-1080 cells with the pIRES-CEA plasmid (kind gift of Dr. G. Prud'homme, St. Michael's Hospital, Toronto, Canada) using lipofectamine according to manufacturer's instructions. Transfected cells were selected by growth for 7 days in MEM with 0.75 mg/mL G418.
Antibody fragment production
Secretion vectors for shMFE and sm3E containing a C-terminal His6 tag have been described previously [17]. Disulfide stabilized scFvs were produced by mutating residues R44 and G234 to cysteine using the QuikChange Kit (Stratagene) according to manufacturer's instructions. Mutations were confirmed by sequencing. Plasmids were transformed into the YVH10 strain of yeast using the EZ Yeast Kit (Zymo Research) and plated on SD-CAA media supplemented with 40 μg/mL tryptophan. Individual colonies were grown in 1 L flasks and secretion induced for 48 h at 37°C as described previously [17]. The cleared supernatant was concentrated using Millipore 10 kDa ultrafiltration membranes and the His-tagged proteins purified using BD Talon metal affinity resin following the manufacturer's batch-column protocol. Eluted proteins were further purified by anion exchange chromatography on a Hi-Q column equilibrated with 20 mM Tris buffer at pH 8.25 and size exclusion chromatography on a Superdex 75 column (GE Healthcare). Samples were run on a 12% Bis–Tris gel with or without 100 mM DTT and Coomassie stained to check size and purity. Fab fragments were produced from IgG M85151a by papain digestion using the ImmunoPure Fab Preparation Kit (Pierce) according to manufacturer's instructions. Purified antibody fragments were conjugated to Alexa-488 fluorophores using the Microscale Protein Labeling Kit (Invitrogen) following manufacturer's instructions. Protein concentration and degree of fluorophore labeling were determined from UV absorbance at 280 and 490 nm as described in the Microscale Kit. The immunoreactive fraction was estimated by incubating scFvs with a fivefold molar excess of soluble CEA (Fitzgerald) and determining the fraction of antibody that elutes as a complex on size exclusion chromatography.
Linker cleavage and protease stability
The (Gly4Ser)3 linker connecting the VH and VL domains of the scFvs was cleaved by incubating the antibody fragments with 0.02 units/mL subtilisin in digestion buffer (20 mM Tris–HCl, 5 mM calcium chlorate, pH 7.5) for 90 min at 37°C. Digested samples were run on a 12% Bis–Tris gel with or without 100 mM DTT and Coomassie stained. For functional protease stability assays, Alexa-488 labeled scFvs were incubated with increasing concentrations of subtilisin in digestion buffer for 60 min at 37°C. Trypsinized LS174T cells were labeled with the digested antibody fragments at subsaturating concentrations for 20 min on ice and mean cellular fluorescence measured on an EPICS Coulter XL flow cytometer (Beckman Coulter, Inc.)
Cell–surface binding
Trypsinized LS174T cells were fixed with Cytofix Buffer (BD Biosciences) for 20 min at 4°C to prevent antibody trafficking. For KD measurements, fixed cells were incubated with Alexa-488 labeled antibodies or antibody fragments at a range of concentrations for 16–20 h at 37°C. To avoid antibody depletion effects, a tenfold molar excess of antibody over antigen was maintained throughout. Labeled cells were washed twice with PBS + 0.1% BSA, and the mean Alexa-488 cellular fluorescence measured on an EPICS Coulter XL cytometer. The normalized signal was fit to the equation MFU = Bmax ([Ab]/([Ab] + KD)) to determine the KD. For koff measurements, fixed cells were labeled overnight with saturating concentrations of Alexa-488 conjugated scFvs. Cells were washed and resuspended at 37°C in PBS–BSA containing 100 nM unlabeled sm3E to prevent rebinding of dissociated antibodies. At each time point, cells were washed and the mean fluorescence quantified by flow cytometry. Fluorescence values were normalized by the signal at t = 0 and fit to an exponential decay to determine the koff. The number of antibodies bound per cell at saturation was determined by labeling cells with saturating concentrations of Alexa-488 conjugated antibodies or antibody fragments and converting the fluorescence intensity to a number of molecules using Alexa-488 calibration beads and the calculated number of fluorophores per antibody.
Net antibody internalization assays
LS174T cells were subcultured into 96-well plates at a concentration of 105 cells per well and allowed to adhere for 12–16 h. After washing cells once in media, 125 μL of Alexa-488 conjugated antibodies or antibody fragments at a concentration of 20 nM in MEM + FBS was added to each well. Cells were incubated at 37°C in the continuous presence of the labeled antibodies. At each time point, plates were chilled, wells washed twice in PBS–BSA, and cells lifted by incubating with 200 μL cell dissociation buffer (Gibco) for 10 min on ice. Dissociated cells were transferred to microfuge tubes by pipetting and sedimented at 14,000×g. Cell pellets were resuspended in PBS–BSA with or without 25 μg/mL anti-Alexa-488 quenching IgG (molecular probes) for 25 min on ice. The mean fluorescence of the surface quenched and unquenched cells was measured on an XL cytometer and used to calculate the relative amounts of surface and internal fluorescence as described previously [4]. In brief, surface fluorescence was determined as (unquenched MFU − quenched MFU)/ε, where ε is the quenching efficiency determined by comparing the quenched and unquenched signals of cells that had been labeled briefly on ice to prevent internalization. The internal fluorescence was then calculated as total MFU − surface MFU. Non-specific internalization of antibodies due to fluid phase uptake was measured using cells where the CEA was pre-blocked with unlabeled antibody and subtracted from the internal signal. The corrected internal MFU values were plotted against the integral of the surface fluorescence determined using the trapezoidal rule and fit to a linear curve, the slope of which is the internalization rate ke [26]. scFv uptake experiments in the LIM1215, SW-12222, and HT-1080-CEA cell lines were performed essentially as described above except trypsin–EDTA was used in place of cell dissociation buffer to lift the cells from the plates at each time point.
Surface decay
LS174T cells subcultured in 96-well plates as above were surface labeled with saturating concentrations of Alexa-488 labeled sm3E, ds-shMFE-M, or M85151a IgG for 1 h on ice. Unbound antibody was washed from each well and cells were incubated in media at 37°C. At each time point, cells were chilled and transferred to microfuge tubes as described above. Cells were then surface labeled on ice with PE conjugated secondary and tertiary antibodies to determine the amount of anti-CEA antibody remaining on the surface. Goat anti-mouse PE (1:50 dilution) was used for M85151a and anti-His biotin (1:70 dilution) followed by streptavidin-PE (1:100 dilution) for the scFvs. Cells were analyzed by flow cytometry to measure the 488 signal (total cell-associated antibody) and PE signal (surface accessible antibody). Cells were also imaged for 488 and PE colocalization using a Deltavision deconvolution microscope (Applied Precision, Inc.)
Fluorescence imaging
LS174T cells were subcultured at ~2 × 105 cells per well on 8-well glass coverslip bottom dishes (Nunc). After attachment, cells were incubated overnight at 37°C with 10 nM anti-CEA scFvs conjugated to either Alexa-488 or Alexa-594. Cells were then labeled for 1 h at 37°C with fluorescent markers of endocytosis including 1 μg/mL Cholera toxin subunit B-594, 20 μg/mL transferrin-488, 10 μM dextran-488, or 100 nM LysoTracker red. Cells were washed and imaged on a Deltavision deconvolution microscope to determine 488 and 594 colocalization.
CEA downregulation
LS174T cells subcultured into 96-well plates as above were incubated at 37°C in media with or without 50 nM unlabeled anti-CEA antibodies or antibody fragments. At each time point, cells were chilled, washed twice with cold CO2 independent media, and labeled for 40 min on ice with 20 nM of a non-competitive Alexa-488 labeled anti-CEA antibody. Cells were then washed, lifted with cell dissociation buffer, and analyzed by flow cytometry as above. The Alexa-488 signal of cells incubated with unlabeled antibody was normalized by the signal of cells incubated with media alone to determine the degree of antigen downregulation.
Biotinylated CEA turnover
LS174T cells were subcultured into 12-well plates at a density of 2 × 106 cells per well and grown for 24 h at 37°C. Cells were washed and surface biotinylated with 1 mg/mL NHS-SS-biotin (Pierce) in PBS, pH 8.0. The labeling reaction was quenched after 30 min by the addition of 100 mM Tris–HCl. The cells were then washed twice in media and incubated at 37°C. At each time point, cells were placed on ice, washed twice with PBS–BSA, and incubated in 500 μL of ice cold lysis buffer for 10 min. The cell lysate was cleared by centrifuging at 14,000×g for 15 min and biotinylated proteins pulled down with streptavidin resin (Pierce). The bound proteins were washed and eluted by cleaving the disulfide linker with 100 mM DTT. Eluted samples were run on a 4–12% SDS-PAGE gel, transferred to a nitrocellulose membrane, and blotted for CEA using M85151a IgG (1:1,000 dilution) and goat anti-mouse-HRP secondary (1:1,000 dilution). Band intensities at each time point were quantified using QuantityOne Software (Bio-Rad) and normalized by the band intensity at time zero.
The internalization rate of cell bound antibodies and antibody fragments is predicted to significantly alter the microdistribution, surface accessibility, and retention time of these molecules in tumor tissue. Therefore, we set out to quantitatively measure the internalization of antibodies and antibody fragments directed against the common tumor target CEA. Furthermore, we examined how antibody parameters such as affinity, stability to protease digestion, valency, and target epitope influence these internalization rates.
scFv production
As a model system to examine the effect of antibody properties on cellular uptake, we developed a series of anti-CEA single-chain variable fragments (scFvs) with differences in affinity, stability to protease degradation, and valency. scFvs are ~27 kDa antibody fragments consisting of the VH and VL domains of an IgG connected by a peptide linker. Previous affinity maturation work resulted in two scFvs sm3E and shMFE that bind to the same epitope on CEA with a nearly 300-fold difference in affinity (sm3E KD = 30 pM, shMFE KD = 8.5 nM) [17]. To make fragments with increased stability, cysteine residues were inserted into the VH and VL domains of the proteins to form an interdomain disulfide bond. The formation of this interdomain disulfide has been shown to increase the protease and thermal stability of antibody fragments [11, 31]. Additionally, the disulfide stabilized scFvs (ds-scFvs) are secreted in yeast as a mix of disulfide locked monomers (ds-scFv-M) and dimers (ds-scFv-D) which can be separated to provide fragments with differences in valency.
scFv binding and stability
Binding to fixed LS174T cells
KD (pM)
koff compet (min−1)
t1/2 off compet
26 ± 4
7.5 × 10−5
153.8 h
30 ± 5
2.4 × 10−4
48.1 h
9.6 ± 0.7
9.6 × 10−5
120.1 h
160 ± 24
6.6 × 10−2
10.5 min
9,300 ± 3,300
1.6 × 10−1
4.3 min
85 ± 5
8.0 × 10−2
8.7 min
The contribution of the interdomain disulfide to functional scFv stability was assessed by incubating the antibody fragments in increasing concentrations of the protease subtilisin, then measuring binding to LS174T cells at subsaturating concentrations. As expected, the disulfide stabilized proteins displayed significantly higher binding activity following protease treatment than the native scFvs suggesting improved stability (Fig. 1d). As a whole, the in vitro cell binding assays confirmed that the secreted scFvs exhibited the expected differences in affinity, stability to protease digestion, and valency, and were therefore a useful model system for the effects of variation in these parameters on net internalization.
Internalization rate constant (ke) measurements
Internalization rates were derived from the quenched fluorescence data by plotting the internal fluorescence against the time integral of the surface fluorescence and applying a linear fit (Fig. 2b and Supplemental Fig. 1). Uptake rates were measured in this manner for all scFvs and are reported as internalization half-times in Fig. 2c (where t1/2 = ln(2)/ke). In general, internalization of the anti-CEA scFvs is slow with half-times ranging from 10 to 16 h. Surprisingly, neither antibody affinity, stability to protease digestion, nor valency significantly affect the internalization rate of bound antibody fragments in this assay as there is no significant difference in uptake half-times among the scFvs.
The fluorescence quenching protocol was also used to measure the uptake rates of a pair of commercially available anti-CEA IgGs M111147 and M85151a. These antibodies bind to different (non-competing) epitopes on CEA from the scFvs and each other (data not shown) and have functional affinities of 70 pM for M111147 and 7 pM for M85151a as measured by cell surface titration. While M111147 was internalized with a half-time of 13 h, similar to the scFvs, M85151a was taken up significantly faster with a half-time of 5 h (Fig. 2c). A monovalent Fab fragment of M85151a with a functional affinity of 2.5 nM was internalized with a net uptake rate (t1/2 ~ 14 h) significantly slower than the IgG, suggesting that the bivalency of the full antibody is necessary for its faster uptake (Fig. 2c).
The effect of cell type on anti-CEA antibody fragment internalization was assessed by measuring the internalization of the high affinity scFv ds-sm3E-M in three additional cell lines. LIM1215 and SW-1222 are colon carcinoma cell lines that express CEA at lower levels than LS174T, while HT-1080-CEA is a fibrosarcoma cell line transfected with a CEA expression plasmid resulting in antigen overexpression. LIM1215 and SW-1222 internalized ds-sm3E-M with half-times of 11.5 and 17.2 h, respectively, both similar to the 15.3 h half-time measured in LS174T cells (Fig. 2d). In contrast, HT-1080-CEA cells internalized the scFv at a significantly faster rate with a net uptake half-time of 4 h.
Antibody surface decay
Fluorescence imaging
To better characterize the route of antibody internalization, fluorescence colocalization studies were performed between anti-CEA scFvs and markers of endocytic pathways. Specifically, dextran, transferrin, cholera toxin B, and LysoTracker were used as markers of early endosomes, recycling endosomes, caveolae-mediated internalization, and lysosomes, respectively. In all cases, anti-CEA scFvs internalized at 37°C for 24 h showed partial, but incomplete fluorescent colocalization with the respective marker (Fig. 4b), suggesting that CEA is internalized non-specifically through multiple pathways.
Surface CEA and antibody levels
Antigen concentration and the number of antibody molecules bound per cell at saturation may also influence the tumor microdistribution of antibodies [37]. To quantify these parameters, the cellular fluorescence of LS174T cells labeled with saturating concentrations of Alexa-488 labeled antibodies or antibody fragments was measured and converted to a number of bound molecules per cell using fluorescent calibration beads. As seen in Fig. 5b, ~400,000 molecules of the monovalent scFv ds-sm3E-M are bound to each cell at saturation, while the scFv dimer ds-sm3E-D and IgG M111147 saturate the cell with ~40% fewer molecules due to bivalent antigen binding. Interestingly, both the IgG and Fab versions of M85151a bind with approximately twice as many molecules per cell at saturation as the other antibodies of equivalent valency. The greater cell surface binding at saturation for M85151a compared to M111147 was also observed on HT-1080-CEA cells (Supplemental Fig. 2) while neither antibody binds HT-1080 in the absence of CEA expression suggesting that M85151a's higher cell labeling is mediated by interactions with CEA and is not due to binding other proteins on the cell surface. The difference in cell binding stoichiometry is also not due to heterogeneous antigen that is partially unreactive with the scFvs and M111147 since >95% of soluble CEA elutes as a complex on size exclusion chromatography when incubated with an excess of any of the tested antibodies (data not shown). One potential explanation for the twofold greater cell binding of M85151a is the possibility that this antibody binds to two different epitopes on each CEA molecule (see "Discussion").
Metabolic turnover of CEA
Theoretical analyses of antibody transport in solid tumors suggest that cellular internalization and catabolism of bound antibodies significantly retards penetration of these drugs into the tumor. In order to test these predictions using CEA-specific antibodies as a model system, the rates of antibody and antibody fragment internalization by CEA expressing tumor cells were measured. Fluorescence measurements using flow cytometry provided a quantitative and facile method for measuring trafficking kinetics that was higher throughput than imaging approaches and avoided artifacts of incomplete antibody stripping observed with acid washing protocols.
With the exception of IgG M85151a (discussed below), all anti-CEA antibodies and antibody fragments tested were internalized in LS174T cells slowly with uptake half-times of 10–16 h. This time scale is consistent with the metabolic turnover rate of CEA in the absence of antibody (t1/2 ~ 15 h) suggesting that the antibodies are taken up passively with the antigen and do little to drive or modulate this uptake. The tested antibodies and antibody fragments have no effect on surface levels of CEA following binding, which is also consistent with a passive uptake mechanism. Since CEA is a GPI-linked protein with no cytoplasmic or transmembrane protein domains, the observed uptake is likely the result of bulk membrane turnover rather than specific protein-mediated pathways [19]. Such non-specific trafficking of CEA may explain the results of immunofluorescent microscopy experiments in which internalized anti-CEA scFvs colocalized partially but incompletely with markers of multiple endocytic pathways. Similar slow metabolic turnover has been observed for other antibodies targeting non-receptor cell surface antigens [23].
Slow cellular uptake of the high affinity anti-CEA scFv ds-sm3E-M on the order of 11–17 h was also observed in two additional colon carcinoma cell lines, LIM1215 and SW-1222. In contrast, uptake was significantly faster (t1/2 ~ 4 h) in a fibrosarcoma cell line HT-1080 transfected with a CEA expression plasmid. It is unclear if this faster uptake is due to greater overall cell surface turnover in this cell line or some function of the artificial CEA overexpression.
Cellular trafficking studies in other systems have suggested that the affinity and valency of soluble ligands or antibodies may alter cellular uptake rates by influencing the fraction of molecules that are recycled to the cell surface following endocytosis, or by altering antigen clustering dynamics on the cell surface [12, 30]. In the case of the anti-CEA scFvs examined here, however, no significant difference in the net uptake constant (ke) was observed for molecules with a range of affinity, stability to protease digestion, and valency. The lack of an affinity dependency may suggest that CEA binding occupancy in the endosome has little effect on the fraction of antibodies that are recycled versus degraded, or alternatively, that both the high and low affinity scFvs are able to maintain antigen binding in the endosome due to the high concentration of CEA. Similarly, the equivalent uptake of the monovalent and bivalent scFvs suggests that crosslinking of two CEA molecules on the cell surface has little effect on the antigen's distribution or trafficking. This result is consistent with previous observations that bivalent binding of IgGs against folate receptor, another GPI-linked protein, were insufficient to drive receptor clustering on the surface or increase uptake [28].
Although affinity and valency have little impact on the uptake constant (ke) of anti-CEA antibodies, they may still influence the total amount of internalized antibody which depends on both ke and the amount of antibody bound (d[Ab]internal/dt = ke × [Ab]bound). This distinction is clearly observed with the behavior of the low affinity scFv ds-shMFE-M in the continuous uptake and surface decay experiments. While ds-shMFE-M is internalized when continuously incubated with the cells at a 20 nM concentration, it dissociates from the cells prior to internalization in the surface decay assay. Both of these cases are relevant to in vivo tumor targeting. The continuous uptake experiments are similar to the loading phase of tumor targeting where a high plasma concentration maintains a sufficient antibody concentration in the tumor to drive binding and internalization, while the surface decay assay represents the retention phase when antibody clears from the plasma and tumor. In contrast to the low affinity case, the high affinity and bivalent antibodies have dissociation rates (koff) slower than the internalization rate (ke) such that binding will be essentially irreversible in both targeting regimes [23].
Unlike the remainder of the tested antibodies, IgG M85151a exhibited a distinct trafficking profile with a significantly faster net uptake rate (t1/2 ~ 5 h) and the ability to downregulate surface CEA. These properties were both valency dependent, as a monovalent Fab fragment of M85151a was internalized slowly and had no effect on surface CEA. Both the M85151a IgG and Fab also bind with approximately twice as many molecules per cell at saturation as compared to other antibodies of equivalent valency. One potential explanation for this twofold higher cell binding stoichiometry is the possibility that these antibodies bind to more than one epitope per CEA molecule. Monoclonal antibodies capable of binding multiple epitopes per CEA molecule have been reported previously, a phenonmenon attributed to the high sequence homology of repeat domains within the antigen [20, 29, 40]. If M85151a does in fact bind two epitopes per CEA molecule, it may also provide a mechanism for the faster uptake. Bivalent antibodies that bind to a single epitope per antigen can only crosslink two molecules. In contrast, a bivalent molecule that binds more than one epitope per molecule may be able to crosslink larger clusters of antigens. Previous studies have demonstrated that the formation of large clusters of GPI-linked proteins can increase antigen localization in caveolae, as well as drive greater antigen internalization and downregulation [10, 15, 28].
Although the experiments presented here indicate that the internalization of anti-CEA antibodies and antibody fragments in LS174T cells occurs slowly, this rate is sufficient to significantly impact antibody distribution and retention in the tumor. Thurber and Wittrup have shown that anti-CEA scFvs are able to penetrate significantly farther into LS174T spheroids when incubated at 20°C versus 37°C due to reduced cellular internalization [36]. Similarly, Ackermann et al. have demonstrated that the slowly internalized IgG M111147 is able to penetrate significantly farther into LS174T spheroids than the rapidly internalized IgG M85151a (in preparation). The difference in penetration distance can be quantitatively predicted from the ke values and cell surface binding stoichiometry measured here.
Based on these results and other computational predictions, we suggest that antibodies with slow cellular internalization rates should have advantages for most tumor targeting applications due to their improved penetration and retention in the tumor (with the exception of immunotoxins and antibody-drugs that must be internalized to be cytotoxic [39]). In some cases, it may be possible to engineer more slowly internalized antibodies by either selecting for proteins that are efficiently recycled following endocytosis or by using monovalent antibodies that avoid faster uptake due to antigen clustering. Alternatively, antibody internalization may be reduced by targeting antigens with slower metabolic turnover. One promising target is the colorectal cancer marker A33 which has extended cell surface persistence due to interactions at the tight junction [1].
This work was supported by CA101830 and the NIGMS/MIT Biotechnology Training Program. The authors also thank the Ludwig Institute and Dr. Gerald Prud'homme for cell lines and plasmids.


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Efficient, accurate, service with great selection of products.
The Garden Grocer made our visit to Disney World even better! It was easy and prices were reasonable - will do it again next time!
Garden Grocer was easy to use. Everything was fresh! Already recommended it to other visitors who used the delivery service.


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DIY Label Projects and Free Printables • Tutorials and printables, including these free printable DIY vintage labels by 'Free the Diva'!
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Printable vintage style Christmas tickets. Great for scrapbooking.
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Located in Portland, Oregon, PSU has about 30,000 undergraduate and graduate students. PSU's motto is "Let Knowledge Serve the City," and we provide every student with opportunities to work with businesses, schools and organizations on real-world projects ... more
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Ed O'Loughlin was born in Toronto and raised in Ireland. He reported from Africa for the Irish Times, and was Middle East correspondent for the Sydney Morning Herald and The Age of Melbourne.
His first novel, Not Untrue & Not Unkind was longlisted for the Man Booker Prize 2009 and shortlisted for the Kerry Group Irish Fiction Award.
His second novel, Toploader, a darkly comic vision of the "war against terror", was published by Quercus in 2011. All You Can Eat, a novella satirizing the Irish economic crash, appeared in 2013.
His third novel, Minds of Winter, is published by Riverrun in August 2016.
He lives in Dublin with his wife and two children.
"In both concept and execution the novel is a serious piece of work at once vastly entertaining and ambitious on a scale that leaves much of contemporary Irish fiction looking woefully insubstantial….there will be few better historical novels published this year."
The Sunday Times
"An enthralling series of interlocking mysteries; masterfully evokes the beauty of polar regions."
Gavin Francis, author
"Ed O'Loughlin's Minds of Winter … may well be the Franklin novel to end all Franklin novels. Never have so many different narrative threads been taken up and twined together.. It would seem a daunting task to connect so many historical figures in a single volume, something like trapping demons in a cursed box, but the talisman that O'Loughlin employs is deceptively modest: a single marine chronometer, Arnold 294, which showed up intact at a 2009 auction in the UK when it was supposed to have been issued to Franklin's ships 164 years previously."
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AN HM REVENUE & CUSTOMS CRACKDOWN on tax avoidance and evasion has brought in an additional £23.9bn to the public purse.
The haul marks HMRC's highest since records began, which it said is a result of its investigations.
The figure is almost £1bn higher than the target set by George Osborne in the Autumn Statement in December.
HMRC credited "increased activity" on unpaid tax for the figure.
Of the total amount it had raised, more than £8bn came from large business, £1bn from criminals and £2.7bn from tackling avoidance schemes in courts.
The current tax gap – the difference between estimated tax due and that collected – stands at 7%.
In total, HMRC has said it expects to secure £100bn between May 2010 and March 2015 as a result of its investigations into unpaid tax.
"HMRC will pursue those seeking to avoid their responsibilities and will collect the taxes that are due," said exchequer secretary to Treasury minister David Gauke.
"The government is determined to tackle the minority that seek to avoid paying the taxes they owe," he added.
Related reading
HMRC revises VAT treatment of dwellings
HMRC has outlined a change in VAT policy to the treatment of dwellings that have been formed from either the construction of new buildings, or from the conversion of non-residential buildings
Duke of Westminster and IHT – a tragedy from all perspectives
Let us hope that valuable asset protection vehicles are not made prohibitively burdensome or abolished in the desire to "simplify" IHT
Micro-businesses want more clarity about HMRC's digital tax plans
Freelancers and micro-businesses still need more information about the government's plans to make tax digital
Government presses ahead with non-dom tax changes
The government is pressing ahead with changes to the way it taxes individuals with a foreign domicile


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Barakat began as a partnership between a carpet-weaving business based in Punjab, Pakistan and a carpet-selling business based in Cambridge, Massachusetts. The two founders,Habibullah Karimi, an Afghan refugee living in Pakistan, and Chris Walters, an Ame ... more
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I am a native Spanish Speaker from Valencia Spain. I am also a Spanish Instructor at Georgia Perimeter College. I have been teaching Spanish for 10 years including one year at the middle school level.
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...At IRS, I was known as ?Mr. DIF? because of my leadership, oversight, and hands-on development of IRS? discriminant function (DIF) models for selecting all types of compliance workload. At IRS, I also accumulated extensive experience in: 1) other case selection and resource allocation modeling;...
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Japan Expo continues in Paris, and some kind souls at SQUARE PORTAL captured - fittingly - 13 minutes of gameplay footage from a live demo of the game. We've seen plenty of story, exploration and combat up until this point, but never had a good look at the menu system. In the E3 demo, there was a very limited menu available, but here, we see several pieces of equipment, stats, and even a color palette for customizing Lightning's various outfits. For customization nerds (like me), this is quite exciting. Check it out!
Lightning Returns: Final Fantasy XIII releases February 11th in North America and February 14th in Europe. But you know that by now, right?
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(From France Galop)
Le Havre Triumphs in French Derby
Second in the French 2,000 Guineas on May 10, Le Havre reversed the form in the €1.5m Prix du Jockey Club (French Derby, Fr-I) over a non-staying seventh Silver Frost June 7 at Chantilly.
Le Havre managed to catch the Head family's Fuisse, who hit the front two out but did not stay either. Aidan O'Brien's Westphalia was third in front of the fast finishing Beheshtam.
Le Havre was bred by Anna Sundstrom and Team Hogdala's Haras de Coulonces, who sold him to Jean-Claude Rouget for €100,000 at the 2007 Arqana Deauville August sale.
Racing under French owner Gerard Augustin-Normand's colors and ridden by Christophe Lemaire, he completed a group triple for Jean-Claude Rouget on the day.
It was a first in the French Classic for the leading trainer and the rider Christophe Lemaire. Rouget has now passed the €4m mark in prize money in France in 2009 and leads the table by over €2.5m.
The two other group races on the card also went the way of Jean-Claude Rouget stable courtesy of Quetsche in the Prix de Royaumont (Fr-III) and Oiseau de Feu in the Prix Paul de Moussac (Fr-III).
It was, however, an emotional win for the trainer, based in Pau in the South West of France. The winning owner Gerard Augustin-Normand, a stockbroker, watched the race from his home in Normandy as he is a rather discreet character, but the trainer spoke with him over the phone and declared: "He's a very sensitive man and I can tell you that at his home today, he's the happiest man in the world. The pace was fast enough and we had a clear run. I did have doubts about my horse's staying power and it was his first attempt over this distance.
"But he finished well. I knew he could do it. It is such a hard race to win. It is pretty special to me. I remember the first time I followed the Prix du Jockey Club. It was the 1966 edition, won by Nelcius. I was eating cherries. Since then, I know when cherries are in season."
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According to a report on www.businessday.co.za, bad weather could drive up the cost of salvaging the stranded oil tanker MT Phoenix to $4.5m, citing information from the South African Maritime Safety Authority. The tanker reportedly ran aground north of Durban on Tuesday.
One well-placed industry source cited by the news service estimated the costs could soar even higher.
Phoenix reportedly was stranded off a residential area, making the salvage job that much more challenging.
(Source: www.businessday.co.za)
An injured lobsterman was medically evacuated by U.S. Coast Guard helicopter crew Tuesday roughly 130 nautical miles east of Cape Cod.
At about 10:15 a.m.,
Unifire AB's FlameRanger is a first-of-its-kind robotic nozzle system which automatically detects and extinguishes fires for fixed installations, indoors and outdoors,
A fire that broke out aboard the passenger vessel Caribbean Fantasy at 7:42 a.m. on August 17 has been extinguished, and the vessel has been moored safely in port.


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Great cover for my megacab
Reviewed By Bruce C (Henderson, NV) Reviewed for a 2011 Dodge Ram —8/20/2016 8:48 AM
Ordered this cover for my Dodge ram megacab. Arrived quickly. After putting the cover on a couple of times to get the hang of it, it now goes on in minutes...I have the towing mirrors....once you put the mirror pockets over the mirrors, fold them in, pull the cover over the cab, snug it up over the front bumper...hook the bottom back of the cover to my steprails and its done!..looks great, lightweight, can be locked on with optional cable. 4 year warranty, washable..A bit pricey but works!
Happy Customer
Reviewed By Elizabeth H (BRISTOL, VA) Reviewed for a 2007 Subaru Outback —8/20/2016 8:33 AM
It's a bit slick to handle, but it works wonderfully well. I couldn't be happier. When covering my Outback Wagon, it looks like a giant baking potato in the driveway. I will be ordering another cover soon for my other Subaru vehicle.
Disappointed Buyer
Reviewed By Paul C (Centerville, MA) Reviewed for a 2013 Scion FR-S —8/20/2016 3:17 AM
i previously purchased two Covercraft Noah covers and this Evolution cover is to replace one of the Noah covers, which seem to literally disintegrate after a couple of years of very light use. I was very disappointed when the Evolution arrived since it appears to be exactly the same material as the Noahs. I was told by you that the manufacturer said that it is a different material, which I do not believe. More likely the manufactirer had this custom cover sitting in his warehouse and labele


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There will be another exiting Mixed Reality event taking place on the 15th & 16th November, at the Park Plaza Victoria, London, which is MXR Summit 2016. The gathering "aims to bring together some of the biggest minds from within the…
Applications, Companies, Selected Videos, Systems
3d4medical Presents a Showcase of an AR Body Browser
Here you can see the virtual counterpart of Gunter von Hagens "body worlds", however, much more interactive and a much clearer focus on the subject – the exploration of anatomy. Virtual anatomy models are placed at your preferred learning environment.…
Conferences, General
MICCAI 2016 in Athens, Greece
The Medical Image Computing and Computer Assisted Intervention (MICCAI ) Society has decided to move the event from Istanbul, Turkey to Athens, Greece. The conference is still going to be organized in collaboration with Bogazici, Sabanci, and Istanbul Technical Universities. Exact…
CURAC 2016 in Bern, Switzerland
This year, the annual CURAC conference of the Deutsche Gesellschaft für Computer-und Roboterassistierte Chirurgie e.V. will be held from the 29th of September to the 1st of Oktober 2016 in Bern Switzerland. The event will run under the motto "Translation – From Bench to…
Companies, Systems
Sharing Experience With Hololens
Yesterday, I finally got the chance to test the Augmented Reality see-through head mounted display Microsoft Hololens at the open day event of the Navigated Augmented Reality Visualization System (NARVIS) Lab, a research laboratory the Chair for Computer Aided Medical…
Applications, Companies, Curious
Augmented Reality T-Shirts by Wayuco
Wayuco is a new brand for design t-shirts. But their designs have a special feature. They can be used as tracking targets, which are detected by Wayuco software on smartphone or tablet devices to project an animation onto the design. This turns a still image or written statement into a…
KinectFusion for Anatomy Registration
This video of my former colleague Ben Glocker shows how to use KinectFusion to register virtual with real anatomy in real time. Once both 3D structures are registered in space, Kinect tracking takes over and allows for a combined view onto…
Applications, Companies, Conferences
First AR, VR and 360° Video trade show in Köln, Germany
From the 20th to 25th of September, the very first iteration of the trade show and expo DIGILITY will take place in Köln, Germany covering the topics Virtual Reality, Augmented Reality and 360-Degree-Videos. DIGILITY is a satellite event of the photokina under the motto"New realities. New…
Applications, Conferences
CARS 2016 in Heidelberg, Germany
The next edition of CARS 2016, the 30th International Congress and Exhibition of Computer Assisted Radiology and Surgery, will start soon! This year, the conference will be held in Heidelberg at the Convention Center, June 21 to 25, 2016. Keynote…


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...With my strong academic performance, I'm qualified to teach several math topics, with an emphasis on statistics and calculus. I've had experience tutoring teenagers for GED test prep, and I've also tutored classmates in college. I worked at the Baltimore City Detention Center tutoring inmates for three years.
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