Ecuador has ruled out abandoning the U.S. dollar as a means of confronting the global financial crisis.
Economic Policy Minister Pedro Paez says US$6.3 billion in a dollarization fund and another US$2.5 billion deposited in local banks can be used to cushion the impact on Ecuador.
Paez says the …
A MISSING man is being hunted by police across Essex afterwarnings he has been seen in the Epping area.
James Doe, 37, was charged with a high-value burglary inWorcestershire in 2001, but did not appear for his court case andhas been missing ever since.
During his nine years on the run, he has been spotted indifferent areas of Essex, including Epping, Chelmsford, Harlow, andMaldon.
Police are keen to see him in court to face charges, which relateto the theft of Pounds …
DALLAS - A federal judge on Tuesday blocked the Dallas suburb of Farmers Branch from enforcing a voter-approved law prohibiting apartment rentals to illegal immigrants until a legal challenge is resolved.
U.S. District Judge Sam Lindsay had previously issued a temporary restraining order in May, blocking the law a day before it was to take effect. He issued a preliminary injunction Tuesday.
"Farmers Branch, rather than deferring to the federal government's determination of immigration status, has created its own classification scheme for determining which noncitizens may rent an apartment," Lindsay wrote.
The law would have required apartment managers to verify …
Swine flu is a "sneaky virus" that is likely to keep spreading to new parts of the world and within countries already affected, the head of the World Health Organization said Friday.
At least 42 countries have confirmed cases of the disease, which has sickened 11,168 people and caused 86 deaths, most of them in North America.
"This is a subtle, sneaky virus," WHO's Director-General Margaret Chan said at the close of the global body's annual assembly. "It does not announce its presence or arrival in a new country with a sudden explosion of patients seeking medical care or requiring hospitalization."
Countries need to …
Citing fundraising challenges, Kanawha County Public Libraryofficials no longer expect to begin work by 2011 on all seven of the$50 million worth of modernization projects they have planned.
Because of the slowed pace of fundraising, getting all the moneytogether at once may no longer be possible. Instead, the focus is onbuilding as soon as possible a new $30 million Main Library indowntown Charleston and replacing the tiny Marmet Library with alarger facility.
Besides those two new libraries, library officials had hoped tobuild a new Elk Valley Library in Big Chimney and add on to thelibraries in Cross Lanes, Dunbar, St. Albans and Sissonville.
After three …
Interview Answers in a Flash, by Pat Criscito and Dee Funkhouser 2006. Hauppauge, NY: Barron's Educational Services, Inc. 332 pages, Soft Cover, $12.99
Intended Audience: A, B, D, E, F, G, H, I
How is the book most useful for its intended audience?
It is a great tool for preparing for an interview - offering practice questions and essential tips for preparation.
The top five things you learned from reading this book:
1. A good question to ask interviewers if you do not get the position you interviewed for is, "What was it about the successful candidate that made him or her the ideal choice?"
2. The STAR acronym for Situational/ Behavioral question …
Israel's prime minister is urging Sri Lanka not to give in to terrorism.
Sri Lankan prime minister Ratnasiri Wickramanayake is on a rare visit to the Jewish state. The two countries renewed relations in 2000 after a 30-year lull.
The two share a fight against suicide bombers and other extremists. Israel has been battling Palestinian militants for decades. Sri Lanka's fight …
LOS ANGELES--Researchers have identified a gene that when mutatedcan lead to an elevated but modest risk of breast cancer.
The altered gene, called CHEK2 or CHK2, can double the estimated13 percent lifetime risk of breast cancer that U.S. women face,researchers said.
About 1 percent of people-- both women and men--carry the mutatedgene.
Carrying it does not necessarily mean someone will develop breastcancer, the scientists stressed. Instead, the risk is comparable tothat faced by women who have a mother or sister with breast cancer,who never have children or who experience menopause at a later age.
"CHK2 is not something anyone should go out and get …
Knowledge, Attitudes and Practices of Prenatal Care
Providers in a Province with Low Testing Rates
ABSTRACT
Objective: To describe the knowledge, attitudes and practices of prenatal care providers in relation to prenatal HIV testing.
Methods: A stratified random sample of 784 family physicians, 200 obstetricians and 103 midwives providing prenatal care in 3 health planning regions in Ontario received a questionnaire.
Results: Response was 622/1087 (57%). Almost half of participants (43%) were not aware of Ontario's prenatal HIV testing policy. Eighty-five percent of participants reported that they offered or ordered HIV testing for all pregnant …
Lebanese militant Samir Kantar and four other prisoners crossed into a buffer zone between Lebanon and Israel on Wednesday, part of a dramatic prisoner swap that also involved the return of two dead Israeli soldiers to the Jewish state.
The Lebanese were driven in vehicles to the buffer zone, and were to cross by foot into Lebanon proper. Army crews removed barricades at the border to let the cars in.
…
Kanawha County's budget encountered another strain in an alreadyfinancially challenging year after the Regional Jail Authority votedto raise the amount needed by the counties to house each inmate perday.
In a move that will cost Kanawha County about $160,000 moreannually, the authority raised the per diem from $47.53 to $48.25,which is a raise of 72 cents per inmate, per day. The change takeseffect July 1, 2009.
Commission President Kent Carper said the rate increase might benecessary, but the state needs to help alleviate some of thefinancial stress that counties are placed under.
"The fact that they've raised rates I don't like, but Iunderstand it," Carper …
Pollution in Central Pennsylvania from tiny airborne particles is higher than allowed under new federal rules, but it still is unclear how the state and local governments will respond.
The U.S. Environmental Protection Agency said Dec. 17 that 18 Pennsylvania counties - including Cumberland, Dauphin, Lancaster, Lebanon and York were out of compliance with its new standard for fine-particle pollution, also known as soot. Parts of four other counties also were included.
The EPA long has restricted larger pollution particles, but the new limit is the agency's first venture into regulating the tiny pieces, called particulate matter 2.5 (PM 2.5). Regions that have reduced …
WASHINGTON - Former House Speaker Newt Gingrich said Sunday he could have been a contender, but has no regrets about skipping the race for the Republican presidential nomination in 2008.
Gingrich said he'd had a Web site ready to launch this week and already had received several million dollars in pledges.
"I think we would clearly have been competitive financially within three weeks, and we literally had not even set up the Web site yet," he said. "But what hit me was it would have been an underdog campaign. I mean, clearly, if you were going to come from behind, I think it would have been a real campaign. I think we would have had a chance to win."
Gingrich spokesman Rick Tyler said Saturday that Gingrich opted out of the race, a crowded one with nine candidates, after determining he could not legally explore a bid and stay as head of his tax-exempt political organization.
Besides the potential legal difficulties, Gingrich said running would have wasted the effort spent building up American Solutions for Winning the Future, the tax-exempt political arm of his lucrative empire as an author, pundit and consultant.
"To give up and kill an organization we spent a year on and that had 2,000 sites around the country where people had now invested their time and effort just to look at whether or not you could run, I thought would be irresponsible," he said on ABC's "This Week."
Just last week, Gingrich said he had set an Oct. 21 deadline to raise $30 million in pledges for a possible run, acknowledging that the task was difficult but not impossible.
He said Sunday that the deciding factor was learning he risked violating the McCain-Feingold campaign finance law.
"I thought there was a way that you could continue the momentum of those ideas while I began to prepare a presidential campaign," Gingrich said. "What we learned yesterday morning was, I mean, it's literally a go to jail, criminal activity."
In a taped appearance broadcast on "Fox News Sunday," Gingrich also criticized the law.
"The effect of the McCain-Feingold censorship act has been to weaken the middle class, to make it harder to have a middle-class candidate and to make it much, much harder to raise money and so I think you've got to be realistic about what it takes to campaign," he said.
Gingrich said he would not align himself with any of the candidates.
"I'm not going to endorse anybody. I'm going to try to offer ideas that hopefully all of them can look at," he said.
Gingrich is the latest Republican to decide against joining the race. In early September, Nebraska Sen. Chuck Hagel announced he would not run for the White House or seek a third term.
---
On the Net:
American Solutions for Winning the Future: http://www.americansolutions.com
Last fall's U.S. Senate race between then-U.S. Sen. George Allen and Democrat Jim Webb was a battle between strongly-clashing opponents.
It was a blogger-led "Draft Webb" effort that convinced the decorated Vietnam veteran and former Republican to get into the Senate race against singleterm incumbent George Allen. Blogger Lowell Feld, one the leaders of the Draft Webb movement, even became the campaign's netroots coordinator.
The Allen campaign, on the other hand, had no Internet presence other than a basic website. Alien hired an "eCampaign Manager" in mid-summer, naming somebody who had no connection with political bloggers.
These two approaches collided on August 11, when Alien made his nowfamous attack on Webb campaign staffer S.R. Sidarth, nicknaming the 20-year-old of Indian descent "macaca" before a laughing crowd. The incident was caught on videotape by the Webb campaign, who worked with bloggers to release the video via YouTube.
As the Allen campaign offered explanations that shifted daily, Republican bloggers were left adrift, unable to even keep up with the latest excuse. Democratic bloggers, on the other hand, were able to pick up talking points that Lowell Feld provided on his own blog.
Widely-respected libertarian blogger Jon Henke was brought in by the Allen campaign before the month was out, with just over sixty days to turn things around. A Virginian, Henke already knew the Democratic and Republican bloggers throughout the state, many personally.
Webb won, of course, defeating Alien by the narrowest of margins.
Henke has concluded that campaigns must engage blogs from day one, both to establish credibility and to prevent unfavorable frames from being established. Democratic blogs had spent months putting forth a narrative about Allen that presented him as a bully. "Macaca" served to crystallize that, and the media simply used the existing narrative. Henke believes that, by the time he signed on with the campaign, it was too late to shape the narrative.
Tech-sawy activists throughout the state provided a steady stream of stories, photos and videos to the Webb campaign. And, of course, they carried the message, repeating talking points and keeping the Alien campaign on the defense.
By neglecting blogging opinion-shapers of all political stripes, nobody had Allen's his back when he most needed support.
Senate Republicans recruited Jon Henke as the New Media Director for the Republican Communications Office, where he started last month. Perhaps they've learned their lesson.
READ MORE FROM WALDO JAQUITH AT WWW.WALDOJAQYITH.ORG
ANSA news agency reports that a U.S. military helicopter has crashed in northern Italy, saying there were possible deaths.
Bank of America Corp. Chief Executive Kenneth Lewis says the $25 billion the government is injecting into his bank will be put to work making loans and will be paid back at the latest in five years.
In an interview aired Sunday on CBS' "60 Minutes," Lewis confirmed that top executives were given no option last week when Treasury Secretary Henry Paulson unveiled a plan for the government to pour $125 billion into nine major banks. In return, the government will get ownership stakes in the financial institutions.
Lewis said Paulson told the executives that the money being loaned to the banks to jump-start frozen U.S. lending was "the right thing for the American financial system, and therefore it was the right thing for America."
He told the news program that he was stunned at the amount being loaned, but believes the $25 billion received by the Bank of America, which is based in Charlotte, North Carolina, will be put to good work. However, the Treasury's plan does not demand banks use the money for consumer or corporate lending _ and could instead use it to buy weaker companies.
"We wouldn't want to do it that way, because you can make more money lending," he said. "And, so it _ the intent _ will be to use it to grow loans and to make more net income."
While the government taking an ownership position in banks smacks of socialism to some, Lewis said the situation will not last long.
"Yeah, I think somewhere between three and five years, we'll pay (the government money) off," he said. "And then _ and then you go back to _ more toward capitalism."
There was a point during the meeting with Paulson when some of the bankers resisted any stipulations or caps related to executive compensation. Lewis, who received compensation of $20.4 million last year, argued that Wall Street executives already make too much money.
Council trading standards officials have installed bins for scammailings to be dropped in as part of the fight against con artists.
Bath and North East Somerset Council is supporting the nationwideScamnesty campaign, with bins at libraries at The Podium, Keynshamand Radstock this month. Officials will collect letters or printedemails and analyse them to see if the Office of Fair Trading mightbe able to take action against the senders.
ABSTRACT Hyaluronan is a megadalton glycosaminoglycan composed of repeating units of D-N-acetylglucosamine-[beta]-D-Glucuronic acid. It is known to form a highly hydrated pericellular coat around chondrocytes, fibrosarcoma, and smooth muscle cells. Using environmental scanning electron microscopy we detected fully hydrated hyaluronan pericellular coats around rat chondrocytes (RCJ-P) and epithelial cells (A6). Hyaluronan mediates early adhesion of both chondrocytes and A6 cells to glass surfaces. We show that chondrocytes in suspension establish early "soft contacts" with the substrate through a thick, hyaluronidase-sensitive coat (4.4 + or - 0.7[mu]m). Freshly-attached cells drift under shear stress, leaving hyaluronan "footprints" on the surface. This suggests that chondrocytes are surrounded by a multilayer of entangled hyaluronan molecules. In contrast, A6 cells have a 2.2 + or - 0.4-[mu]m-thick hyaluronidase-sensitive coat, do not drift under shear stress, and remain firmly anchored to the surface. We consider the possibility that in A6 cells single hyaluronan molecules, spanning the whole thickness of the pericellular coat, mediate these tight contacts.
INTRODUCTION
Cell adhesion to the extracellular matrix (ECM) is a complex multicomponent process, which is involved in the regulation of cell motility, proliferation, differentiation, and survival. Cell-ECM contact commonly occurs at specialized sites such as focal adhesions where the interaction is mediated via heterodimeric transmembrane adhesion receptors of the integrin family (Geiger et al, 2001; Martin et al, 2002). These molecules are directly associated, through their extracellular domains, with specific ECM networks, and link them to the actin cytoskeleton (Adams, 2001; Geiger et al, 2001). The transmembrane interactions of integrins with F-actin are mediated by complex networks of plaque proteins that regulate both the assembly and stability of the contacts, and the signaling processes.
Matrix adhesion is a multistage process, involving, in addition to the integrin-mediated adhesion, an integrin-independcnt cell-ECM interaction (Hanein et al., 1993, 1994, 1995; Zimmerman et al, 2002). Using a variety of adhesive surfaces, it was shown that the early stages of attachment of A6 cells (epithelial cells of Xenopus laevis) are resistant to inhibition by RGD, the integrin-specific peptide, and to cytoskeleton-disrupting drugs (Hanein et al., 1993, 1994). This early adhesion was shown to occur at a timescale of seconds, and to be mediated by cell-surface hyaluronan (Zimmerman et al., 2002).
Hyaluronan is a large linear glycosaminoglycan, with typical molecular mass of afew million Daltons (Toole, 2001), composed of a repeating disaccharide of [D-W-acetylglucosamine-[beta]-D-Glucuronic acid] (Lee and Spicer, 2000). Due to the carboxyl group of the glucuronic acid, hyaluronan is highly negatively charged at physiological pH, and behaves in solution as a polyelectrolyte, forming a viscous gel. Dry, surface-grafted hyaluronan layers can swell within a few seconds, adsorbing water to 2.4-fold their initial thickness (Mathe et al., 1999). The ability to adsorb large amounts of water, combined with the repulsion between identically charged groups, makes hyaluronan a good lubricant (Israel-achvili and Wennerstrom, 1996; Tadmor et al., 2002).
Hyaluronan can either be secreted by the cells to the ECM or associated with the plasma membrane. As an ECM component hyaluronan is involved in mediating and modulating cell adhesion as well as in maintaining osmotic balance and reducing friction in tissues such as the synovium, vitreous humor, and cartilage (Toole, 2001). At the cell surface it is known to comprise a pericellular coat, which can be visualized as a hyaluronidase-sensitive area in particle-exclusion assays. Due to intrinsic limitations of this approach only substantial coats can be detected, such as in fibrosarcoma cells (McBride and Bard, 1979), chondrocytes (Lee et al., 1993), and smooth muscle cells (Evanko et al., 1999).
The adhesion of A6 cells to the substrate was shown to be drastically attenuated when the cells are treated with hyaluronidase, which hydrolyzes hyaluronan by randomly cleaving the [beta]-N-acetyl-glucosamine-[1-4] glycosidic bonds (Zimmerman et al., 2002). Addition of exogenous hyaluronan to the treated cells or to the substrate restores cell adhesion, whereas addition of hyaluronan to both the cells and the surface inhibits the adhesion (Zimmerman et al., 2002). This indicates that hyaluronan can enhance or block cell adhesion, depending on whether it is present on the cell surface, on the substrate, or on both.
To understand the mechanism of ECM adhesion mediated by cell-surface hyaluronan, it is important to establish the physical properties, including the thickness, of this layer. The major difficulty in visualizing a hyaluronan layer arises from its highly hydrated nature. Dehydration of such samples, a mandatory step in conventional (non cryo-) electron microscopy techniques, generates a distorted view of cell surfaces, inevitably reducing a hydrated pericellular coat to dispersed fibers. Indeed, the substantial pericellular coat of human smooth muscle cells (Evanko et al., 1999), or of fibrosarcoma cells, appears as a sparse fibrous matrix after air-drying or freeze-drying, respectively (Bard et al., 1983).
Hyaluronan molecules are being simultaneously synthesized and extruded through the cell membrane by a transmembranal glycosyltransferase, hyaluronan synthase. A portion of these hyaluronan molecules remains anchored to the hyaluronan synthase (Weigel et al., 1997), whereas the rest are released from the cells and bind either to integral membrane hyaluronan receptors, mainly CD44 (Bajorath, 2000), or to the ECM. It is not clear whether hyaluronan molecules are attached to receptors on the membrane to form a "de Gennes brush configuration" (de Gennes, 1987; Lee et al., 1993; Toole, 2001), or rather form multilayered gels where nonanchored hyaluronan molecules are entangled with the receptor-grafted molecules.
The striking observation of hyaluronan-mediated adhesion of A6 cells to various surfaces (Hanein et al., 1993, 1994, 1995; Zimmerman et al., 2002) raises the question of the generality of this phenomenon to other cell types and of the ability of a thick hyaluronan pericellular coat, such as that surrounding chondrocytes, to mediate cell-substrate adhesion (Lee et al., 1993).
In this study we visualized the cell-bound hyaluronan of RCJ-P rat chondrocytes and A6 Xenopus epithelial cells, using an environmental scanning electron microscope (ESEM) (Danilatos, 1991) and a particle exclusion assay based on 3D reconstruction from fluorescence microscopy. This examination revealed a thick coat around both cells. We further found that the initial adhesion of rat chondrocytes to glass surfaces is hyaluronan mediated, as is that of A6 cells. The mechanical properties of these early cell adhesions suggest that, whereas in chondrocytes hyaluronan forms thick entangled multilayers, in A6 cells the interaction is mediated by hyaluronan molecules attached directly to the membrane.
MATERIALS AND METHODS
Cell culture
RCJ-P chondrocytes (rat chondrocytes from fetal calvaria, batch 15.01.98; Prochon Biotech, Rehovot, Israel) were cultured at 37[degrees]C in humidified atmosphere of 5% CO2 in air in [alpha]-minimum essential medium (Biological Services, The Weidmann Institute) supplemented with 15% fetal calf serum (Biolab Ltd., Jerusalem, Israel).
A6 cells (kidney epithelial cells from Xenopus laevis, ATCC.CCL 102) were cultured at 27[degrees]C in humidified atmosphere of 5% CO2 in air in Dulbecco's minimum essential medium (Biological Services, The Weizmann Institute) diluted to 85% with water and supplemented with 10% fetal calf serum (Biolab Ltd.).
Glass coverslips, used for cell culturing, were coated with serum by incubation for 3-16 h with 10% fetal calf serum (Biolab Ltd.).
Cell treatment with hyaluronidase
Cells were suspended using trypsin-EDTA (Biological Services, The Weizmann Institute), centrifuged, and resuspended in serum-free medium. Hyaluronidase (hyaluronidase type IV-S from bovine testes, Sigma, St. Louis, MO) was added to the suspended cells to a final concentration of 500 units/ml. Incubation was performed at 37[degrees]C for RCJ-P cells. A6 cells were incubated at 37[degrees]C for the flow experiments and at 27[degrees]C for the ESEM experiments. After treatment, the cells were centrifuged, and washed three times with serum-containing medium to remove residual enzyme and hyaluronan fragments. Cells were then resuspended in serum-containing medium.
Sample preparation for conventional (dry) scanning electron microscopy
Cells were suspended using trypsin-EDTA, centrifuged, and gently resuspended in serum-containing medium. They were then seeded on glass coverslips and incubated for 25 min at 37[degrees]C (RCJ-P) or 10 min at 27[degrees]C (A6). Fixation was performed with 2% glutaraldehyde in 0.1 M cacodylate buffer, 5 mM CaCl^sub 2^, pH 7.2, for 30 min, followed by three rinses (5 min each) with 0.1 M cacodylate buffer. The cells were postfixated for 1 h with 1% osmium tetraoxide in the same buffer. The coverslips were then rinsed, dehydrated with ethanol, and critical point dried with CO2 (Pelco CPD2, Ted Pella, Redding, CA). The samples were sputter-coated with thin film of the order of 10 nm of gold-palladium (S 150, BOC Edwards, Sussex, UK) and examined in the environmental scanning electron microscope, XL 30 ESEM FEG (Philips/FEI, Eindhorn, Netherlands) operated at 10 kV using high vacuum mode.
Preparation of hydrated sample for environmental scanning electron microscopy
Suspended cells were centrifugea, washed, and fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, 5 mM CaCl^sub 2^, pH 7.2, for 30 min. After rinsing with 0.1 M cacodylate buffer for 5 min, and three times with water (5 min each), the cells were incubated for 45 min with 2% uranyl acetate in water at pH 3.5, washed, resuspended in water, and seeded on serum-coated glass coverslips at 4[degrees]C for 16 h. For osmium letraoxide labeling, the fixed cells were rinsed with 0.1 M cacodylate buffer instead of water, and incubated for 45 min with 1% osmium tetraoxide in the same buffer. Finally the cells were washed twice with water and seeded on serum-coated coverslips. The samples were examined in the environmental scanning electron microscope, XL 30 ESEM FEG (Philips/FEI) at 10 kV, using wet mode at 5[degrees]C, 6.4 Torr (867 Pascal), 7.8 mm working distance.
Particle exclusion assay
Suspended RCJ-P cells were centrifugea, washed, and resuspended in serum-containing medium. They were then seeded in 35-mm tissue culture plates on scrum-coated coverslips and incubated for 25 min at 37[degrees]C. The cells were washed and fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, 5 mM CaCl^sub 2^, pH 7.2, for 30 min. They were then rinsed with 0.1 M cacodylate buffer for 5 min, and three times with water for 5 min each. The cells were stained with CY3 reactive dye (#Q13008, Biological Detection Systems, Pittsburgh, PA) in PBS for 3 min, and washed with water to remove excess dye. FITC-labeled 0.4-[mu]m silica beads (kindly provided by Prof. S. Margel, Bar Ilan University, Ramat Gan, Israel) were added to the plate so that the cells were completely immersed in beads. Micrographs were taken with a digital microscope system (DeltaVision, Applied Precision, Inc., Issaqua, WA) as previously described (Zamir et al., 1999). Image acquisition and processing were performed with Resolve3D and Priism programs (Zamir et al., 1999).
For 3D imaging, a series of z-sections were taken at 0.5-[mu]m intervals. The images were reconstructed with the full deconvolulion-based imaging of the Priism software.
Cells under shear flow
Suspended RCJ-P cells were centrifuged, washed, and resuspended in serum-containing medium. They were seeded on 35-mm serum-coated glass coverslips (Marienfeld, Bad Mergentheim, Germany), and incubated for 25 min at 37[degrees]C. The cells were then placed in a parallel plate flow chamber (GlycoTech, Rockville, MD) at 37[degrees]C, and subjected to flow, exerting a shear stress of 6.5 dyne/cm^sup 2^, applied for three minutes by peristaltic pump (Minipuls3, Gilson, Middleton, WI). Time-lapse movies were taken with DeltaVision digital microscope at 2-s intervals.
A6 cells were treated in the same manner, except that uncoated glass coverslips (batch by special order Marienfeld, Bad Mergentheim, Germany) were used. Cells were incubated for 5 min at 27[degrees]C before application of the flow.
Hyaluronan "footprint" labeling
Cells were exposed to flow for 3 min, as described above, then fixed for 20 min with 3% paraformaldehyde in phosphate-buffered saline (PBS) (Biological Services, The Weizmann Institute). The cells were washed three times with PBS, 10 min each, then incubated for 2 h at 37[degrees]C with 1:100 biotinylated hyaluronic acid binding protein (bHABP, 0.5 mg/ml, Seikagaku, Japan). They were then washed three times with PBS, for 15 min each, incubated with 1:200 CY3-conjugated streptavidin (1.8 mg/ml, ENCO, Petach Tiqva, Israel) for 30 min at 37[degrees]C, and washed again. Micrographs were taken with the DeltaVision digital microscope.
RESULTS
Visualization of cell-associated hyaluronan layers using the ESEM
The introduction of the ESEM has revolutionized the observation of biological samples by scanning electron microscopy by enabling direct examination of biological specimens in an aqueous environment. The microscope is designed to operate in atmosphere containing water at a pressure of up to 10 Torr (1333 Pascal). Thus, samples can be imaged in a humid environment in equilibrium with liquid H2O and/or with water vapor. These features are particularly critical for examination of hydrated gels, which undergo radical and irreversible changes upon dehydration. Our primary reference cells for studying the pericellular coat by ESEM were chondrocytes (RCJ-P) whose pericellular coat was reported to be in the range of several [mu]m thick (Lee et al., 1993). Various approaches were undertaken to visualize the pericellular coat, including conventional fixation with glutaraldehyde followed by treatment with osmium tetraoxide. All these treatments failed to reveal cell-associated material outside the plasma membrane (see below). Finally, treatment with uranyl acetate was attempted, with the idea that the heavy uranyl ions UO^sub 2^^sup 2+^ would bind to the negatively charged hyaluronan and concomitantly favor its visualization. Indeed, examination of fixed RCJ-P cells deposited on glass coverslips from suspension and incubated with 2% uranyl acetate at pH 3.5 revealed a 4.4 + or - 0.7-[mu]m-wide, sharply defined halo around the cells (Fig. 1 a). A6 epithelial cells subjected to the same treatment were surrounded by a 2.2 + or - 0.4-[mu]m-thick halo (Fig. 1 b).
Both RCJ-P and A6 cells treated with hyaluronidase to remove cell-bound hyaluronan (before fixation) and then incubated with uranyl acetate, did not display a halo when examined with ESEM (Fig. 1, c and d), supporting the notion that this halo indeed represents a hyaluronan-based pericellular coat.
The hyaluronan coat alone (i.e., without staining with uranyl acetate) is completely transparent to the electron beam, and thus no halo is visible around unstained cells (Fig. 1, e and f). The uranyl-labeled hyaluronan halo appears, however, to be semitransparent to electrons; the image of the cell membrane obtained through the labeled coat appears uniformly blurred, although the depth of field is well above the cell thickness (Fig. 1, a and b). In contrast, hyaluronidase-treated cells and cells that were not incubated with uranyl acetate display sharp borders (Fig. 1, c-f).
Critical point drying of RCJ-P cells results in the disappearance of the hyaluronan gel from the cell surface and exposure of the underlying microvilli (Fig. 1 g). The surface texture of A6 cells is quite different, being dominated by broad lamellae and membrane folds (Fig. 1 K). As expected, the surface-bound gel is not retained upon dehydration.
The ESEM also makes it possible to monitor the dehydration of the hyaluronan coat upon reduction of pressure in the ESEM chamber (Fig. 2). Chondrocytes (RCJ-P) were fixed and labeled with 2% uranyl acetate, pH 5.0, as described above, and examined by the ESEM (Fig. 2 a). Gradual reduction in pressure resulted in slow evaporation of the gel-retained water and, consequently, dehydration of the gel (Fig. 2 b). The dehydration resulted in shrinkage of the cells to 80% of their original projected area, and disappearance of the gel. Traces of uranyl acetate remain associated with the matrix at pH 5.0, thus marking the area that was associated with hyaluronan before dehydration (see below) (Fig. 2 b).
Binding of uranyl ions to the hyaluronan coat of chondrocytes is highly pH-dependent. Cells incubated with uranyl acetate at pH 3.1 (Fig. 3 a) or at pH 4.3 (Fig. 3 c) were positively labeled, but the signal was not as strong as at pH 3.5 (Fig. 3 b). At pH 5.0 uranyl acetate stains the hyaluronan gel, but tends to precipitate within the fixed gel because of reduced solubility (Fig. 3 d). Chondrocytes were also incubated with uranyl acetate oxalate at pH 7.0, the rationale being that at this pH hyaluronan should be fully negatively charged and thus maximally bind the uranyl cations. No staining of the gel was detected under these conditions, however (Fig. 3 e), probably because the solubility of uranyl acetate at this pH is too low.
The standard procedure for preparing samples for scanning electron microscopy includes postfixation with osmium tetraoxide (OsO^sub 4^), which particularly binds to lipids in the cell membrane. Such fixation enhanced the contour of the cell body, but did not highlight the cell-bound hyaluronan gel (Fig. 3 f).
3D visualization of the pericellular hyaluronan coat of chondrocytes by particle exclusion assay
The ESEM images of fixed chondrocytes (RCJ-P) and epithelial cells (A6) directly deposited from suspension showed that these cells are surrounded by a several-[mu]m-thick layer of hyaluronan. ESEM cannot, however, provide direct information on the thickness of the coat in the vertical dimension, on the apical cell surface. In other words, the cells may be entirely coated by hyaluronan or, alternatively, the hyaluronan coat may have "oozed" from the cell surface to the nearby glass surface, at least in part. To better evaluate the thickness of the coat in the vertical dimension, a particle exclusion assay based on fluorescence was employed. Chondrocytes were allowed to adhere to glass coverslips for 25 min before fixation under the same conditions used for the ESEM. The cells were then directly labeled with tetramethyl rhodamine iso-thiocyanate (red). After gentle washing, the cells were incubated with a large excess of FITC-labeled 0.4-[mu]m silica beads (green), such that they were completely immersed in beads, yet the beads would be excluded from the viscous zone of pericellular gel. Serial optical sections 0.5 [mu]m apart in depth were recorded using the digital DeltaVision microscope, which can generate 3D images by deconvolution-based 3D image reconstruction. A top view of such sections confirms that a 5- to 6-[mu]m zone from which beads are excluded surrounds untreated chondrocytes (dark area in Fig. 4 a and Movie 1 a, Supplementary Material), whereas the beads can access the surface of hyaluronidase treated cells (Fig. 4 b and Movie 1 b, Supplementary Material). The deconvoluted and reconstructed image shows an exclusion area, 1.2 [mu]m thick, above the cells (Fig. 4 c and Movie 1 c, Supplementary Material; see figure legend for further technical details). Again, hyaluronidase-treated cells have no excluded volume around them (Fig. 4 d and Movie 1 d, Supplementary Material). These data confirm that the hyaluronan coat indeed surrounds the entire cell, including the apical aspect, where its thickness is >1 [mu]m. The reduced thickness of the gel in the upper part of the cell may be due to the 25-min incubation of the cells before observation, which allows them to undergo at least partial spreading. It was unfortunately impossible to visualize cells directly deposited from suspension, because they are not well anchored on the glass, thus preventing imaging of a z-series.
The role of the hyaluronan coat in regulating the mechanical properties of early adhesions of chondrocytes
We have previously demonstrated that hyaluronan mediates and modulates matrix adhesion of A6 epithelial cells to a variety of surfaces (Zimmerman et al., 2002). To assess whether early adhesion of chondrocytes is also hyaluronan mediated, and to test the mechanical properties of the hyaluronan-mediated early cell adhesions, suspended RCJ-P cells were allowed to adhere for 25 min to serum-coated glass (Fig. 5 c). The cells were then subjected to a continuous flow of medium, which applies to them a constant shear force of 6.5 dyne/cm^sup 2^. Cells before and during the application of force were recorded by time-lapse video microscopy (Fig. 5 d and Movie 2 a, Supplementary Material). Comparison of the number of untreated cells before and after application of flow shows that ~80% of the cells remained firmly attached. In contrast, hyaluronidase-treated cells, lacking the hyaluronan coat, did not bind to the surface, and were thus instantly removed by the flow (Fig. 5, compare a to b and Movie 2 b, Supplementary Material). Only ~1.5% of the hyaluronidase-treated cells remained attached. We conclude that the early stages of chondrocyte adhesion are hyaluronan mediated.
Given the thickness of the hyaluronan coat surrounding cells such as chondrocytes, it is conceivable that the first interactions between the cells and the matrix are mediated by a relatively soft and viscous gel. To determine the mechanical properties of this adhesion, cells were exposed to a constant flow 25 min after seeding, and their forced translocation was recorded by time-lapse phase microscopy at 30 images/min. Examination of these movies showed that many of the attached untreated cells passively drifted in the general direction of flow before detaching from the surface. Tracking of the moving cells pointed to an average translocation of 55.5 + or - 31.6 [mu]m (range: 20.1-130.3 [mu]m), at an average speed of 2.3 + or - 1.6 [mu]m/sec (range: 0.7-3.9 [mu]m/sec). Individual "translocation tracks" can be visualized by comparing images taken at different time points, as in Fig. 5 d' and d' + 1 '. Both are magnifications of the marked area in Fig. 5 d taken at a 60-s interval. In Fig. 5 d' + 1 ' the pathway of each cell was reconstructed from the 30 time-lapse images in between. This behavior of passive translocation under flow is most probably due to rolling or sliding of the cells on a hyaluronan "cushion," as the distance covered is too large to be associated with cells anchored through integrin-mediated adhesions.
Considering its thickness, it is conceivable that the pericellular coat of chondrocytes comprises a multiple layer of entangled hyaluronan molecules. It is thus possible that cells, passively translocating under flow, will leave behind surface-bound hyaluronan footprints. To examine such possibility, chondrocytes were subjected to flow as described above, then fixed and incubated with biotinylated hyaluronan binding proteins (bHABP), followed by streptavidin-CY3. Fluorescence microscopy examination of these specimens revealed tracks of hyaluronan, generally located upstream to the cells (Fig. 6). Additional patches of hyaluronan were scattered on the surface, probably marking sites where cells had drifted and detached (data not shown). The average length of these hyaluronan "footprints" was 71.8 + or - 21.3 [mu]m (ranging from 28 to 105 [mu]m). The larger "footprints" were left by groups of more than one cell, thus the "footprints" correspond in their size to the drifting distance of cells under flow. In contrast to untreated cells, hyaluronidase-treated cells readily detach, and do not drift when subjected to flow.
A6 cells were subjected to the same procedure described above for chondrocytes, except that the time of incubation before application of flow was reduced to 5 min. Upon application of flow, ~80% of the cells remain attached to the glass. In contrast to chondrocytes, these cells do not drift but occasionally vibrate around their attachment centers with maximal dislocations of the order of [mu]m. Even upon application of maximal flow rate, equivalent to a force of 61.8 dynes/cm^sup 2^, essentially all the cells remained anchored to the glass. Posttreatment of the glass with hyaluronan binding protein does not reveal any traces of hyaluronan. Alter hyaluronidase treatment, <45% of the cells remain attached to the glass, confirming that their attachment is hyaluronan mediated.
The behavior of A6 cells indicates that the hyaluronan coat is not displaceable by application of shear force. Alternatively, these cells may rapidly switch from hyaluronan-mediated adhesion to receptor-mediated attachment. We consider the possibility that in A6 cells single hyaluronan molecules span the whole thickness of the coat as a brush emanating directly from attachment sites on the membrane.
DISCUSSION
In this study we have addressed the involvement of the pericellular hyaluronan coat in the adhesion of cells to external surfaces. Hyaluronan and its receptors, primarily CD44, are involved in many cellular processes, among them cell adhesion, motility, proliferation, and signaling (Borland et al., 1998; Toole, 2001). However, unlike the "conventional" ligand-receptor setting, many cells, such as chondrocytes, are surrounded by a thick hyaluronan layer whose properties, as well as its mode of interaction with the plasma membrane, can affect the adhesive process (McBride and Bard, 1979; Lee et al., 1993; Evanko et al., 1999). The first objective of the present study was to visualize and to determine the physical properties of the cell-associated hyaluronan. Performing ESEM on uranyl acetate stained cells, we have visualized a well defined, homogeneous, 4.4 + or - 0.7-[mu]m-thick, hydrated, hyaluronidase-sensitive coat around chondrocytes (RCJ-P) in suspension and a 2.2 + or - 0.4-[mu]m-thick coat around epithelial cells (A6).
Our results support the notion that hyaluronan surrounds both chondrocytes and epithelial cells as a gel phase, the exact concentration of which is yet to be determined. Although the external borders of the hyaluronan gel surrounding individual cells are sharply defined, the coats of cells grouped together merge, forming one uniform layer around and between them (Fig. 3). The pericellular coat appears to be homogeneous and partially transparent to electrons, with sharp and defined borders, whereas the cell membrane appears blurred (Fig. 1). These features are interpretable within the framework of the experimental technique used. The hyaluronan coat becomes visible when the liquid water around the cells is gradually removed by evaporation. If single hyaluronan molecules sprout sparsely from the gel layer at its boundary, the water surface tension will force them to condense at the interface upon evaporation. As a result, hyaluronan coat borders appear sharp.
The image in wet-mode ESEM is acquired with a gas (GSE) detector, which exploits the water molecules saturating the microscope chamber to amplify the signal of the secondary electrons emitted by the sample. In our experimental set up, the uranyl ions introduced in the pericellular coat are used as particularly efficient stimulation of secondary electron emission. As the secondary electron emission is amplified in the pericellular coat, the emission due to the electrons back scattered from the membrane will be also amplified. This will result in a blurred image. In agreement with this interpretation, when the pericellular coat is removed, or in the absence of uranyl acetate treatment, a sharp image of the cell border is obtained (Fig. 1, c and d).
In agreement with the polyelectrolyte properties of the hyaluronan gel, the thickness of the pericellular coat is sensitive to pH (Fig. 2). Surprisingly, at pH 4.3 the hyaluronan coat appeared thinner and less intense than at pH 3.5. Many variables could be affected by the pH and result in reduction of the coat apparent density and size. One such variable is the uranyl ion solubility, which may affect its ability to bind hyaluronan at higher pH. We can furthermore envisage that at higher pH hyaluronan chains may be less entangled because of repulsion between like charges. Hyaluronan molecules that are not directly bound to the membrane receptors may consequently be washed away during sample preparation resulting in a thinner and less dense coat. This suggestion is supported by the proven trail of hyaluronan shed by the cells drifting in a flow, after contact with the substrate has been established.
Finally, in the particle exclusion assay the 0.4-[mu]m silica beads do not penetrate the hyaluronan layer, and the pericellular coat is stable enough to form a few [mu]m-thick layer in the vertical dimension (Fig. 4). We note here that, as hyaluronan is not likely to be affected by glutaraldehyde fixation, cross-linking of hyaluronan-bound proteoglycans (Lee et al., 1993; Evanko et al., 1999; Knudson and Knudson, 2001; Kiani et al., 2002) may contribute to the rigidity of the fixed coat.
All the above evidence is consistent with the pericellular coat being in a gel phase. It is mandatory at this point, to compare the results obtained here with what is known about the physical properties and structure of hyaluronan in vitro and in the extracellular matrix. Hyaluronan forms three-dimensional hydrated gels in vitro. The thickness of surface-grafted hyaluronan as measured by imaging ellipsometry under humid atmosphere (Mathe et al., 1999) is within the range of 100 nm, which corresponds to 200-nm radius of gyration (Laurent, 1987). As expected from polyelectrolyte gels, the layer thickness is sensitive to salt concentration (Albersdorfer and Sackmann, 1999).
The hyaluronan polymer usually consists of 2000-20,000 disaccharides, with a molecular weight range of several million Daltons, depending on the tissue source (Toole, 2001). Concentrated (1 mg/ml) hyaluronan solutions form networks when carefully dried on mica or graphite (Jacoboni et al., 1999). Diluted solutions (1-5 [mu]g/ml) of hyaluronan molecules of 4.2 x 10^sup 6^ Da, visualized with the atomic force microscope, showed separated hairpin-shaped molecules with a typical length of 6-7 [mu]m (Cowman et al., 1998). High molecular weight hyaluronan was suggested to form a three-dimensional network of antiparallel hyaluronan ribbons, stabilized by specific hydrogen bonds between acetamido NH moieties and carboxylate groups on neighboring chains, and by hydrophobic interactions between the sugar aliphatic moieties (CH) (Hadler et al., 1982; Scott, 1992; Scott and Heatley, 1999, 2002). These interactions are proposed to be responsible for the gel-like characteristics of hyaluronan. The pericellular coat of eukaryotic cells contains, besides hyaluronan, also other proteins, among them proteoglycans such as aggrecan (Lee et al., 1993; Knudson and Knudson, 2001; Kiani et al., 2002) and versican (Evanko et al., 1999), which interact with hyaluronan. These can cross-link neighboring hyaluronan molecules, contributing to the stability of the hyaluronan-based gels around cells. The hyaluronan viscoelastic properties are extremely affected by the presence of those proteoglycans. Hyaluronan solutions from bacterial sources, which lack the hyaluronan binding proteoglycans, are viscoelastic liquids with a concentration-dependent viscosity typical of polyelectrolytes with excess salt rather than of gels (Gribbon et al., 2000; Krause et al., 2001).
The thickness of the hyaluronan layer observed around chondrocytes in this study is much larger than the expected molecular radius of gyration. It is thus conceivable that extended hyaluronan molecules anchored to membrane receptors project out from the membrane, forming thick brushes, when supported by a high density of receptors on the membrane. We note that the observed thickness is a moderate estimate, as hyaluronan layers progressively shrink with decreasing environment humidity (Mathe et al., 1999). We are currently testing the dimensions of the hyaluronan gel in cells completely immersed in water, using a novel approach (Thiberge et al., 2002).
The physical properties of the pericellular hyaluronan coat have a major impact on the interaction of cells with external surfaces. Due to its thickness, which is several orders of magnitude above the size of typical membrane proteins, the pericellular coat is most likely the first cellular component that encounters the matrix during the attachment process (Zimmerman et al., 2002). The properties of hyaluronan-mediated adhesion are thus strongly affected by the coat properties. Under shear stress of 6.5 dyne/cm^sup 2^ chondrocytes drifted on the matrix leaving trails of hyaluronan "footprints" (Fig. 6). Thus the thick chondrocyte pericellular coat can be stretched and peeled off the cell, at least in part. Assuming that the hyaluronan chains are not pulled off the receptor by these forces, this is consistent with the hyaluronan molecules being entangled in multiple layers. In contrast to chondrocytes, A6 cells, under a similar shear stress, remained anchored to the matrix. Their motions are limited to vibrations and oscillations around a fixed site, suggesting the model of suspended spheres anchored to the substrate through a mash of long tethers. This behavior is consistent with hyaluronan molecules being attached to A6 cells in a "brush" configuration, where each hyaluronan molecule is directly attached to a receptor in the membrane.
The early, hyaluronan-mediated adhesion sets the stage for the establishment of receptor-mediated interactions between members of the integrin family and corresponding ECM proteins such as fibronectin or vitronectin (Geiger et al., 2001; Martin et al., 2002). There are various possible scenarios for this transition: Cells can extend long dynamic membrane projections (e.g., filopodia) that may protrude beyond the hyaluronan coat, forming a direct membrane contact. Chondrocytes, for example, contain microvilli, ranging in length from 1.14 to 2.84 [mu]m (mean length: 1.76 + or - 0.49 [mu]m), based on transmission electron microscope and SEM measurements (data not shown). The chondrocyte hyaluronan coat is within the range of 3-5.6 [mu]m (average 4.4 + or - 0.7 [mu]m, from ESEM measurements). The coat thickness was measured on chondrocytes that underwent fixation while in suspension. Considering the surface properties and the contribution to the pericellular coat rigidity of hyaluronan-bound proteoglycan cross-linking, we can assume that the measured coat dimensions are representative of those in live chondrocytes. The microvilli are thus hidden within the hyaluronan coat; the latter may shrink upon interaction with the matrix, exposing the microvilli, ready to interact with the matrix and promote inegrin-mediated adhesion. It is noteworthy that the microvilli were not detectable in the wet ESEM, suggesting that they either do not protrude beyond the border of the gel, or collapse during the observation.
An alternative scenario for the transition between hyaluronan-mediated and integrin-mediated adhesion is that the hyaluronan coat may be locally removed, exposing the integrins to the matrix. Removal of the hyaluronan coat could be achieved by lateral diffusion of hyaluronan receptors, by local degradation, by internalization of hyaluronan via the CD44 receptor (Knudson et al., 2002), or by local change of pH or ion concentration, which may lead to hyaluronan shrinkage (Albersdorfer and Sackmann, 1999).
Being hydrated, the pericellular coat provides a stable osmotic environment, thus buffering small instabilities. When chondrocytes were dried in the ESEM chamber by reducing the vapor pressure, the pericellular coat responded to the reduction in pressure with a delay of 10-15 min, whereas water droplets disappeared almost instantaneously. This is reminiscent of the behavior of hyaluronan in cartilage, synovial fluid, and the extracellular matrix, where it has the role of preserving tissue hydration and swelling by maintaining the osmotic pressure (Israelachvili and Wennerstrom, 1996; Knudson and Knudson, 2001; Toole, 2001).
Finally, the concept of cells being surrounded by a several-[mu]m-thick pericellular coat with the properties of a gel is not to be considered lightly. It implies that every interaction with the environment around the cells, soluble or insoluble, will be affected by the presence of such layer that any component, be it signaling molecules, proteolytic enzymes, metabolites, nutrients, or drugs, need to penetrate to reach the cell membrane.
SUPPLEMENTARY MATERIAL
An online supplement to this article can be found by visiting BJ Online at http://www.biophysj.org.
We thank Prof. S. Margel (Bar Ilan University, Ramat Gan, Israel) for providing us with FITC-labeled silica beads. L.A. is an incumbent of the Dorothy and Patrick Gorman Professorial Chair. B.G. is an incumbent of the E. Neter Chair in Tumor and Cell Biology.
This work was supported by a grant from the Ziegler family trust.
[Sidebar]
Biophysical Journal Volume 85 September 2003 1996-2005
[Reference]
REFERENCES
Adams, J. C. 2001. Cell-matrix contact structures. Cell. Mol. Life Sci. 58:371-392.
Albersdorfer, A., and E. Sackmann. 1999. Swelling behavior and viscoelasticity of ultrathin grafted hyaluronic acid films. Eur. Phys. J. B. 10:663-672.
Bajorath, J. 2000. Molecular organization, structural features, and ligand binding characteristics of CD44, a highly variable cell surface glycoprotein with multiple functions. Proteins. 39:103-111.
Bard, J. B., W. H. McBride, and A. R. Ross. 1983. Morphology of hyaluronidase-sensilive cell coats as seen in the SEM after freeze-drying. J. Cell Sci. 62:371-383.
Borland, G., J. A. Ross, and K. Guy. 1998. Forms and functions of CD44. Immunology. 93:139-148. Cowman, M. K., M. Li, and E. A. Balazs. 1998. Tapping mode atomic force microscopy of hyaluronan: extended and intramolecularly interacting chains. Biophys. J. 75:2030-2037.
Danilatos, G. D. 1991. Review and outline of environmental SEM at present. J. Microsc. 162:391-402.
de Gennes, P. G. 1987. Polymers at the interface; a simplified view. Adv. Colloid Interface Sci. 27:189-209.
Evanko, S. P., J. C. Angello, and T. N. Wight. 1999. Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arteriosder. Thromb. Vasc. Biol. 19:1004-1013.
Geiger, B., A. Bershadsky, R. Pankov, and K. M. Yamada. 2001. Transmembrane crosstalk between the extracellular matrix-cyloskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2:793-805.
Grihbon, P., B. C. Heng, and T. E. Hardingham. 2000. The analysis of intermolecular interactions in concentrated hyaluronan. Riochem. J. 350: 329-335.
Hadler, N. M., R. R. Dourmashkin, M. V. Nermut, and L. D. Williams. 1982. Ultrastructure of a hyaluronic acid matrix. Proc. Natl. Acad. Sci. USA. 79:307-309.
Hanein, D., B. Geiger, and L. Addadi. 1994. Differential adhesion of cells to enantiomorphous crystal surfaces. Science. 263:1413-1416.
Hanein, D., B. Geiger, and L. Addadi. 1995. Cell adhesion to crystal surfaces: a model for initial stages in the attachment of cells to solid substrates. Cells and Materials. 5:197-210.
Hanein, D., H. Sabanay, L. Addadi, and B. Geiger. 1993. Selective interactions of cells with crystal surfaces. Implications for the mechanism of cell adhesion. J. Cell Sci. 104:275-288.
Israelachvili, J., and H. Wennerstrom. 1996. Role of hydralion and water structure in biological and colloidal interactions. Nature. 379:219-224.
Jacoboni, I., U. Valdre, G. Mori, D. Quaglino, Jr., and I. Pasquali-Ronchetti. 1999, Hyaluronic acid by atomic force microscopy. J. Struct. Biol. 126:52-58.
Kam, Z., D. A. Agard, and J. W. Sedat. 1997. Three-dimensional microscopy in thick biological samples: a fresh approach for adjusting focus and correcting spherical aberration. Bioimaging. 5:40-49.
Kiani, C., L. Chen, Y. J. Wu, A. J. Yee, and B. B. Yang. 2002. Structure and function of aggrecan. Cell Res. 12:19-32.
Knudson, C. B., and W. Knudson. 2001. Cartilage proteoglycans. Semin. Cell Dev. Biol. 12:69-78.
Knudson, W., G. Chow, and C. B. Knudson. 2002. CD44-medialed uptake and degradation of hyaluranan. Matrix Biol. 21:15-23.
Krause, W. E., E. G. Bellomo, and R. H. Colby. 2001. Rheology of sodium hyaluronate under physiological conditions. Biomacromolecules. 2:65-69.
Laurent, T. C. 1987. Biochemistry of hyaluronan. Ada Ololaryngol. Suppl. 442:7-24.
Lee, G. M., B. Johnstone, K. Jacobson, and B. Caterson. 1993. The dynamic structure of the pericellular matrix on living cells. J. Cell Biol. 123:1899-1907.
Lee, J. Y., and A. P. Spicer. 2000. Hyaluronan: a multifunctional, mega Dalton, stealth molecule. Curr. Opin. Cell Biol. 12:581-586.
Martin, K. H., J. K. Slack, S. A. Boerner, C. C. Martin, and J. T. Parsons. 2002. Integrin connections map: to infinity and beyond. Science. 296:1652-1653.
Mathe, G., A. Albersdorfer, K. R. Neumaier, and E. Sackmann. 1999. Disjoining pressure and swelling dynamics of thin adsorbed polymer films under controlled hydration conditions. Langmuir. 15:8726-8735.
McBride, W. H., and J. B. Bard. 1979. Hyaluronidase-sensitive halos around adherent cells. Their role in blocking lymphocyte-mediated cytolysis. J. Exp. Med. 149:507-515.
Scott, J. E. 1992. Supramolecular organization of extracellular matrix glycosaminoglycans, in vitro and in the tissues. FASEB J. 6:2639-2645.
Scott, J. E., and F. Heatley. 1999. Hyaluronan forms specific stable tertiary structures in aqueous solution: a ^sup 13^C NMR study. Proc. Natl. Acad. Sci. USA. 96:4850-4855.
Scott, J. E., and F. Heatley. 2002. Biological properties of hyaluronan in aqueous solution are controlled and sequestered by reversible tertiary structures, defined by NMR spectroscopy. Biomacromolecules. 3:547-553.
Tadmor, R., N. Chen, and J. Israelachvili. 2002. Thin film rheology and lubricity of hyaluronic acid solution at a normal physiological concentration. J. Biomed. Maler. Res. 61:514-523.
Thiberge, S., E. Moses, and O. Zik. 2002. Electron microscopy of cells in a wet environment: A new high resolution imaging technique. Biophys. J. 82:495a.
Toole, B. P. 2001. Hyaluronan in morphogenesis. Semin. Cell Dev. Biol. 12:79-87.
Weigel, P. H., V. C. Hascall, and M. Tammi. 1997. Hyaluronan symhases. J. Biol. Chem. 272:13997-14000.
Zamir, E., B. Z. Katz, S. Aota, K. M. Yamada, B. Geiger, and Z. Kam. 1999. Molecular diversity of cell-matrix adhesions. J. Cell Sci. 112:1655-1669.
Zimmerman, E., B. Geiger, and L. Addadi. 2002. Initial stages of cell-matrix adhesion can be mediated and modulated by cell-surface hyaluronan. Biophys. J. 82:1848-1857.
[Author Affiliation]
Miriam Cohen,*[double dagger] Eugenia Klein,[dagger] Benjamin Geiger,* and Lia Addadi[double dagger]
*Department of Molecular Cell Biology, [dagger]Chemical Research Services Unit, and [double dagger]Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel
[Author Affiliation]
Submitted January 14, 2003, and accepted for publication April 2, 2003.
Address reprint requests to Lia Addadi, Dept. of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-9344105; Fax: 972-8-9344151; E-mail: lia.addadi@weizmann.ac.il.
(C) 2003 by the Biophysical Society
0006-3495/03/09/1996/10 $2.00
South Africa's players have to overcome the talent of Brazil and their understandable feelings of inferiority if they are to carry the hopes of a nation and reach the Confederations Cup final.
Like any opponent going up against the record five-time World Cup winners, South Africa knows it is as good as beaten if it goes onto the field for Thursday's semifinal match star-struck by former world player of the year Kaka and striker Robinho.
The likes of burly central defender Matthew Booth cannot match the South Americans when it comes to skill, so Bafana Bafana is relying on raucous home support to rouse them to a performance of greater intensity than their opponents can muster.
"We don't want to get carried away putting these guys up on a pedestal," South Africa goalkeeper Rowen Fernandez said. "Yes, they're idols to millions and we respect them all as footballers, but we're going to go out there Thursday and compete on the pitch.
"It's 11 versus 11, obviously, and I'm sure Matthew is dying to get stuck into Robinho and give Kaka a piece of his mind."
Fernandez and Booth are among four players in the South African squad that beat Brazil at the 2000 Sydney Olympics, but an under-23 tournament is quite another thing compared to a full international like Thursday's at Ellis Park.
"Playing for a draw against them isn't going to do us any good," South Africa midfielder Teko Modise said. "They're going to allow us space and time to attack them and to play and we'll look to utilize that. We really want to play our hearts out and attack. We need to score as many goals as we can because we all know they're capable of scoring many.
"When we first came into the camp, we said we want to go to the semis and, now we're here, we also want to go to the final as well. It's possible. The Brazilians are not unbeatable."
But Brazil has racked up 10 goals in group games against World Cup holder Italy, African champion Egypt and the United States, compared to the two South Africa managed in a significantly easier group.
"But by the same token, we might look on the positive side and say they also conceded goals," South Africa striker Katlego Mashego said. "It's a plus for us that they can concede goals. We get them tired, we might sneak in and have one or two chances. It's not impossible. We just have to give our all."
And South Africa could have the right player to take advantage of any slips by a defense that conceded three times in a 4-3 win over Egypt before tightening for 3-0 wins over the United States and Italy.
Bernard Parker made his first mark on the tournament when he stopped his own teammate from scoring a winning goal against Iraq by blocking a shot on the line, but the striker then scored both goals against New Zealand and has hit a tournament-high 10 shots on target.
Spain striker David Villa is way back in second place with seven on-target efforts, albeit with three goals.
Brazil, though, has the statistic that counts.
The South Americans have scored more goals than anyone else, two more than European champion Spain.
With Luis Fabiano, Robinho and Kaka exchanging positions fluidly in attack, Booth and his colleagues could be in for a torrid time.
Even so, Brazil will be without Juan after the key defender was ruled out for the remainder of the tournament with a left thigh injury and the players were not taking anything for granted ahead of a match they are overwhelming favorites to win.
"Tradition doesn't win or lose matches," Luis Fabiano said. "In a semifinal, you have to be careful. To make the final we need to enter the match with the same concentration and motivation that we had against Italy.
"It's going to be a difficult match. They are playing at home and will give their lives against Brazil."
One thing that is guaranteed is that Ellis Park will echo to the near-deafening noise of local supporters blowing their vuvuzela trumpets, something that those unfamiliar with South African football have said can be distracting.
"They are expressing themselves and they have a unique way of getting behind the team," Fernandez said. "The players, the South African ones anyway, are comfortable with it.
"If it works as a psychological benefit for us, I hope the supporters go on Thursday and blow even louder."
Olympic champion Elena Dementieva improved her 2009 winning streak to 12 matches on Thursday, beating Iveta Benesova 6-4, 6-1 to reach the third round of the Australian Open.
Dementieva, who won titles at Auckland and Sydney before the season's first major, struggled with her serve early but recovered to finish the match in 72 minutes.
The 27-year-old Dementieva reached the French Open and U.S. Open finals in 2004, beaten both times by fellow Russians, and has not returned to the championship of a Grand Slam tournament since.
Her highlight last year was a win over fellow Russian Dinara Safina in the gold medal match at the Beijing Olympics. She was ousted in the semifinals at Wimbledon and the U.S. Open in 2008 and has never advanced beyond the fourth round at Melbourne Park.
Amelie Mauresmo, who won here and at Wimbledon in 2006, rallied for a 4-6, 6-3, 6-2 win over Britain's Elena Baltacha. Mauresmo, a former No. 1 now seeded 20th after an injury-prone 2008, advanced when Baltacha double-faulted on match point.
Fellow Frenchwoman Virginie Razzano ousted No. 14 Patty Schnyder of Switzerland 6-3, 6-1.
Other women advancing included No. 13 Victoria Azarenka of Belarus and No. 18 Dominika Cibulkova of Slovakia, a 6-0, 6-2 winner over Taiwan's Chan Yung-jan.
No. 2 Serena Williams, aiming to continue a pattern of winning the Australian Open every odd year since 2003, was playing Gisela Dulko. Her sister Venus Williams has an evening match against Carla Suarez Navarro of Spain.
Men's No. 1 Rafael Nadal takes on Roko Karanusic and No. 4 Andy Murray has the last night match on the Rod Laver Arena.
No. 6 Gilles Simon of France won the opening match on center court at Melbourne Park, beating Australian Chris Guccione 6-7 (5), 6-4, 6-1, 6-2.
For the first time in six years, Amer Delic is still in contention after two rounds.
And to think, he thought he was heading home after he lost 6-0, 6-7 (3), 6-0 to Florian Mayer in his third qualifying match.
The 26-year-old Bosnian-born American got a place in the main draw as a lucky loser and knocked off No. 28-seeded Paul-Henri Mathieu in the second round in five sets Wednesday. His next assignment will be tougher: defending champion Novak Djokovic.
"Things have been going my way," he said. "Why stop it here?"
Given the history in the Balkans, and the mix of its migrants to Australia, the atmosphere for Delic's match against the 21-year-old Serb will have an edge to it.
Delic's run has attracted a group of enthusiastic fans _ unruly according to the father of his vanquished first-round rival, fellow American Taylor Dent; disrespectful by Mathieu's reckoning.
The flag-waving Bosnians who are following Delic traded chants across the court with Serbian fans Wednesday, during, before and after points against his French rival.
"I couldn't control any of that, though I was trying to," Delic said. "I felt bad for Paul and I apologized to him right after."
Any extra tension for Friday's match is what police, local organizers and Delic want to avoid.
He said his family moved to Florida in 1996 to get away from any conflict, his parents wanting to start a new life after the Bosnian war.
"I just hope the next match with Novak doesn't turn into a World War III," he said. "I'm going to try to tell my fans that we don't need to be embarrassing ourselves in front of the world. I'm hoping Novak says something to Serbian fans, also. Leave the politics aside.
"It's not my fault and it's not Novak's fault. We're out here playing tennis and we need to keep it that way."
Donnie Wahlberg, lead singer and "bad boy" of New Kids on theBlock, agreed to make public-service announcements on fire safety anddrugs in return for eventual dismissal of a charge stemming from ahotel fire.
Under an agreement between prosecutors and Wahlberg's lawyers,an arson charge was reduced to criminal mischief. The case will beon hold until the 21-year-old singer completes the public service.
He originally faced up to 20 years in jail on the first-degreearson charge. Wahlberg was arrested March 27 after a hallway fire inthe historic Seelbach Hotel in Louisville, Ky., where he was staying.There were no injuries and only a small patch of carpet was burned.
During a night hearing that was hastily announced, DistrictJudge James M. Green lectured Wahlberg.
"I try not to say much in these situations . . . but this is aterrible situation, and you're a very lucky young man," Green said."This is a great deal. I hope you make us proud." As part of theagreement, Wahlberg "accepted responsibility" for the fire.
"I want to apologize to the people of Louisville," saidWahlberg. "I think, as everyone agrees, this is a most unfortunateincident. It's been a very ugly incident."
He added, "I'm very excited and very much welcome doing thesepublic-service announcements, which is what I've been doing, which isvery much what my career is based on."
Jesse optimistic on S. African trade mission
In a telephone call-in from Johannesburg, S.A. on his weekly radio broadcast at Dr. King's Workshop, the Rev. Jesse L. Jackson Sr. said he hopes his trip will stimulate trade between Africa and African American businesses in a major way.
Leading a three-nation tour, where he was joined by his wife, Jacqueline, son, Jonathan, and a delegation of business persons from his Rainbow PUSH/Wall Street, LaSalle Street Trade Bureaus, Jackson said the trip is designed to stimulate trade between African American firms and Africa.
He's hoping to bridge the digital divide between the two continents.
Having visited Accra, Ghana, Lagos, Nigeria and Johannesburg and Cape Town in South Africa, Jackson said he has focused on debt relief, the devastating impact of AIDS on that continent where he's trying to mobilize world opinion on this issue, as well as business investment.
Jackson wants to engage in trade partnerships with African nations and their leaders. He's scheduled to return to Chicago Thursday.
Jackson told the audience while many will celebrate Valentine's Day, "this year, we'll have 2 million in jail of whom 55 percent are Black with more than half on non-violent drug charge. These laws are insane on one hand and very profitable on the other.
"We will not stop until we rescue our children and reclaim them and turn these jails into museums," he told a cheering audience.
Jackson said this Saturday at Dr. King Workshop, 930 E. 50th St., he's conducting a voter registration drive because it is the last Saturday for residents to register in order to vote in the March 21 primary.
He said South Africa "paid a big price for the right to vote and now they are voting. We paid a big price for the right to vote, and we're not voting."
Jackson also announced that Rubin "Hurricane" Carter, the boxer who was the subject of the movie "Hurricane," will join him Friday where he'll show the film to Cook County Jail inmates. Carter will also be at Dr. King's Workshop, 10 a.m., Saturday Feb. 19th as well.
Jackson, who recently appeared on WVON's Cliff Kelley show, said the theme of his trip is: "Connecting Telecommunications and Media Markets Through U.S. Minority and African Business Partnerships."
Article Copyright Sengstacke Enterprises, Inc.
Photo (Jesse Jackson Sr.)
ABSTRACT:
This article describes a single-subject study of the effects of a contextualized instructional package on the area and perimeter performance of middle school students with emotional and behavioral disorders (EBD). The instructional package included area and perimeter problem-solving activities embedded in contextualized, or real-life, scenarios; use of manipulative techniques to reinforce concepts; and a self-monitoring component, a critical behavior management component to instructional interventions for students with EBD. Results indicated that the instructional package was effective for improving area and perimeter performance among the participants; transfer and maintenance results were mixed.
Students with emotional and behavioral disorders (EBD) experience deficits in mathematics achievement when compared with peers without disabilities (McLaughlin, Krezmien, & Zablocki, 2009; Trout, Nordness, Pierce, & Epstein, 2003; Wagner, Kutash, Duchnowski, Epstein, & Sumi, 2005). In addition, secondary students with EBD are more likely than their peers with other disabilities to receive a poor grade, be retained in a grade, and drop out of school before graduation (Wagner & Cameto, 2004). Furthermore, secondary general educators and special educators reported teaching mostly basic arithmetic skills rather than essential conceptual knowledge and skills to secondary students with EBD (Maccini & Gagnon, 2002). The literature clearly shows that the population of secondary students with EBD has limited access to and limited success with noncomputational mathematics concepts, including knowledge of area and perimeter problem solving.
Noncomputational mathematics includes complex skills and concepts related to geometry and algebra. Recent reforms in mathematics (i.e., National Council of Teachers of Mathematics [NCTM] Principles and Standards, 2000; National Mathematics Advisory Panel, 2008) and changes in general and special education regulations (Individuals with Disabilities Education Improvement Act of 2004 [IDEIA]; No Child Left Behind Act of 2001 [NCLB; 2002]) have identified the need for effective mathematics instruction of noncomputational skills, including the calculation of perimeter and area of polygons, for students with EBD in secondary grades. Area and perimeter are traditionally taught through the use of teacher-directed instruction of formulae. Often, students do not learn or understand the underlying concept associated with the formula and fail to master the formulae. This lack of mastery impairs the ability to generalize the concepts and impedes the learning of more complex geometric skills. As a consequence, students with EBD who do not acquire these foundational skills do not succeed in middle school and high school mathematics and do not meet the rigorous standards mandated by the NCLB (2002).
One of the problems with the current model of teaching geometry to students with EBD is the failure to meld the abstract concepts with the concrete applications of perimeter and area formulae. Situating learning experiences in contextualized and authentic problems can assist secondary students with EBD in understanding noncomputational concepts related to measurement and geometry (Goldman, Hasselbring, & The Cognition and Technology Group at Vanderbilt, 1997). One way to teach conceptual understanding is to give students the opportunity to construct their own knowledge, building meaning from their own background knowledge and using reallife activities to support further understanding (Goldsmith & Mark, 1 999; Maccini & Gagnon, 2002; Schloss, Smith, & Schloss, 2001; Stiff, 2001; Ward, 2001). However, these constructive approaches alone will not be adequate for students with disabilities who typically benefit from direct instruction (Baker, Gersten, & Lee, 2002; Landrum, Tankersley, & Kauffman, 2003; Maccini & Gagnon, 2000). Instead, a balanced approach that includes opportunities for students to develop deep understandings of concepts through embedded meaningful contexts associated with constructivism, while efficiently processing material through direct instruction (Smith & Geller, 2004), is necessary for teaching geometry to students with disabilities.
Currently, mathematics instruction in special education classrooms tends to focus on computation, neglecting conceptual understanding (Gersten & Chard, 1999). Consequently, outcomes are particularly poor for secondary students with EBD. Researchers found that students with EBD did not significantly improve math achievement scores despite 5 years of full-time special education services (Anderson, Kutash, & Duchnowski, 2001) and that students with EBD actually experienced an increase in mathematics deficits over their school careers (Nelson, Benner, Lane, & Smith, 2004).
Despite the known mathematic deficiencies of youth with EBD, little attention has been paid to developing and implementing effective mathematics interventions for secondary students with EBD. In a methodological review of mathematics intervention research for secondary students with EBD, Mulcahy and Krezmien (2008) found that only 1 1 studies have been conducted since 1 975. Furthermore, previous research on math interventions with secondary students with EBD focused primarily on basic skills and computation (Mulcahy & Krezmien, 2008). These earlier investigations did not examine student performance on math skills and concepts required in middle and high school settings and essential to meet national and state standards as required by NCLB (2002), IDEIA (2004), and NCTM (2000). Futhermore, there are currently no published studies on effective interventions for geometry instruction for secondary students with EBD.
The current study was designed to address these gaps in the research literature by applying a package of empirically based practices to a geometry-related topic from the middle school curriculum. Because the research literature is devoid of a set of empirically based interventions for secondary students with EBD, the intervention package was developed based on research of effective interventions for students with other disabilities (Maccini, Mulcahy, & Wilson, 2007). The study, which was conducted with secondary students with EBD, examined the effects of a contextualized instructional package on objectives related to area and perimeter. The intervention involved components of instruction that were found to be effective in previous research: (a) the use of contextualized instruction (Bottge, 1999; Goldman et al., 1997), (b) self-monitoring of student academic and social behavior (Carr & Punzo, 1 993; Reid & Harris, 1993), and (c) the use of manipulative materials and cue cards (Butler, Miller, Crehan, Babbitt, & Pierce, 2003; Cade & Gunter, 2002; Carr & Punzo, 1 993).
The study investigated four research questions:
1. Does a contextualized instructional package to teach the geometric concepts of area and perimeter result in increases in the mathematics accuracy of secondary students with EBD?
2. Do secondary students with EBD maintain performance on geometry-related tasks mastered through the use of a contextualized instructional package over time?
3. Do secondary students with EBD transfer performance on geometry-related tasks learned through the use of a contextualized instructional package to mathematics problems with similar contexts?
4. Do secondary students with EBD transfer performance on geometry-related tasks mastered through the use of a contextualized instructional package to more complex area and perimeter problems?
Method
Participants
Four middle school students with EBD (Carlos, Grace, Steven, and Riley) participated in this study. Two of the initial 4 participants dropped out of the study, and 2 additional students were recruited. The intervention with the initial 2 participants (Carlos and Grace) was replicated with Steven and Riley after Carlos and Grace completed the intervention. This was necessary to improve the power, as recommended by Tawney and Gast (1984). Information obtained from school records regarding demographic characteristics of the participants including gender, race, age, grade level, IQ, and achievement scores at the time of the study are displayed in Table 7. In addition, further description of participant behaviors and performance during the intervention are described in the Results section.
Participants met the following eligibility criteria established prior to the study: (a) They were previously school identified as having an EBD, (b) they received instruction in a selfcontained special education math class at the time of the study, and (c) they scored below 60% on an investigator-developed pretest targeting objectives related to area and perimeter of squares and rectangles.
Instructor and Setting
The intervention was implemented by the first author. The participants were pulled from a social skills class for students with EBD in a public middle school in suburban Maryland. The room used for the intervention, with two exceptions, was a 5-foot � 8-foot vacant room. The school supplied one student desk, two chairs, and two stools for the intervention. All study activities, including dependent measures, were completed in the same room. Sessions 8 and 9 for Riley were conducted at Riley's home because they fell during the school spring break. These sessions were conducted at the dining room table, with the same materials and manipulatives used at school. No other individuals were around, and the space was clear and neat during these sessions.
Measures
Domain probes, objective probes, and transfer and maintenance probes were administered to participants in the study. Domain probes provided a pretest-posttest assessment of student skills across the entire unit, much like a survey curriculum-based assessment (CBA; Hudson & Miller, 2006). Objective probes provided a daily progress monitoring of student performance on individual objectives, similar to an untimed focused CBA (Hudson & Miller, 2006). Transfer and maintenance probes assessed participants' ability to use the skills and concepts they acquire to solve problems of similar context and more complex problems, as well as the ability to continue to successfully solve area and perimeter problems over time. All probes were scored for the percentage of accurate responses.
Prior to the investigation, the domain and objective probes were piloted with a group of middle school students with average to aboveaverage academic achievement from three different middle schools in the same district. The piloted probes were evaluated for their appropriateness for use with the district's middle school population through test-retest reliability measures. Permanent products (probes) were analyzed as repeated measures of percentage of accurate responses and the percentage of items completed on the daily self-monitoring recording sheet. In addition, each student involved in the pilot was individually interviewed following the probe administration for feedback on improving the directions, nature of the questions, and physical layout of the probe.
Domain probes. The domain probes were administered to students as a pretest measure, during baseline, and after the intervention phase. The domain probes measured student performance on prerequisite tasks, such as ordering shapes by area and perimeter, as well as on tasks derived from the middle school curriculum, such as exploring the relationship among area, perimeter, and dimensions of squares and rectangles. Probe items directly related to the school district's curriculum, state curriculum, and NCTM (2000) standards. Problems on the assessment were restricted to those involving rectangles consistent with the instructional unit. The assessments consisted of (a) closed-ended questions, (b) openended questions, (c) one-step problems, and (d) multistep problems (Balanced Assessment Project Team, 1999; Hudson & Miller, 2006; Rectanus, 1997).
Parallel versions of domain probes consisted of 10 curriculum-based problems on area and perimeter, with items sampled from each objective, and totaled 42 points. A minimum of two domain probes were administered to students during baseline. Following the intervention phase, three domain probes were administered on successive school days.
Objective probes. Parallel versions of the objective probes were given only during the intervention phase, at the end of each class session in which the particular objective was taught. Objective probes totaled 10 to 12 points each. If a participant met the criterion (80%) on the objective probe at the end of the lesson, the next consecutive lesson was administered. If the criterion was not met, the same lesson was administered the following session.
Transfer and maintenance probes. Each participant was assessed on his or her ability to generalize, or transfer, the knowledge learned on similar mathematics tasks that involve different story lines than those addressed in the instructional set (near transfer) as well as more complex and different math problems than those targeted for instruction (far transfer). Participants were also assessed on their ability to maintain their new skills over time. A transfer and maintenance probe was administered twice during baseline and then three times following the end of the intervention. The first postintervention transfer and maintenance probe was administered on the school day immediately following the final intervention session. The transfer and maintenance probe was also administered 5 and 10 school days after the end of the intervention phase.
Social validity measure. Social validity measures ensure that research is considered by consumers to be useful (Lloyd & Heubusch, 1996). The participants completed an investigator-developed social validity instrument at the end of the study that involved questions about the appropriateness of the intervention and the participants' perceptions of its effectiveness. The instrument was based on instruments used in previous research (Butler et al., 2003; Calhoon & Fuchs, 2003; Maccini, 1998). Participants responded to 10 questions on a five-point Likert scale concerning the effectiveness of various aspects of the intervention. Participants indicated a score of 1 if they strongly disagreed with a statement, 2 if they disagreed, 3 if they felt neutral, 4 if they agreed, and 5 if they strongly agreed. All of the questions were positively phrased to maintain consistency. In addition, participants responded to six open-ended questions.
Independent Variable
The independent variable consisted of an instructional package designed to teach area and perimeter objectives drawn from the NCTM (2000) standards, the state curriculum, and the district curriculum. The independent variable targeted participants' procedural and conceptual knowledge of area and perimeter through contextualized problem-solving opportunities and included a self-monitoring behavior management component.
Contextualized instruction. Contextualized instruction was presented in the form of real-life applications of meaningful area and perimeter problems (Bottge, 1999; Goldman et al., 1997) provided in each lesson. For example, topics were introduced and practice opportunities were provided related to building patios, designing skate parks, building dog pens, and other real-life contexts.
Procedural knowledge. Procedural knowledge was targeted through the use of cue cards that identified the process learners use to solve problems (Cade & Gunter, 2002; Carr & Punzo, 1993; Hawkins, Brady, Hamilton, Williams, & Taylor, 1994; Hudson & Miller, 2006). Cue cards included definitions of area and perimeter and contained space for students to write in formulae and procedures during instructional sessions. Participants were taught to use the cue cards to assist them in solving problems during the intervention phase.
Conceptual knowledge. Conceptual knowledge was targeted through the use of concrete applications (manipulatives) and pictorial representations during instruction. Participants were required to solve problems using manipulative materials including Geoboards, Post-it notes, inch tiles, and paper clips. Participants identified a variety of rectangle- and squareshaped objects in the room and then measured and calculated the area and perimeter of those shapes using the manipulatives to enable students to develop a conceptual understanding of the relationships between area and perimeter of similar geometric figures. Students then used the conceptual understanding gained from practice with concrete examples to develop and understand the use of formulae at an abstract level. When formulae are understood through this process, participants should be able to solve area and perimeter problems in a more efficient manner (Hudson & Miller, 2006).
Self-monitoring. Self-monitoring of academic performance and behavior was included as a component of the instructional package. Participants were presented with a self-monitoring checklist derived from materials and procedures used in prior research on self-monitoring (Carr & Punzo, 1993; HaIIahan, Lloyd, Kosiewicz, Kauffman, & Graves, 1979; Harris, Friedlander, Saddler, Frizzelle, & Graham, 2005; Reid & Harris, 1 993; Uberti, Mastropieri, & Scruggs, 2004) and were taught to self-record the total number of problems completed and the number of problems completed correctly during independent practice for each session. The selfmonitoring checklist also required students to self-evaluate based on a set of behavioral expectations.
Instruction on self-monitoring occurred at the beginning of the intervention phase. First, each participant was provided with explicit definitions of "academic achievement" and "on-task behavior." Then, behavioral expectations were discussed, including a definition of "appropriate behavior," and examples and nonexamples were provided of desired behaviors. Each participant had an existing behavior contract; the instructor referred to the behavioral expectations in the contracts to provide a rationale for self-monitoring behavior. Each student worked on individual behavioral expectations through use of the contract. Procedures for completing the self-monitoring checklist were modeled by the instructor and then practiced by the participants.
Instructional Procedures
Components of effective instruction (Hudson & Miller, 2006; Rosenshine & Stevens, 1986) were used in instructional delivery. Lesson plans were structured to include each component to ensure a systematic implementation of the lessons. New concepts were presented in short, concrete steps, as recommended by Hudson and Miller (2006) and Rosenshine and Stevens (1986). Constructivistbased activities were infused into each lesson during demonstration and guided practice opportunities throughout the unit.
Instructional unit and lesson plans. The instructional unit, including all lesson plans and activities, was investigator developed and adapted from prerequisite and gradeappropriate concepts and skills within the NCTM (2000) standards, the state curriculum, and the district curriculum, but the instructional content was adapted to meet the academic needs of the participants. Adapting instructional content is often necessary (a) to meet the individual needs of students with EBD, (b) to teach related prerequisite skills, and (c) to access grade-appropriate mathematics standards (Bryant, Kim, Hartman, & Bryant, 2006). For instance, the unit addressed only customary units of inches and feet; the problem examples focused only on rectangular regions and involved two-digit by two-digit computation. These adaptations were implemented to ensure that participants would not experience difficulties with other mathematics skills and concepts that would interfere with their ability to learn area and perimeter.
The instructional unit consisted of 1 1 lessons, including the introduction/self-monitoring lesson. The remaining 10 lessons covered topics across four objective sets. The first 8 lessons (Lessons 1-1 through 3-2) focused on prerequisite skills, whereas the last 3 lessons (Lessons 4-1 through 4-3) dealt with middle school objectives. The length of the intervention was designed to reflect the length of a typical secondary mathematics unit. The intervention included 45-minute instructional periods, 4 days per week, over an average of 1 1 .5 sessions (r = 11-13) per participant.
Experimental Design and Study Procedures
The current research study employed a single-subject multiple probe across 2 participants, replicated by 2-participants design (Horner & Baer, 1 978; Kennedy, 2005; Tawney & Gast, 1984) to evaluate the effects of teaching secondary students a contextualized instructional package with self-monitoring techniques to solve geometry problems. The multiple-probe technique was chosen because it allowed the researcher to conduct infrequent probes of baseline behavior rather than continuous baseline measures in instances in which continuous measurement may be reactive or impractical or when an assumption of baseline stability can be made in advance of the study (Horner & Baer, 1978). In the current study, collecting continuous baseline data was both impractical and unnecessary. Because students did not receive instruction in area and perimeter during the baseline phase, if they did not demonstrate mastery of the skills at pretest, no threat could be assumed of an unstable baseline due to exposure to those or similar topics. In addition, collecting infrequent probes rather than continuous baseline data allowed data collection on a greater number of participants and was necessary because of time restrictions imposed by the school schedule.
Baseline. The baseline was established through administration of the domain probes. To ensure that regular mathematics instruction was not interrupted, the participants were administered the baseline probe sessions and intervention during a daily social skills class period. Throughout the study, participants received mathematics instruction from their special education math teacher in the selfcontained classroom on topics and activities in the district's middle school mathematics curriculum. During the baseline phase, the students had no contact with the instructor except during probe sessions.
Intervention. Each participant received the intervention after the preceding participant completed the intervention phase. Participants' out-of-class time was monitored, and sessions were scheduled so that all participants missed the same amount of the social skills class. Because instruction took place during the students' social skills class, no participants missed regular math instruction.
Booster session. An additional booster session (Montague, 2004) was added to the intervention after low performance on the first postintervention domain probe session by the first two participants. Booster sessions were the same for each participant; each objective was reviewed, and two examples were practiced from each objective set. No objective probe was administered during the booster session. Domain probes were administered in subsequent sessions.
Interrater Reliability and Fidelity of Treatment
Interrater reliability. Interrater reliability was obtained on 100% of the domain probes. A trained research assistant and the investigator independently scored each probe. The percentage of scorer agreement was calculated by dividing the number of agreements of correct responses by the number of disagreements and agreements and multiplying the result by 100% (Kennedy, 2005). Reliability was 100% across all domain probes for all students.
Fidelity of treatment. Fidelity of treatment observations were conducted by independent observers via a checklist that incorporated the components of the intervention. Prior to the investigation, the observers were trained on the components of the intervention and provided the fidelity checklist. Training consisted of an explanation of the instructional procedures, a review of the components of the fidelity checklist, and two mock instructional sessions, in which fidelity data were collected. Observers maintained 100% agreement over the mock sessions.
Fidelity observations were conducted for 33% of the instructional and probe sessions. The independent observer sat in the room with the instructor and the participant. The observer sat in a location that allowed him to see each component of the intervention, and he did not speak to the child or the instructor during the intervention. The fidelity of treatment for each session was calculated by dividing the number of components present (as recorded on the checklist) during the observation by the number of total components on the checklist and multiplying the quotient by 100%. The fidelity of treatment for the study was obtained by adding the percentages for each session and dividing the sum by the number of observations. Treatment fidelity for the study was 100%.
Interobserver agreement was obtained one time for every three observations of the first independent observer. During these sessions, a second independent observer conducted fidelity of treatment (via the checklist) at the same time as the first observer. After each observation, the observers added their completed checklists to a notebook, which was reviewed by the investigator. Interobserver agreement on the fidelity of treatment was calculated by dividing the number of agreements on the checklists from each independent observer by the total number of items on the checklist and multiplying the quotient by 100%. Interobserver agreement for the study was 1 00%.
Results
Academic Measures
Math accuracy. All participants improved their mean percentage accuracy from baseline to postintervention on the domain probes. Figure ? displays the results of percentage accuracy on domain probes during baseline and postintervention phases for the first 2 participants (i.e., Carlos and Grace). Figure 2 displays the results of percentage accuracy on domain probes during baseline and postintervention phases for participants in the replication (i.e., Steven and Riley). Visual analysis of the four graphs reveals marked improvement in level from baseline to postintervention for all participants. Furthermore, there was low variability in data points in both the baseline and postintervention phases, reflecting stable data for each participant. Carlos increased 91 percentage points in mean percentage accuracy, Grace increased 72 percentage points, Steven increased 83 percentage points, and Riley increased 67 percentage points.
An error analysis of the postintervention domain probes revealed that each participant earned point deductions for inappropriate or missing labels (e.g., labeling solution with inches or inches2). Across four postintervention domain probes, Carlos missed 3 points because of mislabeling. Across four postintervention domain probes, Grace missed 6 points because of mislabeling. Across three probes, Steven missed 2 points because of mislabeling. Finally, Riley missed 4 points because of mislabeling on three probes. Errors similar to these were also noted on the pretests; however, the more apparent issues with the pretests were the lack of completion of questions (students attempted very few items on the pretest) and errors in computation or conceptual understanding for the problems that were completed.
Each participant experienced high levels of performance across objective probe administrations. Carlos was the only participant who required repeated lesson sessions to meet 80% criterion on objective probes. He required one repeated session following Lesson 1-2: Ordering by Area. He also required one repeated session following Lesson 2-1: Measuring Perimeter Using Non-Standard Units. Both of these lessons were prerequisite skills and were administered during the 1st week of the intervention. For the remaining sessions, Carlos earned a mean score of 96.6% on objective probes (r = 90%-100%). Grace, Steven, and Riley demonstrated high levels of performance on objective probes throughout the intervention, earning mean scores of 97.5% (r = 90%1 00%), 96.2% (r = 80%-1 00%), and 98% (r = 90%-1 00%) on objective probes, respectively.
Maintenance. The maintenance task was an exercise taken directly from the instructional unit. As demonstrated in Figure 7, Grace showed no improvement on the maintenance task from baseline to postintervention sessions. In contrast, as demonstrated in Figure 2, Steven earned a score of 100% on the maintenance task during each postintervention transfer and maintenance probe session, and Riley earned a score of 0% on the first postintervention maintenance task and then 100% during the 5-day and 10-day postintervention transfer and maintenance probe sessions. Carlos refused to attempt the maintenance task during each of the postintervention transfer and maintenance probe sessions.
Transfer tasks. The near transfer task was similar in nature to tasks completed during the intervention, but participants were not explicitly taught using the exact wording or steps required to solve this particular problem. Grace and Riley each earned a mean score 0% on the near transfer task during postintervention transfer and maintenance probe sessions, and Carlos refused to attempt the near transfer task during each of the postintervention transfer and maintenance probe sessions. Steven, on the other hand, showed improvement on the near transfer task, earning scores of 100%, 83%, and 100%, respectively, on each of the postintervention transfer and maintenance probe sessions.
The far transfer task consisted of a complex, multistep problem using the same concepts and skills covered in the instructional unit. No direct instruction was provided on the far transfer task. Grace, Steven, and Riley each showed improvement on far transfer tasks, albeit limited (r = 50%-58%), from baseline to postintervention (see Figures 7 and 2). Carlos refused to attempt the far transfer task.
Self-monitoring. Each of the participants completed the self-monitoring checklist at the end of each session without instructor prompting or input. In addition, each participant appeared interested and eager to complete the self-monitoring checklist each day. Participants regularly cheered for themselves when they calculated the percentage correct.
Social validity. The average scores on the social validity measure ranged from 4 to 5 (M = 4.7, mode = 5). Overall, participants responded that they strongly agreed that the intervention package helped them to understand area and perimeter better (M = 5), and they would recommend the intervention to others (M = 4.75, r = 4-5). They also responded that they strongly agreed that they feel better about measurement and geometry skills as a result of using the intervention (M = 5). Finally, the participants reported that they strongly agreed that the self-monitoring checklist helped them to solve area and perimeter problems and they would recommend its use to other students (M = 4.75, r = 4-5).
The participants also responded positively to the intervention in the open-ended questions. For example, Riley stated she was interested in learning measurement and geometry skills "so I could help others, and teach them, too." Grace reported that "this is what I'm best at in math." Carlos stated, "If I'm needed, I will be able to help." When asked what they liked best about the intervention, participants' responses were favorable. Riley reported that she "liked using the cubes and the Geoboard," and Steven reported that he liked the cue card best. When asked what they liked least, the response was unanimous: the tests.
Discussion
This study represents the first investigation of the effectiveness of a contextualized instructional package on the area and perimeter performance of middle school students with EBD. The findings from this investigation contribute to the existing research literature base in several ways. First, prior to this study, no research had been published on mathematics interventions for secondary students with EBD in the area of geometry and measurement. Second, the instructional package studied in the investigation represents a unique combination of empirically supported components, delivered within a balanced approach integrating constructivism and explicit instruction (Mulcahy & Gagnon, 2007; National Mathematics Advisory Panel, 2008). Third, the instructional package taught prerequisite and grade-appropriate area and perimeter objectives directly aligned with the NCTM standards, the state curriculum, and the district curriculum. This indicates that the instructional approach implemented in this study was sufficient for meeting the rigorous standards associated with the NCLB but was flexible enough to support the learning of students with severe academic and behavioral deficiencies. Finally, this study is important because half of the participants were female, in contrast to the near absence of female participants in existing mathematics research in this area (Mooney, Epstein, Reid, & Nelson, 2003).
This investigation was able to demonstrate positive results regarding the first research question, which was related to mathematics accuracy on area and perimeter objectives. All participants were able to improve accuracy on both domain probes and objective probes with respect to area and perimeter, and each of the participants earned greater than 90% on at least two postintervention domain probes. The high performance during instruction and during the postintervention domain probe sessions suggests that the instructional package positively affected performance on mathematics accuracy. The consistent changes in outcomes for Carlos and Grace demonstrated that the independent variable had a positive impact on the dependent variable, which establishes a functional relationship (Kennedy, 2005). Replication of these findings with Steven and Riley demonstrated robustness of the experimental control and generality to other participants (Kennedy, 2005).
The findings with regard to maintenance and transfer probes were mixed. Three participants (Grace, Steven, and Riley) were able to maintain some level of performance over time, but for Grace and Riley, the performance did not meet the same level at postintervention as during intervention. Only Steven was able to accurately complete the maintenance task at each of the transfer and maintenance probe sessions. Grace and Riley would have shown commensurate performance on maintenance probes, but they misread the directions and accurately completed the wrong task (i.e., they solved for the largest area, not the smallest area), a mistake often made by typically developing students (Battista, 2003). Nonetheless, the 2 participants demonstrated familiarity with the relationship between area and perimeter, and both accurately completed parts of the far transfer task that involved calculating area and perimeter of a garden.
Carlos refused to complete the maintenance and transfer probes, typical of his behavior throughout the intervention and in the classroom. He refused to attempt novel and challenging tasks, characteristic of students with EBD (Gunter & Denny, 1998). His limited persistence to task and self-efficacy, typical of students with learning disabilities (Lackaye & Margalit, 2006; Meltzer et al., 2004), indicates that these factors may also be associated with poor performance on geometry tasks for students with EBD.
Finally, Steven was able to transfer successfully to a task with similar contexts, but none of the participants could successfully complete the far transfer task in its entirety. The results suggest that although participants were able to demonstrate mastery on perimeter and area objectives, their ability to maintain and transfer performance was limited. The mixed results on the transfer and maintenance measures suggest that students with EBD require explicit and sustained instruction of a variety of contexts to maintain and transfer newly learned geometry skills, a contention supported by previous research (Mooney et al., 2003; National Mathematics Advisory Panel, 2008). There are a number of possible explanations for the limited maintenance and transfer including a misplaced or inaccurate assumption that mastery will lead to maintenance and eventually transfer for students with EBD. This may not be the case for students with EBD on complex noncomputational math tasks. The fear of failure may prevent students with EBD from attempting an unfamiliar problem, even when they have the tools to complete it. This point was demonstrated most clearly by Carlos, who refused to attempt any unfamiliar problems. The other participants attempted the problems, but when a solution did not reflect what they expected, they did not attempt to find a more accurate solution.
It is also possible that the limited length of the intervention phase was insufficient to help participants improve performance on transfer and maintenance tasks. In fact, this intervention was much shorter than typical teachermediated interventions for students with EBD, which average 22 days (Pierce, Reid, & Epstein, 2004). Finally, the limited scope of the intervention, which targeted a narrow topic in geometry, did not include instruction in solving new and novel problems, nor did it teach problem-solving skills generally.
Limitations of the Current Study
The limitations of the current study include attrition, design flaws, and the possible effects of extraneous variables such as the location of the intervention and instructor bias. First, participant attrition led to a design change. The original study proposal involved a multiple-probe design across 4 participants, but 2 of the original participants left the study during the baseline phase because of emotional or behavioral crises including hospitalization. Thus, the study design was revised to the multiple-probe design across 2 participants, replicated by 2 participants. Although the original design was more desirable in terms of power and control, the study demonstrated power and control through replication across participants within the experiment and across experiments with 2 new participants (Kennedy, 2005). Two additional limitations to the design warrant consideration. First, one additional consecutive data point should have been collected with Grace during baseline. Although her baseline was consistent and level, Horner and Baer (1978) recommended collecting at least one additional consecutive, or true, baseline session from the previous participant, to be consistent with multiplebaseline design. Concerns about possible attrition led to the decision to begin the intervention prior to the collection of an additional baseline data point, consistent with recommendations of Tawney and Gast (1 984), who provided examples that did not include an additional consecutive baseline.
Next, changes to the instructional procedures during the study may have affected the findings. Booster sessions were not a component of the original design but became necessary as the first 2 participants were unable to demonstrate proficiency on their initial postintervention domain probes, despite performing at consistently high levels on objective probes throughout the intervention. Their difficulty with the initial domain probes may have been due, in part, to breaks in the schedule and the length of the intervention itself. The booster session served as a review, similar to a review session a classroom teacher would implement prior to a unit test (Montague, 2004). Both participants in the original sample (i.e., Carlos and Grace) increased their performance to 90% or greater after the implementation of the booster session. In the replication (i.e., Steven and Riley), the booster session was implemented in the first session following the end of the intervention. Both participants earned 90% or greater on all postintervention domain probes.
An error analysis of the postintervention domain probes revealed several point deductions due to lack of labeling or mislabeling responses despite having accurate conceptual understanding and computation. This finding suggests that the participants were able to accurately solve the problems but made careless mistakes that resulted in inaccurate solutions to the problems. As a consequence, the outcomes on probes may underrepresent their understanding of the concepts learned as they reflect mistakes that were not directly related to the study objectives. Participants were not explicitly taught the practice of reviewing problems and checking for labels within this intervention. Systematic instruction and explicit reinforcement of this practice are likely necessary for students with EBD. Alternately, scoring based only on accuracy of numeric responses, rather than making deductions for labels, might have been more appropriate.
The current study was conducted in an isolated setting, with a 1-to-1 student-toteacher ratio. There were limited opportunities for distractions from objects of other individuals. In addition, the students had the sole attention of the instructor, which is atypical of a classroom environment. Numerous distractions are typical in classroom settings, and individual students do not typically receive the undivided attention of a teacher. Consequently, the intervention implemented in this study may not be effective in group settings or in typical classrooms.
Recommendations for Future Research
This intervention was successful for the participants in a one-on-one isolated setting, when used with geometry and measurement concepts. Replicating the package with different math topics with other students with EBD at the middle and high school levels would be necessary for generalization. Once the effectiveness of the instructional package has been demonstrated through replications with different populations of students, with a variety of math topics, and in more typical classroom environments, future research should also focus on the effectiveness of individual components of this intervention package. Once this intervention has been established as empirically supported through replication and further research on the components, the important components should be examined using an experimental group design as recommended by Odom and colleagues (2005) and the National Mathematics Advisory Panel (2008).
Recommendations for Practice
The current study has implications for teachers of secondary students with EBD. First, results of this study indicate that students with EBD can master grade-appropriate objectives when presented through the use of an instructional package including contextualized instruction and self-monitoring of behavior and academic performance. The intervention was implemented over a period of time that is reflective of the length of a typical secondary instructional unit, and the instructional package may be effective for inclusive classrooms, special education resource rooms, and selfcontained classrooms, when a teacher is able to provide explicit and sustained instruction to individual students. However, the intervention may be impractical for classrooms in which teachers are pressured to cover a vast amount of grade-level material in a limited amount of time and may be restricted in the amount of prerequisite material that can be covered, such as general education classrooms.
Conclusion
Access to the general education curriculum and high-quality instruction in geometry is necessary for improved mathematics performance among students with EBD. Federal policies support the need to improve mathematics performance for secondary students with EBD (IDElA, 2004; NCLB, 2002; NCTM, 2000), but the field has done little to respond to the specific needs of this population. Prior to this study, research on interventions targeting measurement and geometry with secondary students with EBD was absent from the research base. This study provides seminal evidence that these students can improve performance on grade-appropriate measurement and geometry objectives when instruction is delivered through a contextualized instructional package, despite academic and behavioral difficulties. The participants in this study also demonstrated limited success in maintaining performance over time and transferring performance to novel and more challenging tasks.
Continued research is critical to identify effective approaches to teaching geometry to secondary students with EBD. A set of empirically supported instructional approaches for this population of students will likely contribute to more favorable outcomes, including improved performance in math classes, increased high school completion rates, and improved performance on national and international mathematics assessments. With increased demands for mathematics proficiency in school and in the workplace, secondary teachers of students with EBD can no longer focus efforts on basic computation skills to the exclusion of teaching students the concepts and problem-solving skills associated with noncomputational mathematics. Secondary students with EBD can master complex abstract mathematics, but innovative and comprehensive instructional approaches must be developed, studied, and implemented to achieve this end. The field laudably continues to dedicate resources toward research to prevent the development of mathematic deficiencies for primary students, but secondary students with EBD continue to struggle in mathematics and fail to meet standards and expectations in algebra and geometry. In light of what we know about long-term outcomes for students with EBD, the failure to dedicate adequate energy and resources to interventions for secondary students is no longer acceptable.
[Reference]
REFERENCES
Anderson, J., Kutash, K., & Duchnowski, A. J. (2001). A comparison of the academic progress of students with EBD and students with LD. Journal of Emotional and Behavioral Disorders, 9(2), 106-116.
Baker, S., Gersten, R., & Lee, D. (2002). A synthesis of empirical research on teaching mathematics to low-achieving students. Elementary School Journal, 703(1), 51-73.
Balanced Assessment Project Team. (1 999). Middle grades assessment. White Plains, NY: Dale Seymour Publications.
Battista, M. (2003). Understanding students' thinking about area and volume measurement. In D. H. Clements, & C. Bright (Eds.), Learning and teaching measurement: 2003 yearbook (pp. 1 22-1 42). Restan, VA: National Council of Teachers of Mathematics.
Bottge, B. (1999). Effects of contextualized math instruction on problem solving of average and below-average achieving students. Journal of Special Education, 33(2), 81-92.
Bryant, D. P., Kim, S. A., Hartman, P., & Bryant, B. R. (2006). Standards-based mathematics instruction and teaching middle school students with mathematical disabilities. In M. Montague, & A. Jitendra (Eds.), Teaching mathematics to middle school students with learning disabilities (pp. 7-28). New York: Guilford Press.
Butler, F. M., Miller, S. P., Crehan, K., Babbitt, B., & Pierce, T. (2003). Fraction instruction for students with mathematics disabilities: Comparing two teaching sequences. Learning Disabilities Research and Practice, 18(2), 99-111.
Cade, T, & Gunter, P. L. (2002). Teaching students with severe emotional or behavioral disorders to use a musical mnemonic technique to solve basic division calculations. Behavioral Disorders, 27, 208-214.
Calhoon, M. B., & Fuchs, L. S. (2003). The effects of peer-assisted learning strategies and curriculumbased measurement on the mathematics performance of secondary students with disabilities. Remedial and Special Education, 24, 235-245.
Carr, S. C, & Punzo, R. P. (1 993). The effects of selfmonitoring of academic accuracy and productivity on the performance of students with behavioral disorders. Behavioral Disorders, 18, 241-250.
Gersten, R., & Chard, D. (1999). Number sense: Rethinking arithmetic instruction for students with mathematical disabilities. Journal of Special Education, 350), 18-28.
Goldman, S. R., Hasselbring, T. S., The Cognition and Technology Group at Vanderbilt. (1997). Achieving meaningful mathematics literacy for students with learning disabilities. Journal of Learning Disabilities, 30, 1 98-208.
Goldsmith, L. T., & Mark, J. (1999). What is a standards-based mathematics curriculum? Educational Leadership, 57(3), 40-44.
Gunter, P. L., & Denny, R. K. (1998). Trends and issues in research regarding academic instruction of students with emotional and behavioral disorders. Behavioral Disorders, 24, 44-50.
Hallahan, D. P., Lloyd, J., Kosiewicz, M. M., Kauffman, J. M., & Graves, A. W. (1979). Selfmonitoring of attention as a treatment for a learning disabled boy's off-task behavior. Learning Disability Quarterly, 2(3), 24-32.
Harris, K. R., Friedlander, B. D., Saddler, B., Frizzelle, R., & Graham, S. (2005). Selfmonitoring of attention versus self-monitoring of academic performance: Effects among students with ADHD in the general education classroom. Journal of Special Education, 39, 145-156.
Hawkins, J., Brady, M. P., Hamilton, R., Williams, R. E., & Taylor, R. D. (1994). The effects of independent and peer guided practice during instructional pauses on the academic performance of students with behavioral disorders. Education and Treatment of Children, 77(1), 1-28.
Horner, R. D., & Baer, D. M. (1978). Multiple-probe technique: A variation of the multiple baseline. Journal of Applied Behavior Analysis, 7 7(1), 189-196.
Hudson, P., & Miller, S. P. (2006). Designing and implementing mathematics instruction for students with diverse learning needs. Boston, MA: Pearson Education, Inc.
Individuals with Disabilities Education Improvement Act of 2004 (IDEIA), P.L. 108-446, 20 U.S.C. � 1 400 et. seq.
Kennedy, C. H. (2005). Single-case designs for educational research. Boston, MA: Pearson Education, Inc.
Lackay, T. D., & Margalit, M. (2006). Comparisons of achievement, effort, and self-perceptions among students with learning disabilities and their peers from different achievement groups. Journal of Learning Disabilities, 39, 432-446.
Landrum, T. J., Tankersley, M., & Kauffman, J. M. (2003). What is special about special education for students with emotional or behavioral disorders? journal of Special Education, 37, 148-156.
Lloyd, J. W., & Heubusch, D. (1996). Issues of social validation in research on serving individuals with emotional or behavioral disorders. Behavioral Disorders, 22(M, 8-14.
Maccini, P. (1998). Effects of an instructional strategy Incorporating concrete problem representation on the introductory algebra performance of secondary students with learning disabilities. Unpublished doctoral dissertation, Pennsylvania State University.
Maccini, P., & Gagnon, J. C. (2000). Best practices for teaching mathematics to secondary students with special needs. Focus on Exceptional Children, 32(5), 1-23.
Maccini, P., & Gagnon, J. C. (2002). Perceptions and application of NCTM standards by special and general education teachers. Exceptional Children, 68, 325-344.
Maccini, P., Mulcahy, C. A., & Wilson, M. G. (2007). A follow-up of mathematics interventions for secondary students with learning disabilities. Learning Disabilities Research and Practice, 220), 58-74.
McLaughlin, M. )., Krezmien, M., & Zablocki, M. (2009). Special education in the new millennium: Achieving educational equity for students with learning and behavior disabilities. In T. Scruggs, & M. Mastropieri (Eds.), Advances in learning and behavior disabilities, (pp. 1-32). Bingley, UK: Emerald.
Meltzer, L., Reddy, R., Pollica, M. S., Roditi, B., Sayer, )., & Theokas, C. (2004). Positive and negative self-perceptions: Is there a cyclical relationship between teachers' and students' perceptions of effort, strategy use, and academic performance? Learning Disabilities Research & Practice, 79(3), 33-44.
Montague, M. (2004). Math problem solving for middle school students with disabilities. Washington, DC: The Access Center: Improving Outcomes for All Students K-8. Internet site: http://www.k8accesscenter.org/ (Accessed May 3, 2007).
Mooney, P., Epstein, M. H., Reid, R., & Nelson, J. R. (2003). Status of and trends in academic intervention research for students with emotional disturbance. Remedial and Special Education, 24, 273-287.
Mulcahy, C. A., & Gagnon, J. C. (2007). Teaching mathematics to secondary students with emotional/behavioral disorders. In L. M. Bullock & R. A. Gable (Eds.), Seventh CCBD mini-library series: Ensuring a brighter future for troubled children/youth: Challenges and solutions (pp. 1-47). Arlington, VA: Council for Children with Behavioral Disorders.
Mulcahy, C, & Krezmien, M. (2008). A methodological review of mathematics interventions for secondary students with EBD: 7 975 to 2008. Unpublished manuscript.
National Council of Teachers of Mathematics. (2000). Principles and standards for school mathematics: An overview. Internet site: www. nctm.org/standards/ (Accessed October 19, 2005).
National Mathematics Advisory Panel. (2008). Foundations for success: The final report of the National Mathematics Advisory Panel. Washington, DC: U.S. Department of Education.
Nelson, J. R., Benner, G. J., Lane, K., & Smith, B. W. (2004). Academic achievement of K-12 students with emotional and behavioral disorders. Exceptional Children, 77(1), 59-73.
No Child Left Behind Act of 2001, Pub. L. No. 107110, 115 Stat. 1425 (2002).
Odom, S. L., Brantlinger, E., Gersten, R., Horner, R. H., Thompson, B., & Harris, K. R. (2005). Research in special education: Scientific methods and evidence-based practices. Exceptional Children, 77(2), 137-148.
Pierce, C. D., Reid, R., & Epstein, M. H. (2004). Teacher-mediated interventions for children with EBD and their academic outcomes: A review. Remedial and Special Education, 25, 1 75-1 88.
Rectanus, C. (1997). Math by all means: Area and perimeter grades 5-6. White Plains, NY: Math Solutions Publications.
Reid, R., & Harris, K. H. (1993). Self-monitoring of attention versus self-monitoring of performance: Effects on attention and academic performance. Exceptional Children, 60(1), 2940.
Rosenshine, B., & Stevens, R. (1986). Teaching functions. In M. C. Wittrock (Ed.), Handbook of research on teaching (3rd ed., pp. 376-391). New York: MacMillan.
Schloss, P. )., Smith, M. A., & Schloss, C. N. (2001). Instructional methods for secondary students with learning and behavior problems (3rd ed.). Boston, MA: Allyn & Bacon.
Smith, K. S., & Geller, C. (2004). Essential principles of effective mathematics instruction: Methods to reach all students. Preventing School Failure, 48(4), 22-29.
Stiff, L. V. (2001). Constructivist mathematics and unicorns. Internet site: www.nctm.org/news/ pastpresident/2001 -0708presidenthtm (Accessed March 23, 2006).
Tawney, J. W., & Gast, D. L. (1984). Single-subject research in special education. Columbus, OH: Charles E. Merrill.
Trout, A. L., Nordness, P. D., Pierce, C. D., & Epstein, M. H. (2003). Research on the academic status of children with emotional and behavioral disorders: A review of the literature from 1961 to 2000. Journal of Emotional and Behavioral Disorders, 11, 198-210.
Uberti, H. Z., Mastropieri, M., & Scruggs, T. E. (2004). Check it off: Individualizing a math algorithm for students with disabilities via selfmonitoring checklists. Intervention in School and Clinic, 39, 269-275.
Wagner, M., & Cameto, R. (2004). The characteristics, experiences, and outcomes of youth with emotional disturbances. NLTS Data Brief, 3(2).
Wagner, M., Kutash, K., Duchnowski, A. J., Epstein, M. H., & Sum�, C. W. (2005). The children and youth we serve: A national picture of the characteristics of students with emotional disturbances receiving special education. Journal of Emotional and Behavioral Disorders, 13, 79-96.
Ward, C. D. (2001). Under construction: On becoming a constructivist in view of the Standards. Mathematics Teacher, 94(2), 94-96.
[Author Affiliation]
Candace A. Mulcahy
State University of New York at Binghamton
Michael P. Krezmien
University of Massachusetts, Amherst
[Author Affiliation]
AUTHORS' NOTE
Address correspondence to Candace Mulcahy, School of Education, State University of New York at Binghamton, PO Box 6000, Binghamton, NY 13902; email: cmulcahy@binghamton.edu.
MANUSCRIPT
Initial Acceptance: 6/19/09
Final Acceptance: 7/6/09
