An Approach to Improving Student Success in Science, Technology, Engineering, and Mathematics (STEM) Career Pathways.

In this article, we report on an 11-year study that explores approaches to improve student success in college by a five-week summer program in Mathematics and Language Arts for entering freshmen. To recruit students into the program, we invited students accepted at the university and listed as underrepresented and economically disadvantaged (Pell-eligible) by the Office of Institutional Research at California State University, Northridge. The program consisted of all-day Math and English enhancement in mixed ability groups. Results of this program examining Math and English performance at California State University, Northridge showed that students completing the summer programs during the 11-year study period had improved pass rates in Math and English at California State University, Northridge compared with students in a control group who did not participate in the summer program. The results show that intensive pre-college enhancement for entering freshmen can improve student success in college. Student graduation data from the early cohorts (2010, 2011, 2012) were obtained from Institutional Research. The summary results showed that students from the accepted/attending group had substantially increased GPAs and graduation rates, essentially closing the achievement gap. Increased interest in biomedical research careers was also developed by the program, as demonstrated by a five-fold number of summer enrichment participants entering the PhD, MARC (Minority Access to Research Careers) and RISE (Research Initiative for Scientific Enhancement) programs than students who did not attend summer enrichment.

Terminal alpha-d-mannosides are critical during sea urchin gastrulation.

The sea urchin embryo is a United States National Institutes of Health (NIH) designated model system to study mechanisms that may be involved in human health and disease. In order to examine the importance of high-mannose glycans and polysaccharides in gastrulation, Lytechinus pictus embryos were incubated with Jack bean α-mannosidase (EC 3.2.1.24), an enzyme that cleaves terminal mannose residues that have α1-2-, α1-3-, or α1-6-glycosidic linkages. The enzyme treatment caused a variety of morphological deformations in living embryos, even with α-mannosidase activities as low as 0.06 U/ml. Additionally, formaldehyde-fixed, 48-hour-old L. pictus embryos were microdissected and it was demonstrated that the adhesion of the tip of the archenteron to the roof of the blastocoel in vitro is abrogated by treatment with α-mannosidase. These results suggest that terminal mannose residues are involved in the adhesion between the archenteron and blastocoel roof, perhaps through a lectin-like activity that is not sensitive to fixation.

Involvement of l(-)-rhamnose in sea urchin gastrulation. Part II: α-l-Rhamnosidase.

The sea urchin embryo is recognized as a model system to reveal developmental mechanisms involved in human health and disease. In Part I of this series, six carbohydrates were tested for their effects on gastrulation in embryos of the sea urchin Lytechinus pictus. Only l-rhamnose caused dramatic increases in the numbers of unattached archenterons and exogastrulated archenterons in living, swimming embryos. It was found that at 30 h post-fertilization the l-rhamnose had an unusual inverse dose-dependent effect, with low concentrations (1-3 mM) interfering with development and higher concentrations (30 mM) having little to no effect on normal development. In this study, embryos were examined for inhibition of archenteron development after treatment with α-l-rhamnosidase, an endoglycosidase that removes terminal l-rhamnose sugars from glycans. It was observed that the enzyme had profound effects on gastrulation, an effect that could be suppressed by addition of l-rhamnose as a competitive inhibitor. The involvement of l-rhamnose-containing glycans in sea urchin gastrulation was unexpected, since there are no characterized biosynthetic pathways for rhamnose utilization in animals. It is possible there exists a novel l-rhamnose-containing glycan in sea urchins, or that the enzyme and sugar interfere with the function of rhamnose-binding lectins, which are components of the innate immune system in many vertebrate and invertebrate species.

Involvement of L(-)-rhamnose in sea urchin gastrulation: a live embryo assay.

The sea urchin embryo is a National Institutes of Health model system that has provided major developments, and is important in human health and disease. To obtain initial insights to identify glycans that mediate cellular interactions, Lytechinus pictus sea urchin embryos were incubated at 24 or 30 h post-fertilization with 0.0009-0.03 M alpha-cyclodextrin, melibiose, L(-)-rhamnose, trehalose, D(+)-xylose or L(-)-xylose in lower-calcium artificial sea water (pH 8.0, 15°C), which speeds the entry of molecules into the interior of the embryos. While α-cyclodextrin killed the embryos, and L(-)-xylose had small effects at one concentration tested, L(-)-rhamnose caused substantially increased numbers of unattached archenterons and exogastrulated embryos at low glycan concentrations after 18-24 h incubation with the sugar. The results were statistically significant compared with the control embryos in the absence of sugar (P < 0.05). The other sugars (melibiose, trehalose, D(+)-xylose) had no statistically significant effects whatsoever at any of the concentrations tested. In total, in the current study, 39,369 embryos were examined. This study is the first demonstration that uses a live embryo assay for a likely role for L(-)-rhamnose in sea urchin gastrula cellular interactions, which have interested investigators for over a century.

Microbead analysis of cell binding to immobilized lectin. Part II: Quantitative kinetic profile assay for possible identification of anti-infectivity and anti-cancer reagents.

There has been a re-emergence of the use of lectins in a variety of therapeutic venues. In addition lectins are often responsible for the binding of pathogens to cells and for cancer cell clumping that increases their escape from body defenses. It is important to define precisely the activity of inhibitors of lectin-binding that may be used in anti-infection and anti-cancer therapeutics. Here we describe a kinetic assay that measures the activity of saccharide inhibitors of lectin binding using a model system of yeast (Saccharomyces cerevisiae) and lectin (Concanavalin A, Con A) derivatized agarose microbeads that mimics pathogen-cell binding. We show that old methods (part I of this study) used to identify inhibitor activity using only one sugar concentration at one time point can easily provide wrong information about inhibitor activity. We assess the activity of 4 concentrations of 10 saccharides at 4 different times in 400 trials and statistically evaluate the results. We show that d-melezitose is the best inhibitor of yeast binding to the lectin microbeads. These results, along with physical chemistry studies, provide a solid foundation for the development of drugs that may be useful in anti-infectivity and anti-cancer therapeutics.

A role for polyglucans in a model sea urchin embryo cellular interaction.

The enzymatic activities of commercially prepared glycosidases were verified by direct chemical assays using defined substrates and fixed and live sea urchin (Lytechinus pictus) embryos to determine if a model cellular interaction of interest to developmental biologists for over a century (interaction of archenteron tip and roof of the blastocoel) was mediated by glycans. Glycosidases (active and denatured) were incubated with microdissected archenterons and blastocoel roofs in a direct assay to learn if their enzymatic activities could prevent the normal adhesive interaction. Of the five glycosidases tested only ß-amylase (an exoglycosidase) immediately inhibited the interaction at relatively low unit activity. α-Amylase (an endoglycosidase) had no measurable effect, while other glycosidases (α-glucosidase, ß-glucosidase, ß-galactosidase) only substantially inhibited adhesion after a 12-h incubation. We demonstrated that the five glycosidases were active (not inhibited) in the presence of embryo materials, and that cleaved sugars could be detected directly after incubation of some enzymes with the embryos. The biochemical purity of the enzymes was examined using gel electrophoresis under denaturing conditions, and the absence of contaminating proteases was confirmed using Azocoll™ substrate. As we cannot entirely rule out the presence of minor contaminating enzymatic activities, only inhibitions of adhesion after very short incubations with enzyme were considered significant and biologically relevant. Although glycans in indirect experiments have been implicated in mediating the interaction of the tip of the archenteron and roof of the blastocoel, to our knowledge, this is the first study that directly implicates polyglucans with terminal 1,4-linked glucose residues in this adhesive event.

A glycobiology review: carbohydrates, lectins and implications in cancer therapeutics.

This review is intended for general readers who would like a basic foundation in carbohydrate structure and function, lectin biology, and the implications of glycobiology in human health and disease, particularly in cancer therapeutics. These topics are among the hundreds included in the field of glycobiology and are treated here because they form the cornerstone of glycobiology or the focus of many advances in this rapidly expanding field.

Use of specific glycosidases to probe cellular interactions in the sea urchin embryo.

We present an unusual and novel model for initial investigations of a putative role for specifically conformed glycans in cellular interactions. We have used alpha- and ss-amylase and alpha- and ss-glucosidase in dose-response experiments evaluating their effects on archenteron organization using the NIH designated sea urchin embryo model. In quantitative dose-response experiments, we show that defined activity levels of alpha-glucosidase and ss-amylase inhibited archenteron organization in living Lytechinus pictus gastrula embryos, whereas all concentrations of ss-glucosidase and alpha-amylase were without substantial effects on development. Product inhibition studies suggested that the enzymes were acting by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectable protease contamination in the active enzyme samples. The results provide evidence for a role of glycans in sea urchin embryo cellular interactions with special reference to the possible structural conformation of these glycans based on the differential activities of the alpha- and ss-glycosidases.

Exogenous hyalin and sea urchin gastrulation. Part IV: a direct adhesion assay - progress in identifying hyalin's active sites.

In Strongylocentrotus purpuratus the hyalins are a set of three to four rather large glycoproteins (hereafter referred to as 'hyalin'), which are the major constituents of the hyaline layer, the developing sea urchin embryo's extracellular matrix. Recent research from our laboratories has shown that hyalin is a cell adhesion molecule involved in sea urchin embryo-specific cellular interactions. Other laboratories have shown it to consist of 2-3% carbohydrate and a cloned, sequenced fragment demonstrated repeat domains (HYR) and non-repeat regions. Interest in this molecule has increased because HYR has been identified in organisms as diverse as bacteria, flies, worms, mice and humans, as well as sea urchins. Our laboratories have shown that hyalin appears to mediate a specific cellular interaction that has interested investigators for over a century, archenteron elongation/attachment to the blastocoel roof. We have shown this finding by localizing hyalin on the two components of the cellular interaction and by showing that hyalin and anti-hyalin antibody block the cellular interaction using a quantitative microplate assay. The microplate assay, however, has limitations because it does not directly assess hyalin's effects on the adhesion of the two components of the interaction. Here we have used an elegant direct assay that avoids the limitations, in which we microdissected the two components of the adhesive interaction and tested their re-adhesion to each other, thereby avoiding possible factors in the whole embryos that could confound or confuse results. Using both assays, we found that mild periodate treatment (6 h to 24 h in sodium acetate buffer with 0.2 M sodium periodate at 4 degrees C in the dark) of hyalin eliminates its ability to block the cellular interaction, suggesting that the carbohydrate component(s) may be involved in hyalin's specific adhesive function. This first step is important in identifying the molecular mechanisms of a well known cellular interaction in the NIH-designated sea urchin embryo model, a system that has led to the discovery of scores of physiological mechanisms, including those involved in human health and disease.

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos.

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.

Hyalin is a cell adhesion molecule involved in mediating archenteron-blastocoel roof attachment.

The US National Institutes of Health has designated the sea urchin embryo as a model organism because around 25 discoveries in this system have led to insights into the physiology of higher organisms, including humans. Hyalin is a large glycoprotein in the hyaline layer of sea urchin embryos that functions to maintain general adhesive relationships in the developing embryo. It consists of the hyalin repeat domain that has been identified in organisms as diverse as bacteria, worms, flies, mice, sea urchins and humans. Here we show, using a polyclonal antibody raised against the 11.6 S species of hyalin, that it localizes at the tip of the archenteron and on the roof of the blastocoel exactly where these two structures bond in an adhesive interaction that has been of interest for over a century. In addition, the antibody blocks the interaction between the archenteron tip and blastocoel roof. These results, in addition to other recent findings from this laboratory that will be discussed, suggest that hyalin is involved in mediating this cellular interaction. This is the first demonstration that suggests that hyalin functions as a cell adhesion molecule in many organisms, including humans.

Carbohydrate-based experimental therapeutics for cancer, HIV/AIDS and other diseases.

This review, primarily for general readers, briefly presents experimental approaches to therapeutics of cancer, HIV/AIDS and various other diseases based on advances in glycobiology and glycochemistry. Experimental cancer and HIV/AIDS vaccines are being developed in attempts to overcome weak immunological responses to carbohydrate-rich surface antigens using carriers, adjuvants and novel carbohydrate antigen constructs. Current carbohydrate-based vaccines are used for typhus, pneumonia, meningitis; vaccines for anthrax, malaria and leishmaniasis are under development. The link between O-linked beta-N-acetylglucosamine glycosylation and protein phosphorylation in diseases including diabetes and Alzheimer's disease is also explored. Carbohydrate-associated drugs that are in current use or under development, such as heparan sulfate binders, lectins, acarbose, aminoglycosides, tamiflu and heparin, and technologies using carbohydrate and lectin microarrays that offer improved diagnostic and drug development possibilities, are described. Advances in carbohydrate synthesis, analysis and manipulation through the emerging fields of glycochemistry and glycobiology are providing new approaches to disease therapeutics.

Lectin binding and effects in culture on human cancer and non-cancer cell lines: examination of issues of interest in drug design strategies.

By using a non-cancer and a cancer cell line originally from the same tissue (colon), coupled with testing lectins for cell binding and for their effects on these cell lines in culture, this study describes a simple multi-parameter approach that has revealed some interesting results that could be useful in drug development strategies. Two human cell lines, CCL-220/Colo320DM (human colon cancer cells, tumorigenic in nude mice) and CRL-1459/CCD-18Co (non-malignant human colon cells) were tested for their ability to bind to agarose microbeads derivatized with two lectins, peanut agglutinin (Arachis hypogaea agglutinin, PNA) and Dolichos biflorus agglutinin (DBA), and the effects of these lectins were assessed in culture using the MTT assay. Both cell lines bound to DBA-derivatized microbeads, and binding was inhibited by N-acetyl-D-galactosamine, but not by L-fucose. Neither cell line bound to PNA-derivatized microbeads. Despite the lack of lectin binding using the rapid microbead method, PNA was mitogenic in culture at some time points and its mitogenic effect displayed a reverse-dose response. This was also seen with effects of DBA on cells in culture. While this is a simple study, the results were statistically highly significant and suggest that: (1) agents may not need to bind strongly to cells to exert biological effects, (2) cell line pairs derived from diseased and non-diseased tissue can provide useful comparative data on potential drug effects and (3) very low concentrations of potential drugs might be initially tested experimentally because reverse-dose responses should be considered.