Qatar Foundation Annual Research Forum Volume 2010 Issue 1

In the past decade, biodegradable elastomeric polymers have gained considerable attention due to the renewed interest in their applications in the fields of biomedical tissue engineering and implantable drug delivery systems. Elastomers can be regarded as one of the best biomaterials for such applications because their mechanical properties can be manipulated in a manner that makes them as soft as body tissues, they have the ability to recover and withstand the mechanical challenges upon implantation in a mobile part of the body and they have also proven to be well suited to drug controlled drug delivery applications.

Our lab has recently reported on the successful preparation and characterization of a novel family of poly (diol-co-tricarballylate) elastomers, using visible light photo initiated polymerization. This new patented family of elastomers possess many structural, mechanical and physicochemical properties that make them superior to the currently available biodegradable elastomers.

The purpose of this presentation is to shed light on the preparation, characterization and in vivo animal biocompatibility studies conducted on these new elastomers. In addition, a short illustration on their application in controlled drug delivery and tissue engineering and the current and future scope of work planned utilizing the latest Qatar National Research Fund – National Priorities Research Program 3rd cycle support received will also to be presented.

Oligopeptidase B (opdB, EC 3.4.21.83) is a member of the prolyl oligopeptidase family of serine peptidases and unrelated to the trypsin and subtilisin families. It is a potential processing enzyme of prokaryotes to produce biologically active products, being very specific for the basic amino acid pairs of polypeptides. Bacterial oligopeptidase B cleaves globular proteins, albeit in a highly restricted fashion. While most members of this peptidase family hydrolyse peptide bonds at the C-terminal side of proline residues, oligopeptidase B exhibits a trypsin-like substrate specificity, cleaving peptides after basic residues (arginine or lysine). Oligopeptidase B was first cloned and characterized from Escherichia coli, and has also been described in other prokaryotes. Similar enzymes have been found in plants and some other higher organisms. We report the isolation of two different new oligopeptidase B bacterial strains producers. We identified the two genes, designated opdB1 and opB2. The opdB genes encodes a 703-residue peptide with high homology to the oligopeptidase B family in prokaryotes. The isolated opdBs gave the highest similarity score to oligopeptidase B of Stenotrophomonas maltophilia strain K279a (GenBank AM743169). To reveal the structural and kinetic properties of oligopeptidase B in more detail, we have cloned, expressed, and purified the enzymes to produce sufficient material to help in physical investigations, including NMR and x-ray crystallographic measurements. We also carried out a molecular characterization study of the two enzymes.

The thrombospondin type 1 repeat (TSR) is an ancient extracellular protein domain that is commonly found in invertebrate and vertebrate proteins. The ADAMTSL4 protein, also known as TSRC1, belongs to the TSR superfamily and has multiple thrombospondin repeats, most of which are clustered at the C-terminus.

It has been reported that some TSP1 domain-containing proteins, e.g. thrombospondin 1 and thrombospondin 2, could induce apoptosis of endothelial cells but it is not clear how these proteins operate in death pathways.

Our recent work shows that mutations in ADAMTSL4 are responsible for autosomal-recessive isolated ectopia lentis, i.e. abnormal positioning of the lens of the eye and affect the development of the zonular fiber. However, little is known about the function of ADAMTSL4. To shed more light on the function of the ADMTSL4 and its roles in different tissues we extended our work to carry out molecular characterization of this gene and its variants.

We have obtained cDNA clones encoding the full ADAMTSL4 protein and its truncated isoform. Both are subcloned into the pET28a vector for expression in E. coli. In anticipation of possible expression challenges in this host, e.g. formation of inclusion bodies or lack of expression, we subcloned each fragment into the yeast vector, pYES2. The gene in both cases has been fused in frame with a region encoding an N-terminal His-Tag to facilitate the purification of the recombinant protein. DNA analysis indicates that each fragment has been correctly cloned into the pYES2 vector. Each construct was transformed into Saccharomyces cerevisiae for protein expression. Our preliminary analysis indicates that one of the genes is expressed in S. cerevisiae but at a very low level. Work to optimize the expression of ADAMTSL4 in yeast as well as in E. coli is in progress.

We also extracted the seven domains of the ADAMTSL4 using PCR. Each domain was subcloned into the E. coli overexpression vector, pET28a. Expression studies of all the constructs have shown that the seven domains have been successfully overexpressed in E. coli. The overexpression was confirmed using Western blot techniques. The recombinant protein of each domain is purified for NMR and x-ray studies.

Metal toxicity is a global health concern. We summarize the evidence for metal interactions with the nervous system with an emphasis on synaptic transmission. The appropriate functioning of synaptic transmission is crucial for the information transfer in any neural network.

Presynaptically, metal ions modulate transmitter release through their interaction with neurotransmitter (NT) synthesis, fusion of synaptic vesicles, signaling cascades and ion channels. Ca²⁺ entry through voltage-gated channels is impaired by Pb²⁺, Cd²⁺ or Zn²⁺, therefore all processes which depend on Ca²⁺, including NT release, will be affected. Furthermore, some metals interact with intracellular pathways e.g. Pb²⁺ inhibits PKC enzymes through its catalytic domains, and Ni²⁺ causes a decline in the transcription of two isoforms of PKC (prkcc and prkz) and two regulatory binding proteins (prkcbp1 and prkcdb) affecting most functions of PKC. Cd and Hg inhibit adenylate cyclase activity, while the extent of inhibition depends on exposure time and brain area. Exocytosis is impaired by Pb²⁺ and Cu²⁺, which interact with Synaptotagmin I.

Postsynaptically, processes associated with binding of NT to their receptors, activation of the channels and degradation of NT are changed by metal. Zn²⁺, Pb²⁺, Cu²⁺, Cd²⁺, Ni²⁺, Co²⁺, Li³⁺, Hg⁺ and methylmercury modulate NMDA, AMPA/Kainate and/or GABA receptor's activity. These effects are more or less specific e.g. Zn²⁺ and Cu²⁺ modulate all three types of receptors, while Zn²⁺ is more potent (IC50= 0.77μM) compared to Cu²⁺ (IC50=15μM) at NMDA-Rs, but both have similar potencies at GABA-R. For the most part, metal interactions depend on the subunit composition of the NMDA-R, while less data are available for other targets, possibly underestimating their importance.

These modulations change the synaptic efficiency and therefore impair long-term potentiation (LTP). Consequently, metals such as Al, Pb or Cd result in various cognitive deficits. In addition the generation and maintenance of LTP is reduced by metal actions on phosphorylation of transcription factors like CREB as well as by reduction of nitric oxide synthase (NOS) that change retrograde signaling.

Overall, there are multiple effects of metals based on the forms of the metals, their concentrations and the types of neurons involved.

Calreticulin is a conserved Ca²⁺ binding chaperone protein that is localized to the lumen of the endoplasmic reticulum (ER). The protein is implicated in many cellular functions such as the regulation of intracellular Ca²⁺ homeostasis, the regulation of gene expression, the folding of the newly synthesized proteins, cell adhesion, cancer and auto-immunity. The role of calreticulin in Ca²⁺ homeostasis regulation through Ca2+ storage and signaling might be the key to explaining the involvement of the protein in many biological functions inside and outside the ER. In this study, we examined whether calreticulin is responsible for regulating store-operated channels (SOC) on the plasma membrane by assessing changes in Ca²⁺ levels and SOC activity in mouse embryonic fibroblasts that are deficient in calreticulin. Wild type and calreticulin deficient fibroblasts were loaded with fura2-AM and the release of Ca²⁺ from the ER stores was induced using Thapsigargin or Ionomycin, and Ca²⁺ levels were measured using InC lm2 computer program. Calreticulin-defiecint fibroblasts showed a marked decrease in Ca²⁺ influx through SOC compared to wild type. To investigate the mechanism that underlines this decrease in SOC activity we performed western blots using antibodies for ORAI (Calcium release-activated calcium channel protein) and STIM (stromal interacting molecule). Our data showed a significant decrease in the expression of ORAI in the calreticulin-deficient fibroblasts compared to wild type while STIM expression in calreticulin-defiecint fibroblasts did not significantly differ from wild type. We concluded from our data that calreticulin controls SOC activity and this seems to be through regulating the expression of ORAI on the plasma membrane.

The Cohen's lab research focuses mainly on the regulation of mammalian meiosis in mouse models, which include several induced mutants that have helped in broadening our knowledge of meiotic recombination and the gametogenesis. The lab also studies several DNA mismatch repair (MMR) proteins and their subsequent effects on meiotic recombination. Thus, our research serves two major purposes; first, to understand the genetics of re-combination and thereby to understand how such events can fail in human gametogenesis and secondly, to further elucidate the mechanisms of DNA repair and genome stability in an important in vivo system.

Extending from these aims, one of these genes is the MLH3 gene, evolutionarily conserved in many species such as mice, humans, and worms and which can solve the infertility problems in MLH3 mutant mice.

The focus of our project is to investigate the functions of several genes involved in meiotic prophase, using the technique of chromosome spreading. This method allows us to visualize the different stages of prophase I, and see the staining pattern of several antibodies that are specific to meiotic proteins. Using a battery of specific antibodies, we set out to understand the meiotic roles of a number of poorly characterized proteins involved in DNA repair and ubiquitination pathways. These antibodies of interest are those that are involved in the ubiquitination pathway of the sex chromosomes such as antibodies raised against Rnf 8, Rad 18, and Rnf 168, all of which are involved in ubiquitination pathways, as well as other antibodies involved in ubiquitination/deubiquitinatio. A group of other antibodies that include mdc λ, HR9B, MRαB, USP2B, and UBQ 2 is also being studied. Our goal was to first characterize the accumulation of these proteins on meiotic chromosomes in order to identify which, if any of these proteins might be functionally relevant for meiotic recombination and prophase | progression.

The project started with studying the staining pattern of the antibodies listed above in adult male wild type mice, then younger male wild type mice to see both the early and late stages of meiosis. The project is progressing to studying the staining pattern in the MLH3 and Ago 4 mutant mice to see any possible co-localizations between MMR genes and the antibodies considered by this study

Cancer accounts for the death of thousands of people each year and brings pain and suffering to thousands more. Among cancers that specifically affect women, ovarian cancer has the highest fatality rate as 7 out of 10 women die within 5 years of surgery. As highlighted in numerous recent publications, the role of the microenvironment in the development and progression of cancer is critical, albeit not entirely understood and should therefore be a focal point for further inquiry. The goal of our study was to define the metastatic properties enhanced by the interaction between ovarian cancer cells and mesenchymal stem cells. We studied the interaction between ovarian cancer cells and mesenchymal stem cells (MSC) using two different ovarian cancer cell lines (OVCAR3 and SKOV3) as well as different MSC cell lines derived from specific tissue. We studied using cytokine array as well as kinase array the modifications of the MSC upon their interaction with ovarian cancer cells. We demonstrated increased expression of cytokines that might be implicated in ovarian cancer metastasis (IL6 and IL8). Moreover MSC helped the ovarian cancer cells to grow in a tri-dimensional cell culture system. Finally we were able to define transcriptomic modification in the MSC and ovarian cancer cells upon their interaction.

Over 14,000 babies are born in Qatar each year, and it is the State's intention to provide each with a health screen at birth for the timely diagnosis of inborn errors of metabolism. And since the population is characterized by a high consanguinity (estimates vary between 25–70%) from first-cousin marriages, congenital and genetic disorders are responsible for a major proportion of infant mortality, morbidity, and handicap birth defects and are relatively common among the population. Accurate and reliable quantification of amino acid (AA), generally in a plasma sample, allows early diagnosis of disorders such as phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia and citrullinemia. A deficiency of cystathionine B-synthase (CBS) can cause an autosomal recessive disorder of methionine and homocysteine (Hcy) metabolism known as homocystinuria which results in elevated concentrations of Hcy in plasma and urine. Clinical symptoms in untreated patients include progressive myopia and lens dislocation, thromboembolism, epilepsy, and mental retardation. Hcy is implicated as a wide variety of natal and other disorders – including Alzheimer's. The aim of this study is to develop novel screens for AA levels – and in particular Hcy - in the blood using novel approaches in capillary zone electrophoresis (CZE). In order to quantify relevant amino acids it is necessary to deplete proteins from a complex biological sample such as plasma. In this investigation two protein depletion protocols were investigated on human plasma containing Hcy by label-free intrinsic imaging in the UV using CZE. As with a majority of amino acids, Hcy has very little or no UV absorption and for this reason analysis of samples were performed on an advanced high performance capillary electrophoresis (HPCE) platform, by multiple-pixel multiple-imaging indirect UV measurements. Two complementary analysis workflows were deployed to take advantage of the time-development of the AAs in the analysis allowing accurate quantification and minimum inherent bias.

Studies have been made with pure AAs and also with samples spiked in known quantities in blood which offer real clinical advantages. We have also developed methodologies for the simultaneous detection of several amino acids in plasma samples and limits of detection (LOD) and limits of quantification (LOQ) will be presented. These have been shown to be greatly improved with a pre-concentration technique known as ‘stacking’. Work has been undertaken in our labs in Qatar Science & Technology Park and in collaboration with Hamad Medical Center.

Epithelial ovarian carcinoma (EOC) is the sixth most common malignancy in women and the leading cause of death from gynecological cancer in the world. One of the main differences between ovarian cancers and other neoplasm is burden of local extension. Hence the majority of mortality in ovarian cancer is due to extensive peritoneal disease, with a high rate of mortality and an overall survival rate ranging from 20% to 30% five years after surgery according to various studies. As for any metastatic process, the tumor cells have to go through the steps of detaching from the primary tumor, adhering to the peritoneal surface and then invading the peritoneum. Each of these steps might be critical in the development of a metastatic lesion. Therefore it is essential to understand the molecular background of peritoneal adhesion and invasion in order to define new therapeutic targets. It may be that classic 2-dimensional cultures do not represent an ideal model for the initiation of metastasis, and therefore studies using only 2-dimensional cell cultures might not replicate the reality of ovarian cancer physiology. The goal of our study was to define new 3-dimensional culture models that will mimic the peritoneal tissue. The constraints were; to use an easily accessible tissue, in relevant quantities, as close as possible to the peritoneum, with great manipulability. We demonstrated that we could keep the amniotic membrane in culture and mimic adhesion and the early invasion of ovarian cancer cell aggregates. We were then able to follow the invasive process within the membrane and determine different cell behavior.

Developing reproducible 3-dimensional models of ovarian cancer aggregates and early adhesion and invasion will help us gain a more accurate understanding of the molecular mechanisms involved in the ovarian cancer metastatic process and define potential new therapeutic targets hindered by the use of classic 2-dimensional models.