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Browsing by Author "Malik, Ahmed M."
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Item Abnormal mineralization of the Ts65Dn Down syndrome mouse appendicular skeleton begins during embryonic development in a Dyrk1a-independent manner(Elsevier, 2015-05) Blazek, Joshua D.; Malik, Ahmed M.; Tischbein, Maeve; Arbones, Maria L.; Moore, Clara S.; Roper, Randall J.; Biology, School of ScienceThe relationship between gene dosage imbalance and phenotypes associated with Trisomy 21, including the etiology of abnormal bone phenotypes linked to Down syndrome (DS), is not well understood. The Ts65Dn mouse model for DS exhibits appendicular skeletal defects during adolescence and adulthood but the developmental and genetic origin of these phenotypes remains unclear. It is hypothesized that the postnatal Ts65Dn skeletal phenotype originates during embryonic development and results from an increased Dyrk1a gene copy number, a gene hypothesized to play a critical role in many DS phenotypes. Ts65Dn embryos exhibit a lower percent bone volume in the E17.5 femur when compared to euploid embryos. Concomitant with gene copy number, qPCR analysis revealed a ~1.5 fold increase in Dyrk1a transcript levels in the Ts65Dn E17.5 embryonic femur as compared to euploid. Returning Dyrk1a copy number to euploid levels in Ts65Dn, Dyrk1a+/− embryos did not correct the trisomic skeletal phenotype but did return Dyrk1a gene transcript levels to normal. The size and protein expression patterns of the cartilage template during embryonic bone development appear to be unaffected at E14.5 and E17.5 in trisomic embryos. Taken together, these data suggest that the dosage imbalance of genes other than Dyrk1a is involved in the development of the prenatal bone phenotype in Ts65Dn embryos.Item EMBRYONIC BONE DEVELOPMENT AND NFAT EXPRESSION IN THE TS65DN MOUSE MODEL FOR DOWN SYNDROME(Office of the Vice Chancellor for Research, 2012-04-13) Malik, Ahmed M.; Blazek, Joshua D.; Roper, Randall J.Down syndrome (DS) is a common genetic disorder that occurs in ap-proximately 1 out of every 750 live births. DS phenotypes include cognitive deficits, altered craniofacial features, muscle hypotonia, heart defects, and abnormal bone structure. The Ts65Dn mouse model is the most common or-ganismal model used to study DS phenotypes. This model exhibits a number of phenotypic traits comparable to those of humans with DS, including bone anomalies. Past studies have shown that Ts65Dn mice exhibit weaker tra-becular bone due to less trabeculae. They have also been shown to have less bone mineral density and bone mineral content at 6 weeks of age when compared to their euploid counterparts, with the severity of these defects lessening by 16 weeks. No studies of bone development have yet decisively identified the origin of these defects. We hypothesized that abnormal endochondral ossification is responsible for the presence of these deficien-cies in bone mineral content and bone mineral density. Aberrant expression of Nfat has been implicated as the molecular cause of many DS-related phe-notypes, and activity of Nfat can be determined based upon its localization. Specifically, Nfat has been shown to control many aspects of bone develop-ment, which makes it of special interest to this research. To test our hypoth-esis of a bone deficit present during embryonic development of Ts65Dn em-bryos, we are comparing cartilaginous template characteristics, progression of the mineralization front, osteoclast activity, percent bone volume, and Nfat localization in euploid and trisomic mouse femurs at embryonic day 17.5. Our preliminary data show lower percent bone volumes in trisomic fe-murs, suggesting that endochondral ossification in Ts65Dn mice lags behind that of their euploid counterparts. These results indicate that DS bone phe-notypes do indeed originate during embryonic development and create a foundation for future work on their treatment. Supported by: National Science Foundation GK-12 Fellowship; Jerome Lejeune FoundationItem Putative Benzoylformate Decarboxylases and the Annotation Problem(Office of the Vice Chancellor for Research, 2013-04-05) Malik, Ahmed M.; Logsdon, Matthew G.; Andrews, Forest H.; McLeish, Michael J.Benzoylformate decarboxylases (BFDCs) are a relatively uncommon class of thiamin diphosphate-dependent enzymes of commercial interest that catalyze the decarboxylation of benzoylformate, with BFDC from Pseudomonas putida being the most extensively studied among them. Based upon sequence homology, the recently established Thiamin Enzyme Engineering Database (TEED) has identified dozens of sequences in a variety of other microorganisms and annotated them as BFDCs. Interestingly, the majority of these putative BFDCs share >40% sequence identity with PpBFDC but many lack certain amino acids thought to be important in its function. Further, most of the annotated sequences are from organisms with no known pathway involving benzoylformate. To determine the integrity of these sequence annotations, these previously unstudied enzymes must be functionally characterized to determine if they are, in fact, true BFDCs. Currently, we are studying putative BFDCs from Polynucleobacter necessaries and Mycobacterium smegmatis, both of which share most of the same catalytic residues as PpBFDC but have alterations in residues involved in substrate specificity. We have successfully expressed, purified these supposed BFDCs, and characterized them by assaying with a variety of both metabolic and unnatural 2-ketoacid substrates. Comparison of their activity with that of PpBFDC suggests that these two sequences were both incorrectly annotated. We are currently in the process of trying to identify their natural substrate.