Browsing by Author "LaCombe, Jonathan M."

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    Down syndrome mouse models have an abnormal enteric nervous system
    (American Society for Clinical Investigation, 2019-04-18) Schill, Ellen M.; Wright, Christina M.; Jamil, Alisha; LaCombe, Jonathan M.; Roper, Randall J.; Heuckeroth, Robert O.; Biology, School of Science
    Children with trisomy 21 (Down syndrome [DS]) have a 130-fold increased incidence of Hirschsprung Disease (HSCR), a developmental defect where the enteric nervous system (ENS) is missing from distal bowel (i.e., distal bowel is aganglionic). Treatment for HSCR is surgical resection of aganglionic bowel, but many children have bowel problems after surgery. Post-surgical problems like enterocolitis and soiling are especially common in children with DS. To determine how trisomy 21 affects ENS development, we evaluated the ENS in two DS mouse models, Ts65Dn and Tc1. These mice are trisomic for many chromosome 21 homologous genes, including Dscam and Dyrk1a, which are hypothesized to contribute to HSCR risk. Ts65Dn and Tc1 mice have normal ENS precursor migration at E12.5 and almost normal myenteric plexus structure as adults. However, Ts65Dn and Tc1 mice have markedly reduced submucosal plexus neuron density throughout the bowel. Surprisingly, the submucosal neuron defect in Ts65Dn mice is not due to excess Dscam or Dyrk1a, since normalizing copy number for these genes does not rescue the defect. These findings suggest the possibility that the high frequency of bowel problems in children with DS and HSCR may occur because of additional unrecognized problems with ENS structure.
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    DYRK1A-Related Trabecular Defects in Male Ts65Dn Mice Emerge During a Critical Developmental Window
    (2021-08) LaCombe, Jonathan M.; Roper, Randall; Goodlett, Charles; Li, Jiliang; Wallace, Joseph; Meyer, Jason
    Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population. The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. The gene Dual-specificity tyrosine-regulated kinase 1a (Dyrk1a) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic Dyrk1a is related to the emergence of trabecular defects at P30, expression of Dyrk1a in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30. The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background. These data led to the hypothesis that reduction of Dyrk1a gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce Dyrk1a gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic Dyrk1a does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.
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    Sex-specific trisomic Dyrk1a-related skeletal phenotypes during development in a Down syndrome model
    (The Company of Biologists, 2024) LaCombe, Jonathan M.; Sloan, Kourtney; Thomas, Jared R.; Blackwell, Matthew P.; Crawford, Isabella; Bishop, Flannery; Wallace, Joseph M.; Roper, Randall J.; Biology, School of Science
    Skeletal insufficiency affects all individuals with Down syndrome (DS) or trisomy 21 and may alter bone strength throughout development due to a reduced period of bone formation and early attainment of peak bone mass compared to those in typically developing individuals. Appendicular skeletal deficits also appear in males before females with DS. In femurs of male Ts65Dn DS model mice, cortical deficits were pronounced throughout development, but trabecular deficits and Dyrk1a overexpression were transitory until postnatal day (P) 30, when there were persistent trabecular and cortical deficits and Dyrk1a was trending toward overexpression. Correction of DS-related skeletal deficits by a purported DYRK1A inhibitor or through genetic means beginning at P21 was not effective at P30, but germline normalization of Dyrk1a improved male bone structure by P36. Trabecular and cortical deficits in female Ts65Dn mice were evident at P30 but subsided by P36, typifying periodic developmental skeletal normalizations that progressed to more prominent bone deficiencies. Sex-dependent differences in skeletal deficits with a delayed impact of trisomic Dyrk1a are important to find temporally specific treatment periods for bone and other phenotypes associated with trisomy 21.
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    Skeletal Dynamics of Down Syndrome: A Developing Perspective
    (Elsevier, 2020-04) LaCombe, Jonathan M.; Roper, Randall J.; Biology, School of Science
    Individuals with Down syndrome (DS) display distinctive skeletal morphology compared to the general population, but disparate descriptions, methodologies, analyses, and populations sampled have led to diverging conclusions about this unique skeletal phenotype. As individuals with DS are living longer, they may be at a higher risk of aging disorders such as osteoporosis and increased fracture risk. Sexual dimorphism has been suggested between males and females with DS in which males, not females, experience an earlier decline in bone mineral density (BMD). Unfortunately, studies focusing on skeletal health related to Trisomy 21 (T21) are few in number and often too underpowered to answer questions about skeletal development, resultant osteoporosis, and sexual dimorphism, especially in stages of bone accrual. Further confounding the field are the varied methods of bone imaging, analysis, and data interpretation. This review takes a critical look at the current knowledge of DS skeletal phenotypes, both from human and mouse studies, and presents knowledge gaps that need to be addressed, differences in research methodologies and analyses that affect the interpretation of results, and proposes guidelines for overcoming obstacles to understand skeletal traits associated with DS. By examining our current knowledge of bone in individuals with T21, a trajectory for future studies may be established to provide meaningful solutions for understanding the development of and improving skeletal structures in individuals with and without DS.
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    Successful Integration of Distributed Drug Discovery (D3) Components: Computational, Synthetic, and Biological Evaluation of Phenylalanine Derivatives as Potential Biofilm Inhibitors
    (Office of the Vice Chancellor for Research, 2013-04-05) Abraham, Milata M.; LaCombe, Jonathan M.; Carnahan, Jon M.; O'Donnell, Martin J. O.; Scott, William L.; Denton, Ryan E.; Samaritoni, J. Geno; Harper, Richard; Anderson, Gregory G.; Marrs, Kathleen A.; Coffey, Barbara M.
    Distributed Drug Discovery (D3) is a multidisciplinary approach to identifying molecules that exhibit activity in the treatment of neglected diseases such as malaria, leishmaniasis, and tuberculosis as well as recalcitrant cystic fibrosis (CF) airway infections. D3 seeks to accomplish this task by combining computational chemistry, synthetic chemistry, and biological screening all within an educational framework. Recent reports suggest that D-amino acids are effective in the disassembly and inhibition of bacterial biofilms, which are important for a number of bacterial infections, including those in the CF lung. Utilizing chemical drawing software, we constructed (enumerated) target phenylalanine derivatives from commercially available benzyl halides by substitution at the α position of an amino acid scaffold. A subset of these enumerated molecules was computationally selected for synthesis based on chemical properties. These compounds were synthesized using simple, solid-phase techniques in an undergraduate organic chemistry laboratory class. The resulting racemic unnatural amino acid derivatives were then screened for activity in a biofilm assay. The results show biofilm inhibition with synthesized phenylalanine derivatives. Analysis of the results reveals a trend between lipophilicity and the degree of biofilm inhibition. These new molecules may lead to an avenue for therapy for those CF individuals suffering with bacterial lung infection. As a part of the undergraduate curriculum, this work provides the first example of D3-linked undergraduate student computational analysis, synthesis, and biological evaluation.
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    Using Amino Acid Derivatives to Inhibit Pseudomonas aeruginosa Biofilm Formation on Cystic Fibrosis Bronchial Epithelia Cells
    (Office of the Vice Chancellor for Research, 2014-04-11) LaCombe, Jonathan M.; Anderson, Gregory G.; Marrs, Kathleen A.
    Cystic Fibrosis is a genetic disease caused by a mutation which inhibits the proper transport of sodium and chloride ions across epithelium. Improper ion transport results in the accumulation of thick mucus in critical organs such as the lungs, pancreas, liver, and intestines. The genetic mutation is incurable, but treating the symptoms can vastly increase life expectancy. CF patients are often afflicted with bacterial infections which colonize the excess mucus within the lungs. The most prevalent pathogen associated with CF lung infection is Pseudomonas aeruginosa, a Gram-negative bacterium found in soil and water. Pseudomonas aeruginosa exists in two forms: planktonic (free-swimming) and sessile (immobile within a biofilm community). The planktonic form is about 1,000x more susceptible to antibiotics and immune cells than the sessile form. Biofilm communities of sessile bacteria are protected by an exopolysaccharide layer outside of the cell wall. Small molecules which inhibit biofilm formation or initiate biofilm disassembly can dramatically increase the effectiveness of drugs and the immune system. In order to identify novel biofilm-inhibitory molecules, we assessed the activity of a library of small molecules in biofilm assays. Active compounds were then screened for activity on living Cystic Fibrosis bronchial epithelial cells infected with Pseudomonas aeruginosa. Compounds which successfully inhibit biofilm formation without affecting the Cystic Fibrosis bronchial epithelium cells can potentially be a new drug for treating Cystic Fibrosis infections.