Faculty Bibliography

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent KM values were determined with 10.12muM, 4.85muM and 19.95muM, respectively for NAT3 and 1.74muM, 2.44muM and 29.83muM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton-nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1-7 and TM8-14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure-function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.

Mouse models have increased our understanding of the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor that often forms in the cerebellum. A major goal of ongoing research is to better understand the early stages of tumorigenesis and to establish the genetic and environmental changes that underlie MB initiation and growth. However, studies of MB progression in mouse models are difficult due to the heterogeneity of tumor onset times and growth patterns and the lack of clinical symptoms at early stages. Magnetic resonance imaging (MRI) is critical for noninvasive, longitudinal, three-dimensional (3D) brain tumor imaging in the clinic but is limited in resolution and sensitivity for imaging early MBs in mice. In this study, high-resolution (100 mum in 2 hours) and high-throughput (150 mum in 15 minutes) manganese-enhanced MRI (MEMRI) protocols were optimized for early detection and monitoring of MBs in a Patched-1 (Ptch1) conditional knockout (CKO) model. The high tissue contrast obtained with MEMRI revealed detailed cerebellar morphology and enabled detection of MBs over a wide range of stages including pretumoral lesions as early as 2 to 3 weeks postnatal with volumes close to 0.1 mm(3). Furthermore, longitudinal MEMRI allowed noninvasive monitoring of tumors and demonstrated that lesions within and between individuals have different tumorigenic potentials. 3D volumetric studies allowed quantitative analysis of MB tumor morphology and growth rates in individual Ptch1-CKO mice. These results show that MEMRI provides a powerful method for early in vivo detection and longitudinal imaging of MB progression in the mouse brain.

Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-gamma in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor gammat protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.

Scaling up HIV prevention programming among key populations (female sex workers and men who have sex with men) has been a central strategy of the Government of India. However, state governments have lacked the technical and managerial capacity to oversee and scale up interventions or to absorb donor-funded programs. In response, the national government contracted Technical Support Units (TSUs), teams with expertise from the private and nongovernmental sectors, to collaborate with and assist state governments. In 2008, a TSU was established in Karnataka, one of 6 Indian states with the highest HIV prevalence in the country and where monitoring showed that its prevention programs were reaching only 5% of key populations. The TSU provided support to the state in 5 key areas: assisting in strategic planning, rolling out a comprehensive monitoring and evaluation system, providing supportive supervision to intervention units, facilitating training, and assisting with information, education, and communication activities. This collaborative management model helped to increase capacity of the state, enabling it to take over funding and oversight of HIV prevention programs previously funded through donors. With the combined efforts of the TSU and the state government, the number of intervention units statewide increased from 40 to 126 between 2009 and 2013. Monthly contacts with female sex workers increased from 5% in 2008 to 88% in 2012, and with men who have sex with men, from 36% in 2009 to 81% in 2012. There were also increases in the proportion of both populations who visited HIV testing and counseling centers (from 3% to 47% among female sex workers and from 6% to 33% among men who have sex with men) and sexually transmitted infection clinics (from 4% to 75% among female sex workers and from 7% to 67% among men who have sex with men). Changes in sexual behaviors among key populations were also documented. For example, between 2008 and 2010, the proportion of surveyed female sex workers in 9 districts reporting that they used a condom at last intercourse rose from 60% to 68%; in 6 districts, the proportion of surveyed men who have sex with men reporting that they used a condom at last anal sex increased from 89% to 97%. The Karnataka experience suggests that TSUs can help governments enhance managerial and technical resources and leverage funds more effectively. With careful management of the working and reporting relationships between the TSU and the state government, this additional capacity can pave the way for the government to improve and scale up programs and to absorb previously donor-funded programs.

A beetle pheromone that lures nematode worms to an insect host can also stop their development or even kill them outright.

Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.

Lrp4, the muscle receptor for neuronal Agrin, is expressed in the hippocampus and areas involved in cognition. The function of Lrp4 in the brain, however, is unknown, as Lrp4-/- mice fail to form neuromuscular synapses and die at birth. Lrp4-/- mice, rescued for Lrp4 expression selectively in muscle, survive into adulthood and showed profound deficits in cognitive tasks that assess learning and memory. To learn whether synapses form and function aberrantly, we used electrophysiological and anatomical methods to study hippocampal CA3-CA1 synapses. In the absence of Lrp4, the organization of the hippocampus appeared normal, but the frequency of spontaneous release events and spine density on primary apical dendrites were reduced. CA3 input was unable to adequately depolarize CA1 neurons to induce long-term potentiation. Our studies demonstrate a role for Lrp4 in hippocampal function and suggest that patients with mutations in Lrp4 or auto-antibodies to Lrp4 should be evaluated for neurological deficits.