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1.  Cerebellar Zonal Patterning Relies on Purkinje Cell Neurotransmission 
The Journal of Neuroscience  2014;34(24):8231-8245.
Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity.
PMCID: PMC4051975  PMID: 24920627
ataxia; circuitry; connectivity; gene expression; inhibition; physiology
2.  Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA 
Scientific Reports  2014;4:4878.
Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.
PMCID: PMC4007081  PMID: 24786974
3.  Survivin Expression Induced by Endothelin-1 Promotes Myofibroblast Resistance to Apoptosis 
Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and – independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.
PMCID: PMC3241828  PMID: 22041029
Fibrosis; Rho-kinase; Inhibitor of Apoptosis; Mesenchymal Cell; Focal Adhesion Kinase; AKT
4.  Postnatal development of cerebellar zones revealed by neurofilament heavy chain protein expression 
The cerebellum is organized into parasagittal zones that control sensory-motor behavior. Although the architecture of adult zones is well understood, very little is known about how zones emerge during development. Understanding the process of zone formation is an essential step toward unraveling how circuits are constructed to support specific behaviors. Therefore, we focused this study on postnatal development to determine the spatial and temporal changes that establish zonal patterns during circuit formation. We used a combination of wholemount and tissue section immunohistochemistry in mice to show that the cytoskeletal protein neurofilament heavy chain (NFH) is a robust marker for postnatal cerebellar zonal patterning. The patterned expression of NFH is initiated shortly after birth, and compared to the domains of several known zonal markers such as zebrin II, HSP25, neurogranin, and phospholipase Cβ4 (PLCβ4), NFH does not exhibit transient expression patterns that are typically remodeled between stages, and the adult zones do not emerge after a period of uniform expression in all lobules. Instead, we found that throughout postnatal development NFH gradually reveals distinct zones in each cerebellar lobule. The boundaries of individual NFH zones sharpen over time, as zones are refined during the second and third weeks after birth. Double labeling with neurogranin and PLCβ4 further revealed that although the postnatal expression of NFH is spatially and temporally unique, its pattern of zones respects a fundamental and well-known molecular topography in the cerebellum. The dynamics of NFH expression support the hypothesis that adult circuits are derived from an embryonic map that is refined into zones during the first 3-weeks of life.
PMCID: PMC3648691  PMID: 23675325
purkinje cells; patterning; topography; circuit; development
5.  How semantic categorization influences inhibitory processing in middle-childhood: An Event Related Potentials study 
Brain and cognition  2011;76(1):77-86.
Throughout middle-childhood, inhibitory processes, which underlie many higher order cognitive tasks, are developing. Little is known about how inhibitory processes change as a task becomes conceptually more difficult during these important years. In adults, as Go/NoGo tasks become more difficult there is a systematic decrease in the P3NoGo response, indicating the use of effective inhibitory strategies (Maguire et al., 2009). This paper investigates the age at which children employ similar inhibitory strategies by studying behavioral and Event Related Potential (ERP) measures of response inhibition for three Go/NoGo tasks. Seventeen 7z8 year-olds and twenty 10–11-year-olds completed three Go/NoGo tasks that differed in the level of categorization necessary to respond. Both age groups displayed slower reaction times as the tasks became more difficult. Further, both groups displayed the predicted Go versus NoGo P3 amplitude differences in the two simplest tasks, but no significant P3 differences for the most complex task. The reason for this pattern of responses was different in the different age groups. Similar to adults in previous work, the oldest children showed an attenuation of the P3 NoGo response with task difficulty, and no corresponding changes in the Go amplitude. The younger children displayed the opposite pattern, a significant increase in the Go amplitude with task difficulty, and no changes in the NoGo response. These response patterns indicate that efficient inhibitory strategies are developing throughout middle-childhood.
PMCID: PMC3086752  PMID: 21440972
Response Inhibition; Middle Childhood; Event Related Potentials; Categorization
6.  Architecture and development of olivocerebellar circuit topography 
The cerebellum has a simple tri-laminar structure that is comprised of relatively few cell types. Yet, its internal micro-circuitry is anatomically, biochemically, and functionally complex. The most striking feature of cerebellar circuit complexity is its compartmentalized topography. Each cell type within the cerebellar cortex is organized into an exquisite map; molecular expression patterns, dendrite projections, and axon terminal fields divide the medial-lateral axis of the cerebellum into topographic sagittal zones. Here, we discuss the mechanisms that establish zones and highlight how gene expression and neural activity contribute to cerebellar pattern formation. We focus on the olivocerebellar system because its developmental mechanisms are becoming clear, its topographic termination patterns are very precise, and its contribution to zonal function is debated. This review deconstructs the architecture and development of the olivocerebellar pathway to provide an update on how brain circuit maps form and function.
PMCID: PMC3534185  PMID: 23293588
inferior olive; circuitry; topography; climbing fibers; cerebellum; zones
7.  Bud23 Methylates G1575 of 18S rRNA and Is Required for Efficient Nuclear Export of Pre-40S Subunits▿  
Molecular and Cellular Biology  2008;28(10):3151-3161.
BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5′ internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Δ mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.
PMCID: PMC2423152  PMID: 18332120
8.  Empowerment of women and mental health promotion: a qualitative study in rural Maharashtra, India 
BMC Public Health  2007;7:225.
The global burden of mental illness is high and opportunities for promoting mental health are neglected in most parts of the world. Many people affected by mental illness live in developing countries, where treatment and care options are limited. In this context, primary health care (PHC) programs can indirectly promote mental health by addressing its determinants i.e. by enhancing social unity, minimising discrimination and generating income opportunities. The objectives of this study were to: 1. Describe concepts of mental health and beliefs about determinants of mental health and illness among women involved with a PHC project in rural Maharashtra, India; 2. Identify perceived mental health problems in this community, specifically depression, suicide and violence, their perceived causes, and existing and potential community strategies to respond to them and; 3. Investigate the impact of the PHC program on individual and community factors associated with mental health
We undertook qualitative in-depth interviews with 32 women associated with the PHC project regarding: their concepts of mental health and its determinants; suicide, depression and violence; and the perceived impact of the PHC project on the determinants of mental health. The interviews were taped, transcribed, translated and thematically analysed.
Mental health and illness were understood by these women to be the product of cultural and socio-economic factors. Mental health was commonly conceptualised as an absence of stress and the commonest stressors were conflict with husbands and mother-in-laws, domestic violence and poverty. Links between empowerment of women through income generation and education, reduction of discrimination based on caste and sex, and promotion of individual and community mental health were recognised. However, mental health problems such as suicide and violence were well-described by participants.
While it is essential that affordable, accessible, appropriate treatments and systems of referral and care are available for people with mental illness in developing country settings, the promotion of mental health by addressing its determinants is another potential strategy for reducing the burden of mental illness for individuals and communities in these settings.
PMCID: PMC2222163  PMID: 17761003

Results 1-8 (8)