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1.  Sex-specific effects of the DAF-12 steroid receptor on aging in Caenorhabditis elegans 
Sex differences in longevity and aging are seen throughout the animal kingdom. These are likely to result, in part, from sex differences in endocrinology. In the nematode C. elegans, males are the longer-lived sex. Here we explore the possibility that sex differences in insulin/IGF-1 and steroid endocrinology contribute to this sex difference in aging, studying C. elegans populations in liquid culture. We report that in hermaphrodite populations, mutational loss of the DAF-12 steroid receptor affected lifespan as in previous plate culture studies: mutant longevity is suppressed in a weak daf-2 insulin/IGF-1 receptor mutant, but enhanced in a stronger daf-2 mutant. However, in males mutation of daf-12 had little effect on aging in either weak or strong daf-2 mutants. Moreover, while mutation of daf-12 marginally reduced lifespan in daf-2(+) hermaphrodites, as in plate cultured populations, it did not in daf-2(+) males. These results could imply that in C. elegans, as in mammals, sex differences in steroid endocrinology contribute to sex differences in aging.
doi:10.1196/annals.1404.018
PMCID: PMC4335189  PMID: 18056973
C. elegans; aging; gender; steroid; insulin/IGF-1 signaling
2.  Supporting the Mental Health of Mothers Raising Children in Poverty 
Poverty increases maternal stress by heightening exposure to negative life events, job loss, chronic strains, poor housing, dangerous neighborhoods, and conflict with partners, culminating in crippling depressive symptoms, the most prevalent mental health threat. Depressive symptoms interfere with the provision of the strong maternal support needed to counter the hardships of poverty, thus placing infants and toddlers at risk for delayed language, social, and emotional development. Initial clinical trials in high-risk mothers have shown promise, and successive tests of interventions will be strengthened if mothers who have mental health risks can be accurately targeted for inclusion. This article reports on a sequential, data-driven process by which high-risk mothers were targeted for intervention in two trials currently in progress to reduce depressive symptoms. An iterative process of using data to identify at-risk mothers and validate the presence of risk factors helped hone the recruitment and design of the intervention trials. This report also offers guidance for further study.
doi:10.1196/annals.1425.008
PMCID: PMC4225774  PMID: 17954677
mothers; depression; poverty; low income; infants/toddlers; targeting; intervention trials
3.  Plasma membrane Ca2+-ATPases as dynamic regulators of cellular calcium handling 
Annals of the New York Academy of Sciences  2007;1099:10.1196/annals.1387.023.
Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provides a dynamic system for a cell to respond to changing needs of Ca2+ regulation.
doi:10.1196/annals.1387.023
PMCID: PMC3873821  PMID: 17446463
Calcium homeostasis; calcium pump; calcium signaling; PMCA; splice variant
4.  Rapid downregulation of NCX and PMCA in hippocampal neurons following H2O2 oxidative stress 
Annals of the New York Academy of Sciences  2007;1099:10.1196/annals.1387.005.
Na+/Ca2+ exchangers (NCXs) and plasma membrane Ca2+ pumps (PMCAs) are crucial for intracellular Ca2+ homeostasis and Ca2+ signaling. Elevated [Ca2+]i is a hallmark of neurodegenerative disease and stroke. Here we studied the short-term effect of oxidative stress on the plasma membrane Ca2+ extrusion systems in hippocampal neurons and found that after 2–3 hours exposure to 300 μM H2O2, all NCXs and PMCAs were significantly downregulated at the RNA (NCX) and protein (PMCA) level. Rapid internalization and aggregation of the PMCA was also observed. Our data show that the plasma membrane calcium extrusion systems are sensitive early targets of neurotoxic oxidative stress.
doi:10.1196/annals.1387.005
PMCID: PMC3873835  PMID: 17446483
Calcium extrusion; hippocampal neurons; Na+/Ca2+ exchanger; NCX; oxidative stress; PMCA
5.  Emergence of Human Angiohematopoietic Cells in Normal Development and from Cultured Embryonic Stem Cells 
Human hematopoiesis proceeds transiently in the extraembryonic yolk sac and embryonic, then fetal liver before being stabilized in the bone marrow during the third month of gestation. In addition to this classic developmental sequence, we have previously shown that the aorta-gonad-mesonephros (AGM) embryonic territory produces stem cells for definitive hematopoiesis from 27 to 40 days of human development, through an intermediate blood-forming endothelium stage. These studies have relied on the use of traditional markers of human hematopoietic and endothelial cells. In addition, we have recently identified and characterized a novel surface molecule, BB9, which typifies the earliest founders of the human angiohematopoietic system. BB9, which was initially identified with a monoclonal antibody raised to Stro-1+ bone marrow stromal cells, recognizes in the adult the most primitive Thy-1+ CD133+ Lin−, non-obese diabetic—severe combined immunodeficiency disease (NOD–SCID) mouse engrating hematopoietic stem cells (HSCs). In the 3- to 4-week embryo,BB9expression typifies a subset of splanchnopleural mesodermal cells that migrate dorsally and colonize the ventral aspect of the aorta where they establish a population of hemogenic endothelial cells. We have indeed confirmed that hematopoietic potential in the human embryo, as assessed by long-term culture-initiating cell (LTC-IC) and SCID mouse reconstituting cell (SRC) activities, is confined to BB9-expressing cells. We have further validated these results in the model of human embryonic stem cells (hESCs) in which we have modeled, through the development of hematopoietic embryoid bodies (EBs), primitive and definitive hematopoieses. In this setting, we have documented the emergence of BB9+ hemangioblast-like clonogenic angiohematopoietic progenitors that currently represent the earliest known founders of the human vascular and blood systems.
doi:10.1196/annals.1392.010
PMCID: PMC3510976  PMID: 17360801
hematopoietic stem cell; embryonic stem cell; hemangioblast; embryo; blood vessel
6.  Ethics Analysis of Neuroimaging in Alzheimer’s Disease 
This article focuses on the prospects and ethics of using neuroimaging to predict Alzheimer’s disease (AD). It is motivated by consideration of the historical roles of science in medicine and society, and considerations specifically contemporary of capabilities in imaging and aging, and the benefits and hope they bring. A general consensus is that combinations of imaging methods will ultimately be most fruitful in predicting disease. Their roll-out into translational practice will not be free of complexity, however, as culture and values differ in terms of what defines benefit and risk, who will benefit and who is at risk, what methods must be in place to assure the maximum safety, comfort, and protection of subjects and patients, and educational and policy needs. Proactive planning for the ethical and societal implications of predicting diseases of the aging brain is critical and will benefit all stakeholders— researchers, patients and families, health care providers, and policy makers.
doi:10.1196/annals.1379.030
PMCID: PMC3265384  PMID: 17413029
neuroimaging; Alzheimer’s disease; aging; prediction; neuroethics
7.  Tracking Alzheimer’s Disease 
Population-based brain mapping provides great insight into the trajectory of aging and dementia, as well as brain changes that normally occur over the human life span. We describe three novel brain mapping techniques, cortical thickness mapping, tensor-based morphometry (TBM), and hippocampal surface modeling, which offer enormous power for measuring disease progression in drug trials, and shed light on the neuroscience of brain degeneration in Alzheimer’s disease (AD) and mild cognitive impairment (MCI).We report the first time-lapse maps of cortical atrophy spreading dynamically in the living brain, based on averaging data from populations of subjects with Alzheimer’s disease and normal subjects imaged longitudinally with MRI. These dynamic sequences show a rapidly advancing wave of cortical atrophy sweeping from limbic and temporal cortices into higher-order association and ultimately primary sensorimotor areas, in a pattern that correlates with cognitive decline. A complementary technique, TBM, reveals the 3D profile of atrophic rates, at each point in the brain. A third technique, hippocampal surface modeling, plots the profile of shape alterations across the hippocampal surface. The three techniques provide moderate to highly automated analyses of images, have been validated on hundreds of scans, and are sensitive to clinically relevant changes in individual patients and groups undergoing different drug treatments. We compare time-lapse maps of AD, MCI, and other dementias, correlate these changes with cognition, and relate them to similar time-lapse maps of childhood development, schizophrenia, and HIV-associated brain degeneration. Strengths and weaknesses of these different imaging measures for basic neuroscience and drug trials are discussed.
doi:10.1196/annals.1379.017
PMCID: PMC3197831  PMID: 17413023
MRI; Alzheimer’s disease; aging; MCI; dementia; brain degeneration; PET
8.  Regulation of Ncx1 Gene Expression in the Normal and Hypertrophic Heart 
The Na+/Ca2+ exchanger (NCX1) is crucial in the regulation of [Ca2+]i in the cardiac myocyte. The exchanger is upregulated in cardiac hypertrophy, ischemia, and failure. This upregulation can have an effect on Ca2+ transients and possibly contribute to diastolic dysfunction and an increased risk of arrhythmias. Studies from both in vivo and in vitro model systems have provided an initial skeleton of the potential signaling pathways that regulate the exchanger during development, growth, and hypertrophy. The Ncx1 gene is upregulated in response to α-adrenergic stimulation. We have shown that this is via p38α activation of transcription factors binding to the Ncx1 promotor at the −80 CArG element. Interestingly, most of the elements, including the CArG element, which we have demonstrated to be important for regulation of Ncx1 expression are in the proximal 184 bp of the promotor. Using a transgenic mouse, we have shown that the proximal 184 bp is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for upregulation in response to pressure overload.
doi:10.1196/annals.1387.058
PMCID: PMC3096001  PMID: 17446459
Na+/Ca2+ exchanger; NCX1; cardiac hypertrophy; regulation of gene expression; signal transduction; transgenic mice
9.  Genomic Targets in Saliva 
Saliva, the most accessible and noninvasive biofluid of our body, harbors a wide spectrum of biological analytes informative for clinical diagnostic applications. While proteomic constituents are a logical first choice as salivary diagnostic analytes, genomic targets have emerged as highly informative and discriminatory. This awareness, coupled with the ability to harness genomic information by high-throughput technology platforms such as genome-wide microarrays, ideally positions salivary genomic targets for exploring the value of saliva for detection of specific disease states and augmenting the diagnostic and discriminatory value of the saliva proteome for clinical applications. Buccal cells and saliva have been used as sources of genomic DNA for a variety of clinical and forensic applications. For discovery of disease targets in saliva, the recent realization that there is a transcriptome in saliva presented an additional target for oral diagnostics. All healthy subjects evaluated have approximately 3,000 different mRNA molecules in their saliva. Almost 200 of these salivary mRNAs are present in all subjects. Exploration of the clinical utility of the salivary transcriptome in oral cancer subjects shows that four salivary mRNAs (OAZ, SAT, IL8, and IL1b) collectively have a discriminatory power of 91% sensitivity and specificity for oral cancer detection. Data are also now in place to validate the presence of unique diagnostic panels of salivary mRNAs in subjects with Sjöogren's disease.
doi:10.1196/annals.1384.002
PMCID: PMC2910758  PMID: 17435127
human saliva transcriptome analysis; mRNA biomarkers; noninvasive disease detection
10.  Keynote Address: Revaluing the Orbital Prefrontal Cortex 
The importance of orbitofrontal cortex (OFC) in human behavioral regulation is no longer a matter of dispute, though its precise role remains a matter of ongoing investigation. It is ironic that this revaluation of OFC required a major departure from a historical nadir, during which it was viewed as redundant or “silent cortex,” a situation that prevailed even up to the latter half of the 20th century. The increasing wealth of data from diverse fields within neuroscience now provides an unambiguous testament to the importance of this cortical region in behavioral regulation and cognition in general.
doi:10.1196/annals.1401.020
PMCID: PMC2864434  PMID: 17846153
OFC; lobotomy; value
11.  A “Good Parent” Function of Dopamine: Transient Modulation of Learning and Performance during Early Stages of Training 
While extracellular dopamine (DA) concentrations are increased by a wide category of salient stimuli, there is evidence to suggest that DA responses to primary and conditioned rewards may be distinct from those elicited by other types of salient events. A reward-specific mode of neuronal responding would be necessary if DA acts to strengthen behavioral response tendencies under particular environmental conditions or to set current environmental inputs as goals that direct approach responses. As described in this review, DA critically mediates both the acquisition and expression of learned behaviors during early stages of training, however, during later stages, at least some forms of learned behavior become independent of (or less dependent upon) DA transmission for their expression.
doi:10.1196/annals.1390.017
PMCID: PMC2827849  PMID: 17360799
learning; reinforcement; D1; D2; Parkinson; habit; electrophysiology; single unit; VTA; SN; LTP; glutamate; SCH23390; raclopride
12.  Glucocorticoid Hyper- and Hypofunction 
It is now well documented that both increased and decreased stress responses can profoundly affect cognition and behavior. This mini review presents possible neural mechanisms subserving stress effects on memory and aggression, particularly focusing on glucocorticoid (GC) hyper- and hypofunction. First, uncontrollable stress impedes hippocampal memory and long-term potentiation (LTP). Because the hippocampus is important for the stability of long-term memory and because LTP has qualities desirable of an information storage mechanism, it has been hypothesized that stress-induced alterations in LTP contribute to memory impairments. Recent evidence suggests a neural–endocrine network comprising amygdala, prefrontal cortex (PFC), and glucocorticoids may be involved in regulating stress effects on hippocampal mnemonic functioning. Second, antisocial aggressiveness correlates with chronically decreased glucocorticoid production, and this condition leads in rats to behavioral–autonomic deficits reminiscent of the human disorder. Glucocorticoid deficiency-induced antisocial aggressiveness results from functional changes in the PFC, medial and central amygdala, and altered serotonin and substance P neurotransmissions. Accordingly, a neurobiological understanding of how stress and glucocorticoid deficiency alter brain, cognition, and behavior is an important challenge facing modern neuroscience with broad implications for individual and social well-being.
doi:10.1196/annals.1391.014
PMCID: PMC2756062  PMID: 17513462
learning; memory; aggression; corticosterone; hippocampus; amygdala; prefrontal cortex; synaptic plasticity
13.  The Structure and Function of Francisella Lipopolysaccharide 
A key factor in the biology of Francisella spp. is lipopolysac-charide (LPS). Francisella LPS has many unique structural properties and poorly activates proinflammatory responses due to its lack of interaction with toll-like receptor 4 (TLR4). The LPS of this organism can be modified by various carbohydrates including glucose, mannose and galactosamine, which affect various aspects of virulence. Spontaneously occurring colony variants of F. tularensis have altered LPS. This altered LPS may account for the novel phenotypes of these variants that include effects on susceptibility to killing by normal human serum, intracellular survival and animal virulence. Mutants devoid of O-antigen (directed mutants in O-antigen biosynthetic genes) show reduced intracellular survival and mouse virulence. Thus, this surface component is important in F. tularensis pathogenesis, and the inability of the LPS to alarm the immune system coupled with its frequent modification/alteration likely aid the success of this pathogen during human infection
doi:10.1196/annals.1409.006
PMCID: PMC2742961  PMID: 17395723
LPS; lipopolysaccharide; gray variant; phase variation; LPS modification; LPS structure; lipid A; O-antigen
14.  ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood–testes barrier function and testicular immune privilege 
The transcription factor Ets-variant gene 5 (ETV5) is essential for spermatogonial stem cell (SSC) self-renewal, as targeted deletion of the Etv5 gene in mice (Etv5−/−) results in only the first wave of spermatogenesis. Reciprocal transplants of neonatal germ cells from wild type and Etv5−/− testes were performed to determine the role of ETV5 in Sertoli cells and germ cells. ETV5 appears to be needed in both cell types for normal spermatogenesis. In addition, Etv5−/− recipients displayed increased interstitial inflammation and tubular involution after transplantation. Preliminary studies suggest that the blood-testis-barrier (Sertoli-Sertoli tight junctional complex) is abnormal in the Etv5−/− mouse.
doi:10.1196/annals.1411.005
PMCID: PMC2733827  PMID: 17911411
Ets-Related Molecule; ERM; Spermatogonial Stem Cell; Sertoli Cell; Germ Cell Transplantation
15.  Noncoding RNAs of the Mammalian Testis: The Meiotic Transcripts Nct1 and Nct2 Encode piRNAs 
In eukaryotic cells, the vast majority of transcribed sequences are extragenic with no known functions. Translin is a DNA/RNA-binding protein involved in mRNA transport and translation in postmeiotic male germ cells. In an effort to identify meiotic target RNAs of Translin, reversible RNA protein cross-linking and immunoprecipitations with an affinity purified antibody to Translin were performed. Four new meiotically expressed mRNAs and one noncoding RNA with Translin binding sites were identified. Following sequencing, the noncoding RNA, Nct1, was 100% identical to a site on mouse chromosome 2. A second partially homologous sequence, Nct2, was detected nearby. Nct 1 and 2 contained sequences identical to piRNAs. Nct1 and 2 appear to be male germ cell-specific transcripts and are predominantly detected in pachytene spermatocytes. Focusing on the abundant single-copy PIWI-interacting RNA (piRNA), germline small RNA (gsRNA10) (the gsRNA10 sequence is identical to 29 nt in Nct1), we find that gsRNA10 increases greatly as spermatogenesis proceeds with concomitant decreases in Nct1 and 2. The piRNA gsRNA10 binds to the germ cell-specific Y-box protein, MSY2, but not to Translin. Although the size of the primary transcript(s) encoding the piRNAs in the locus on chromosome 2 is not known, we propose that Nct1 and 2 are part of a piRNA precursor.
doi:10.1196/annals.1411.002
PMCID: PMC2713573  PMID: 17905928
spermatogenesis; testis; meiosis; gene regulation; male germ cells; piRNAs; gsRNAs; noncoding RNAs
16.  Methamphetamine Disposition in Oral Fluid, Plasma, and Urine 
This review of the disposition of methamphetamine in oral fluid, plasma, and urine is based on a comprehensive controlled dosing study involving five healthy, drug-free research volunteers who resided on a closed clinical ward for 12 weeks. Subjects were administered four low (10 mg) and high (20 mg) daily oral doses of methamphetamine in two separate sessions. Near-simultaneous collections of oral fluid and plasma were performed on the first day of each low- and high-dose session. Thereafter, oral fluid was provided on each day of dosing by different oral fluid collection methods. All urine specimens were collected on an ad libitum basis throughout the study. Specimens were analyzed by gas-chromatography mass spectrometry for methamphetamine and the metabolite, amphetamine, with a limit of quantification of 2.5 ng/mL for each analyte. Methamphetamine and metabolite concentrations in oral fluid appeared to follow a similar time course in oral fluid as in plasma and were dose-proportional, but oral fluid concentrations exceeded plasma concentrations. Urine drug concentrations were substantially higher than those in oral fluid. Some drug accumulation was noted with daily dosing, but generally did not markedly influence detection times or detection rates of oral fluid tests. Detection times and detection rates for oral fluid and urine were determined at cessation of 4 days of dosing. Generally, detection times and rates for urine were longer than those observed for oral fluid at conventional cutoff concentrations. When contemplating selection of oral fluid as a test matrix, the advantages of oral fluid collection should be weighed against its shorter time of detection compared to that of urine.
doi:10.1196/annals.1384.038
PMCID: PMC2709797  PMID: 17332086
methamphetamine; collection; oral fluid; saliva; urine; plasma
17.  Interpretation of Oral Fluid Tests for Drugs of Abuse 
Oral fluid testing for drugs of abuse offers significant advantages over urine as a test matrix. Collection can be performed under direct observation with reduced risk of adulteration and substitution. Drugs generally appear in oral fluid by passive diffusion from blood, but also may be deposited in the oral cavity during oral, smoked, and intranasal administration. Drug metabolites also can be detected in oral fluid. Unlike urine testing, there may be a close correspondence between drug and metabolite concentrations in oral fluid and in blood. Interpretation of oral fluid results for drugs of abuse should be an iterative process whereby one considers the test results in the context of program requirements and a broad scientific knowledge of the many factors involved in determining test outcome. This review delineates many of the chemical and metabolic processes involved in the disposition of drugs and metabolites in oral fluid that are important to the appropriate interpretation of oral fluid tests. Chemical, metabolic, kinetic, and analytic parameters are summarized for selected drugs of abuse, and general guidelines are offered for understanding the significance of oral fluid tests.
doi:10.1196/annals.1384.037
PMCID: PMC2700061  PMID: 17332074
oral fluid; saliva; interpretation; testing; advantages; limitations
18.  Osteogenic Differentiation of Human Mesenchymal Bone Marrow Cells in Silk Scaffolds Is Regulated by Nitric Oxide 
Bone marrow-derived mesenchymal stem cells (BMSC) are a powerful tool for tissue engineering and can be used in the regeneration of bone and other tissues. Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) plays an important role in bone development and healing. We hypothesized that NO plays a role in osteogenic differentiation of BMSC cultured in three-dimensional silk scaffolds. eNOS protein was measured by Western Analysis and its activity was assessed by measuring nitrite in culture supernatants. Mineralization was evaluated through calcium deposition and the expression of genes associated with osteogenic differentiation (collagen I, RUNX2, and osteocalcin) was quantified using real-time RT-PCR. eNOS was consistently expressed with minor fluctuations, but NO production significantly increased at later time points (weeks 4 and 5). Addition of a competitive NOS inhibitor (L-NAME) resulted in a modest decrease in calcium deposition, which became statistically significant in week 5. This was preceded by a dramatic decrease in RUNX2 and osteocalcin expression in week 4. These results support our hypothesis and implicate NO as an important player in bone tissue engineering.
doi:10.1196/annals.1402.038
PMCID: PMC2698961  PMID: 17656569
nitric oxide; mesenchymal stem cells; cell differentiation; osteogenesis; tissue engineering
19.  Synovial Joint Formation during Mouse Limb Skeletogenesis: Roles of Indian Hedgehog Signaling 
Indian hedgehog (Ihh) has been previously found to regulate synovial joint formation. To analyze mechanisms, we carried out morphological, molecular and cell fate map analyses of interzone and joint development in wild type and Ihh−/− mouse embryo long bones. We found that Ihh−/− cartilaginous digit anlagen remained fused and lacked interzones or mature joints, whereas wrist skeletal elements were not fused but their joints were morphologically abnormal. E14.5 and E17.5 wild type digit and ankle anlagen expressed hedgehog target genes including Gli1 and Gli2 and interzone-associated genes including Gdf5, Erg and tenascin-C, but expression of all these genes was barely detectable in mutant joints. For cell fate map analysis of joint progenitor cells, we mated Gdf5-Cre+/−/Rosa R26R+/− double transgenic mice with heterozygous Ihh+/− mice and monitored reporter β-galactosidase activity and gene expression in triple-transgenic progeny. In control Gdf5-Cre+/−/R26R+/−/Ihh+/− limbs, reporter-positive cells were present in developing interzones, articulating layers and synovial lining tissue and absent from underlying growth plates. In mutant Gdf5-Cre+/−/R26R+/−/Ihh−/− specimens, reporter-positive cells were present also. However, the cells were mostly located around the prospective and uninterrupted digit joint sites and, interestingly, still expressed Erg, tenascin-C and Gdf5. Topographical analysis revealed that interzone and associated cells were not uniformly distributed, but were much more numerous ventrally. A similar topographical bias was seen for cavitation process and capsule primordia formation. In sum, Ihh is a critical and possibly direct regulator of joint development. In its absence, distribution and function of Gdf5-expressing interzone-associated cells are abnormal, but their patterning at prospective joint sites still occurs. The joint-forming functions of the cells appear to normally involve a previously unsuspected asymmetric distribution along the ventral-to-dorsal plane of the developing joint.
doi:10.1196/annals.1402.063
PMCID: PMC2673545  PMID: 18083924
synovial joint patterning; limb skeletogenesis; Indian hedgehog; interzone; joint progenitor cells; hedgehog signaling; Gli proteins; transcription factor Erg
20.  Axin1 and Axin2 Are Regulated by TGF-β and Mediate Cross-talk between TGF-β and Wnt Signaling Pathways 
Chondrocyte maturation during endochondral bone formation is regulated by a number of signals that either promote or inhibit maturation. Among these, two well-studied signaling pathways play crucial roles in modulating chondrocyte maturation: transforming growth factor-beta (TGF-β)/Smad3 signaling slows the rate of chondrocyte maturation, while Wingless/INT-1-related (Wnt)/β-catenin signaling enhances the rate of chondrocyte maturation. Axin1 and Axin2 are functionally equivalent and have been shown to inhibit Wnt/β-catenin signaling and stimulate TGF-β signaling. Here we show that while Wnt3a stimulates Axin2 in a negative feedback loop, TGF-β suppresses the expression of both Axin1 and Axin2 and stimulates β-catenin signaling. In Axin2 −/− chondrocytes, TGF-β treatment results in a sustained increase in β-catenin levels compared to wild-type chondrocytes. In contrast, overexpression of Axin enhanced TGF-β signaling while overexpression of β-catenin inhibited the ability of TGF-β to induce Smad3-sensitive reporters. Finally, the suppression of the Axins is Smad3-dependent since the effect is absent in Smad3 −/− chondrocytes. Altogether these findings show that the Axins act to integrate signals between the Wnt/β-catenin and TGF-β/Smad pathways. Since the suppression Axin1 and Axin2 expression by TGF-β reduces TGF-β signaling and enhances Wnt/β-catenin signaling, the overall effect is a shift from TGF-β toward Wnt/β-catenin signaling and an acceleration of chondrocyte maturation.
doi:10.1196/annals.1402.082
PMCID: PMC2647987  PMID: 18083923
axin; axin1; axin2; TGF-β; Wnt; β-catenin; chondrogenesis
21.  Brain Aging and Its Modifiers 
Aging is marked by individual differences and differential vulnerability of cognitive operations and their neural substrates. Cross-sectional studies of brain volume reveal greater age-related shrinkage of the prefrontal cortex (PFC) and the hippocampus than in the entorhinal and primary visual cortex. Longitudinal studies of regional brain shrinkage indicate that when individual differences are controlled, larger and broader shrinkage estimates are evident, with most polymodal cortices affected to the same extent. The mechanisms of age-related shrinkage are unclear. Vascular risk factors may exacerbate brain aging and account for some of the observed declines as both the PFC and the hippocampus show elevated vulnerability to hypertension. MRI techniques that are sensitive to small vessels function, tissue oxygenation, and perfusion may be especially well suited to study brain aging and its vascular modifiers. We present an example of one such technique, susceptibility weighted imaging (SWI), that allows direct measurement of T2* values that reflect deoxy- to oxyhemoglobin fraction in blood vessels and iron deposits in cerebral tissue. The T2* shortening is associated with advanced age, but the effect is significantly stronger in the PFC and the hippocampus than the entorhinal and visual cortices. Moreover, T2* is shorter in hypertensive participants than in their matched normotensive counterparts, and the difference is especially prominent in the hippocampus, thus mirroring the findings of the neuromorphometric studies. Future research on brain aging would benefit from combining structural and metabolic techniques in a longitudinal design, as such studies will allow examination of leading–trailing effects of those factors.
doi:10.1196/annals.1379.018
PMCID: PMC2630248  PMID: 17413014
aging; MRI; brain; longitudinal; vascular risk; susceptibility weighted imaging; deoxyhemoglobin; iron
22.  Poverty, Gender Inequities, and Women’s Risk of Human Immunodeficiency Virus/AIDS 
Entrenched economic and gender inequities together are driving a globally expanding, increasingly female, human immunodeficiency virus (HIV)/AIDS epidemic. To date, significant population-level declines in HIV transmission have not been observed, at least in part because most approaches to prevention have presumed a degree of individual control in decision making that does not speak to the reality of women’s and girls’ circumstances in many parts of the world. Such efforts have paid insufficient attention to critical characteristics of the risk environment, most notably poverty and gender power inequities. Even fewer interventions have addressed specific mechanisms through which these inequities engender risky sexual practices that result in women’s disproportionately increased vulnerabilities to HIV infection. This article focuses on identifying those mechanisms, or structural pathways, that stem from the interactions between poverty and entrenched gender inequities and recommending strategies to address and potentially modify those pathways. We highlight four such structural pathways to HIV risk, all of which could be transformed: (1) lack of access to critical information and health services for HIV/sexually transmitted infection (STI) prevention, (2) limited access to formal education and skill development, (3) intimate partner violence, and (4) the negative consequences of migration prompted by insufficient economic resources. We argue for interventions that enhance women’s access to education, training, employment, and HIV/STI prevention information and tools; minimize migration; and by working with men and communities, at the same time reduce women’s poverty and promote gender-equitable norms. In conclusion, we identify challenges in developing and evaluating strategies to address these structural pathways.
doi:10.1196/annals.1425.013
PMCID: PMC2587136  PMID: 17954681
poverty; gender; HIV risk; structural pathways
23.  Neural Correlates of Inflexible Behavior in the Orbitofrontal–Amygdalar Circuit after Cocaine Exposure 
Addiction is characterized by compulsive or inflexible behavior, observed both in the context of drug-seeking and in contexts unrelated to drugs. One possible contributor to these inflexible behaviors may be drug-induced dysfunction within circuits that support behavioral flexibility, including the basolateral amygdala (ABL) and the orbitofrontal cortex (OFC). Here we describe data demonstrating that chronic cocaine exposure causes long-lasting changes in encoding properties in the ABL and the OFC during learning and reversal in an odor-guided task. In particular, these data suggest that inflexible encoding in ABL neurons may be the proximal cause of cocaine-induced behavioral inflexibility, and that a loss of outcome-expectant encoding in OFC neurons could be a more distal contributor to this impairment. A similar mechanism of drug-induced orbitofrontal–amygdalar dysfunction may cause inflexible behavior when animals and addicts are exposed to drug-associated cues and contexts.
doi:10.1196/annals.1401.014
PMCID: PMC2587372  PMID: 17846156
addiction; cocaine; orbitofrontal cortex; basolateral amygdala; reversal; associative learning
24.  Integrated Microfluidic Platform for Oral Diagnostics 
While many point-of-care (POC) diagnostic methods have been developed for blood-borne analytes, development of saliva-based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip-based immunoassays (<3–10 min) with low sample volume requirements (10 μL) and appreciable sensitivity (nM–pM). Our microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment (filtering, enrichment, mixing) with electrophoretic immunoassays to quickly measure analyte concentrations in minimally pretreated saliva samples. The microfluidic chip has been integrated with miniaturized electronics, optical elements, such as diode lasers, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device and methods are being tested by detecting potential biomarkers in saliva samples from patients diagnosed with periodontal disease. Our microchip-based analysis can readily be extended to detection of biomarkers of other diseases, both oral and systemic, in saliva and other oral fluids.
doi:10.1196/annals.1384.004
PMCID: PMC2572166  PMID: 17435142
microfluidics; periodontal disease; diagnostics; point-of-care; POC; immunoassay; lab-on-a-chip; saliva
25.  Oral Fluid-Based Biomarkers of Alveolar Bone Loss in Periodontitis 
Periodontal disease is a bacteria-induced chronic inflammatory disease affecting the soft and hard supporting structures encompassing the teeth. When left untreated, the ultimate outcome is alveolar bone loss and exfoliation of the involved teeth. Traditional periodontal diagnostic methods include assessment of clinical parameters and radiographs. Though efficient, these conventional techniques are inherently limited in that only a historical perspective, not current appraisal, of disease status can be determined. Advances in the use of oral fluids as possible biological samples for objective measures of current disease state, treatment monitoring, and prognostic indicators have boosted saliva and other oral-based fluids to the forefront of technology. Oral fluids contain locally and systemically derived mediators of periodontal disease, including microbial, host-response, and bone-specific resorptive markers. Although most biomarkers in oral fluids represent inflammatory mediators, several specific collagen degradation and bone turnover-related molecules have emerged as possible measures of periodontal disease activity. Pyridinoline cross-linked carboxyterminal telopeptide (ICTP), for example, has been highly correlated with clinical features of the disease and decreases in response to intervention therapies, and has been shown to possess predictive properties for possible future disease activity. One foreseeable benefit of an oral fluid-based periodontal diagnostic would be identification of highly susceptible individuals prior to overt disease. Timely detection and diagnosis of disease may significantly affect the clinical management of periodontal patients by offering earlier, less invasive, and more cost-effective treatment therapies.
doi:10.1196/annals.1384.028
PMCID: PMC2570328  PMID: 17435132
periodontal disease; oral fluids; saliva; disease progression; diagnosis; bone resorption

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