Methylphenidate is a psychostimulant medication that produces improvements in functions associated with multiple neurocognitive systems. To investigate the potentially distributed effects of methylphenidate on the brain’s intrinsic network architecture, we coupled resting state imaging with multivariate pattern classification. In a within-subject, double-blind, placebo-controlled, randomized, counterbalanced, cross-over design, 32 healthy human volunteers received either methylphenidate or placebo prior to two fMRI resting state scans separated by approximately one week. Resting state connectomes were generated by placing regions of interest at regular intervals throughout the brain, and these connectomes were submitted for support vector machine analysis. We found that methylphenidate produces a distributed, reliably detected, multivariate neural signature. Methylphenidate effects were evident across multiple resting state networks, especially visual, somatomotor, and default networks. Methylphenidate reduced coupling within visual and somatomotor networks. In addition, default network exhibited decoupling with several task positive networks, consistent with methylphenidate modulation of the competitive relationship between these networks. These results suggest that connectivity changes within and between large-scale networks is potentially involved in the mechanisms by which methylphenidate improves attention functioning.
methylphenidate; fMRI; resting state; multivariate pattern classification; connectome; intrinsic connectivity networks
Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.
Signaling inputs from multiple pathways are essential for the establishment of distinct cell and tissue types in the embryo. Therefore, multiple signals must be integrated to activate gene expression and confer cell fate, but little is known about how this occurs at the level of target gene promoters. During early embryogenesis, Wnt and Nodal signals are required for formation of the Spemann organizer, which is essential for germ layer patterning and axis formation. Signaling by both Wnt and Nodal pathways is required for the expression of multiple organizer genes, suggesting that integration of these signals is required for organizer formation. Here, we demonstrate transcriptional cooperation between the Wnt and Nodal pathways in the activation of the organizer genes Goosecoid (Gsc), Cerberus (Cer), and Chordin (Chd). Combined Wnt and Nodal signaling synergistically activates transcription of these organizer genes. Effectors of both pathways occupy the Gsc, Cer and Chd promoters and effector occupancy is enhanced with active Wnt and Nodal signaling. This suggests that, at organizer gene promoters, a stable transcriptional complex containing effectors of both pathways forms in response to combined Wnt and Nodal signaling. Consistent with this idea, the histone acetyltransferase p300 is recruited to organizer promoters in a Wnt and Nodal effector-dependent manner. Taken together, these results offer a mechanism for spatial and temporal restriction of organizer gene transcription by the integration of two major signaling pathways, thus establishing the Spemann organizer domain.
Wnt; Nodal; Organizer; Siamois; Twin; FoxH1; Smad2/3; Signaling; Transcription; Embryo; Xenopus; Gastrula
In this article, we report results from a new study that surveyed a large, national sample of American adults about their willingness to pay for health reform. As in previous work, we find that self-identified Republicans, older Americans, and high-income Americans are less supportive of reform. However, these basic findings mask three important features of public opinion. First, income has a substantial effect on support for reform, even holding political affiliation constant. Indeed, income is the most important determinant of support for reform. Second, the negative effects of income on support for reform begin early in the income distribution, at annual family income levels of $25,000 to $50,000. Third, although older Americans have a less favorable view of reform than the young, much of their opposition is due to dislike of large policy changes than to reform per se.
The Spemann organizer is an essential signaling center in Xenopus germ layer patterning and axis formation. Organizer formation occurs in dorsal blastomeres receiving both maternal Wnt and zygotic Nodal signals. In response to stabilized βcatenin, dorsal blastomeres express the closely related transcriptional activators, Siamois (Sia) and Twin (Twn), members of the paired homeobox family. Sia and Twn induce organizer formation and expression of organizer-specific genes, including Goosecoid (Gsc). In spite of the similarity of Sia and Twn sequence and expression pattern, it is unclear whether these factors function equivalently in promoter binding and subsequent transcriptional activation, or if Sia and Twn are required for all aspects of organizer function. Here we report that Sia and Twn activate Gsc transcription by directly binding to a conserved P3 site within the Wnt-responsive proximal element of the Gsc promoter. Sia and Twn form homodimers and heterodimers by direct homeodomain interaction and dimer forms are indistinguishable in both DNA-binding and activation function. Sequential chromatin immunoprecipitation reveals that the endogenous Gsc promoter can be occupied by either Sia or Twn homodimers or Sia-Twn heterodimers. Knockdown of Sia and Twn together, but not individually, results in a failure of organizer gene expression and a disruption of axis formation, consistent with a redundant role for Sia and Twn in organizer formation. Furthermore, simultaneous knockdown of Sia and Twn blocks axis induction in response to ectopic Wnt signaling, demonstrating an essential role for Sia and Twn in mediating the transcriptional response to the maternal Wnt pathway. The results demonstrate the functional redundancy of Sia and Twn and their essential role in direct transcriptional responses necessary for Spemann organizer formation.
Siamois; Twin; Goosecoid; Organizer; Transcription; Wnt; Homeodomain
In this paper, we estimate the effect of the tax preference for health insurance on health care spending using data from the Medical Expenditure Panel Surveys from 1996–2005. We use the fact that Social Security taxes are only levied on earnings below a statutory threshold to identify the impact of the tax preference. Because employer-sponsored health insurance premiums are excluded from Social Security payroll taxes, workers who earn just below the Social Security tax threshold receive a larger tax preference for health insurance than workers who earn just above it. We find a significant effect of the tax preference, consistent with previous research.
tax preference; health insurance; health spending
In this paper, we use publicly available data from the Medical Expenditure Panel Survey - Insurance Component (MEPS-IC) to investigate the effect of Massachusetts’ health reform plan on employer-sponsored insurance premiums. We tabulate premium growth for private-sector employers in Massachusetts and the United States as a whole for 2004 – 2008. We estimate the effect of the plan as the difference in premium growth between Massachusetts and the United States between 2006 and 2008—that is, before versus after the plan—over and above the difference in premium growth for 2004 to 2006. We find that health reform in Massachusetts increased single-coverage employer-sponsored insurance premiums by about 6 percent, or $262. Although our research design has important limitations, it does suggest that policy makers should be concerned about the consequences of health reform for the cost of private insurance.
Health care providers may vertically integrate not only to facilitate coordination of care, but also for strategic reasons that may not be in patients’ best interests. Optimal Medicare reimbursement policy depends upon the extent to which each of these explanations is correct. To investigate, we compare the consequences of the 1997 adoption of prospective payment for skilled nursing facilities (SNF PPS) in geographic areas with high versus low levels of hospital/SNF integration. We find that SNF PPS decreased spending more in high integration areas, with no measurable consequences for patient health outcomes. Our findings suggest that integrated providers should face higher-powered reimbursement incentives, i.e., less cost-sharing. More generally, we conclude that purchasers of health services (and other services subject to agency problems) should consider the organizational form of their suppliers when choosing a reimbursement mechanism.
Health economics; Industrial organization; Reimbursement policy
This paper reports the results of a 2009 national survey that quantifies Americans’ willingness to pay to expand health insurance coverage. We asked respondents whether they would support a Medicaid expansion, a subsidy for low-income people, or a subsidy for the chronically ill, if they had to pay more income taxes to cover the program’s costs. Based on respondents’ reported income, we told them approximately how much, in dollar terms, their tax increases would be. Our results reflect a tension in public opinion recognized by previous investigators: a desire for reform but limited willingness to pay for it.
Variation in the use of hospital and physician services among Medicare beneficiaries is well documented. However, less is known about the younger, commercially insured population. Using data from the Community Tracking Study to investigate this issue, we found significant variation in the use of both inpatient and outpatient services across twelve metropolitan areas. HMO insurance reduces, but does not eliminate, the extent of this variation. Our results suggest that health plan spending to better organize delivery systems and manage care may be efficient, and regulations that arbitrarily cap plans’ spending on administration, such as minimum medical loss ratios, could undermine efforts to achieve better value in health care.
This article examines the possibilities for health care reform in the 111th Congress. It uses a simple model of policy making to analyze the failure of Congress to pass the Clinton health plan in 1993 – 1994. It concludes that the factors that created gridlock in the 103rd Congress are likely to have a similar impact in the present.
Establishment of the embryonic mesoderm is dependent on integration of multiple signaling and transcriptional inputs. We report that the transcriptional regulator Foxd3 is essential for dorsal mesoderm formation in zebrafish, and that this function is dependent on the Nodal pathway. Foxd3 gain-of-function results in expanded dorsal mesodermal gene expression, including the Nodal-related gene cyclops, and body axis dorsalization. Foxd3 knockdown embryos displayed reduced expression of cyclops and mesodermal genes, axial defects similar to Nodal pathway loss-of-function, and Nodal pathway activation rescued these phenotypes. In MZoep mutants inactive for Nodal signaling, Foxd3 did not rescue mesoderm formation or axial development, indicating that the mesodermal function of Foxd3 is dependent on an active downstream Nodal pathway. A previously identified foxd3 mutant, sym1, was described as a predicted null mutation with neural crest defects, but no mesodermal or axial phenotypes. We find that Sym1 protein retains activity and can induce strong mesodermal expansion and axial dorsalization. A subset of sym1 homozygotes display axial defects and reduced cyclops and mesodermal gene expression, and penetrance of the mesodermal phenotypes is enhanced by Foxd3 knockdown. Therefore, sym1 is a hypomorphic allele, and reduced Foxd3 function results in a reduction of cyclops expression, and subsequent mesodermal and axial defects. These results demonstrate that Foxd3 is an essential upstream regulator of the Nodal pathway in zebrafish dorsal mesoderm development and establish a broadly conserved role for Foxd3 in vertebrate mesodermal development.
Foxd3; Nodal; Forkhead; Mesoderm; Transcription; Zebrafish
We investigate whether the removal of high-cost individuals from private insurance markets leads to greater coverage for individuals who are similar but not as high cost. Using data on insurance coverage from the Panel Study of Income Dynamics, we estimate the effect of the extension of Medicare to the disabled on the private insurance coverage of non-disabled individuals. We find that the insurance coverage of individuals who had a health condition that limited their ability to work increased significantly in states with high versus low rates of disability.
In this study we used a Random Forest-based approach for an assignment of small guanosine triphosphate proteins (GTPases) to specific subgroups. Small GTPases represent an important functional group of proteins that serve as molecular switches in a wide range of fundamental cellular processes, including intracellular transport, movement and signaling events. These proteins have further gained a special emphasis in cancer research, because within the last decades a huge variety of small GTPases from different subgroups could be related to the development of all types of tumors. Using a random forest approach, we were able to identify the most important amino acid positions for the classification process within the small GTPases superfamily and its subgroups. These positions are in line with the results of earlier studies and have been shown to be the essential elements for the different functionalities of the GTPase families. Furthermore, we provide an accurate and reliable software tool (GTPasePred) to identify potential novel GTPases and demonstrate its application to genome sequences.
cancer; machine learning; classification; Random Forests; proteins
Chromatin immunoprecipitation (ChIP) is a powerful method for analyzing the interaction of regulatory proteins with genomic loci, but has been difficult to apply to studies on early embryos due to the limiting amount of genomic material in these samples. Here, we present a comprehensive technique for performing ChIP on blastula and gastrula stage Xenopus embryos. We also describe methods for optimizing crosslinking and chromatin shearing, verifying antibody specificity, maximizing PCR sensitivity, and quantifying PCR results, allowing for the use of as few as 50 early blastula stage embryos (approximately 5×104 cells) per experimental condition. Finally, we demonstrate the predicted binding of endogenous β-catenin to the nodal-related 6 promoter, binding of tagged Fast-1/FoxH1 to the goosecoid promoter, and binding of tagged Tcf3 to the siamois and nodal-related 6 promoters as examples of the potential application of ChIP to embryological investigations.
chromatin; chromatin immunoprecipitation (ChIP); Xenopus; embryo; transcription factor; histone; DNA; β-catenin; Fast-1/FoxH1; Tcf3; Goosecoid; Xnr6; Siamois
FoxD3 is a forkhead-related transcriptional regulator that is essential for multiple developmental processes in the vertebrate embryo, including neural crest development and maintenance of mammalian stem cell lineages. Recent results demonstrate a requirement for FoxD3 in Xenopus mesodermal development. In the gastrula, FoxD3 functions as a transcriptional repressor in the Spemann organizer to maintain the expression of Nodal-related members of the TGFß superfamily that induce dorsal mesoderm formation. Here we report that the function of FoxD3 in mesoderm induction is dependent on the recruitment of transcriptional corepressors of the TLE/Groucho family. Structure-function analyses indicate that the transcriptional repression and mesoderm induction activities of FoxD3 are dependent on a C-terminal domain, as well as specific DNA-binding activity conferred by the forkhead domain. The C-terminal domain contains a heptapeptide similar to the eh1/GEH Groucho interaction motif. Deletion and point mutagenesis demonstrated that the FoxD3 eh1/GEH motif is required for both repression of transcription and induction of mesoderm, as well as the direct physical interaction of FoxD3 and Grg4 (Groucho-related gene-4). Consistent with a functional interaction of FoxD3 and Grg4, the transcriptional repression activity of FoxD3 is enhanced by Grg4, and reduced by Grg5, a dominant inhibitory Groucho protein. The results indicate that FoxD3 recruitment of Groucho corepressors is essential for the transcriptional repression of target genes and induction of mesoderm in Xenopus.
Induction and patterning of the mesodermal germ layer is a key early step of vertebrate embryogenesis. We report that FoxD3 function in the Xenopus gastrula is essential for dorsal mesodermal development and for Nodal expression in the Spemann organizer. In embryos and explants, FoxD3 induced mesodermal genes, convergent extension movements, and differentiation of axial tissues. Engrailed-FoxD3, but not VP16-FoxD3, was identical to native FoxD3 in mesoderm-inducing activity, indicating that FoxD3 functions as a transcriptional repressor to induce mesoderm. Antagonism of FoxD3 with VP16-FoxD3 or morpholino-knockdown of FoxD3 protein resulted in a complete block to axis formation, a loss of mesodermal gene expression, and an absence of axial mesoderm, indicating that transcriptional repression by FoxD3 is required for mesodermal development. FoxD3 induced mesoderm in a non-cell-autonomous manner, indicating a role for secreted inducing factors in the response to FoxD3. Consistent with this mechanism, FoxD3 was necessary and sufficient for the expression of multiple Nodal-related genes, and inhibitors of Nodal signaling blocked mesoderm induction by FoxD3. Therefore, FoxD3 is required for Nodal expression in the Spemann organizer and this function is essential for dorsal mesoderm formation.
Xenopus; FoxD3; Forkhead; Nodal; mesoderm; transcription
α2-macroglobulin is a major serum protein with diverse functions, including inhibition of protease activity and binding of growth factors, cytokines, and disease factors. We have cloned and characterized Panza, a new Xenopus laevis α2-macroglobulin. Panza has 56–60% amino acid similarity with previously identified Xenopus, mouse, rat and human α2-macroglobulins, indicating that Panza is a new member of the α2-macroglobulin family. Panza mRNA is first detected at the beginning of neurulation in the dorsal endoderm lining the primitive gut (archenteron roof). At the completion of neurulation and continuing through the late tadpole stage, Panza is restricted to a dorsal domain of the gut endoderm adjacent to the notochord and extending along the entire anterior-posterior axis. With outgrowth of the tailbud, Panza expression persists in the chordaneural hinge at the posterior end of the differentiating notochord and extends into the floor plate of the posterior neural tube. As gut coiling commences, Panza expression is initiated in the liver, and the dorsal domain of Panza expression becomes limited to the midgut and hindgut. With further gut coiling, strong Panza expression persists in the liver, but is lost from other regions of the gut. The expression of Panza in endodermal cells adjacent to the notochord points to a potential role for Panza in signal modulation and/or morphogenesis of the primitive gut.
Xenopus laevis; α2-macroglobulin; endoderm; gut; digestive tract; liver
The parvulin-type peptidyl prolyl cis/trans isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study.
Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions.
Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae.
The Fox gene family comprises a large and functionally diverse group of forkhead-related transcriptional regulators, many of which are essential for metazoan embryogenesis and physiology. Defining conserved functional domains that mediate the transcriptional activity of Fox proteins will contribute to a comprehensive understanding of the biological function of Fox family genes.
Systematic analysis of 458 protein sequences of the metazoan Fox family was performed to identify the presence of the engrailed homology-1 motif (eh1), a motif known to mediate physical interaction with transcriptional corepressors of the TLE/Groucho family. Greater than 50% of Fox proteins contain sequences with high similarity to the eh1 motif, including ten of the nineteen Fox subclasses (A, B, C, D, E, G, H, I, L, and Q) and Fox proteins of early divergent species such as marine sponge. The eh1 motif is not detected in Fox proteins of the F, J, K, M, N, O, P, R and S subclasses, or in yeast Fox proteins. The eh1-like motifs are positioned C-terminal to the winged helix DNA-binding domain in all subclasses except for FoxG proteins, which have an N-terminal motif. Two similar eh1-like motifs are found in the zebrafish FoxQ1 and in FoxG proteins of sea urchin and amphioxus. The identification of eh1-like motifs by manual sequence alignment was validated by statistical analyses of the Swiss protein database, confirming a high frequency of occurrence of eh1-like sequences in Fox family proteins. Structural predictions suggest that the majority of identified eh1-like motifs are short α-helices, and wheel modeling revealed an amphipathicity that supports this secondary structure prediction.
A search for eh1 Groucho interaction motifs in the Fox gene family has identified eh1-like sequences in greater than 50% of Fox proteins. The results predict a physical and functional interaction of TLE/Groucho corepressors with many members of the Fox family of transcriptional regulators. Given the functional importance of the eh1 motif in transcriptional regulation, our annotation of this motif in the Fox gene family will facilitate further study of the diverse transcriptional and regulatory roles of Fox family proteins.
The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). We previously reported that the LIM homeobox transcription factor Lmx1b is expressed within the chick IsO where it is sufficient to maintain expression of the secreted factor wnt1 In this paper, we show that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2 in the rostral IsO, and demonstrate that these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8, and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. We propose that Lmx1b.1 and Lmx1b.2 regulation of wnt1, wnt3a, wnt10b, pax8, and fgf8 maintains cell survival in the isthmocerebellar region.
Isthmus; Lmx1b; mesencephalon; metencephalon; Pax; Wnt; zebrafish
The peptidyl prolyl cis/trans isomerase (PPIase) Parvulin (Par14/PIN4) is highly conserved in all metazoans and is assumed to play a role in cell cycle progression and chromatin remodeling. It is predominantly localized to the nucleus and binds to chromosomal DNA as well as bent oligonucleotides in vitro.
In this study we confirm by RT-PCR the existence of a longer Parvulin isoform expressed in all tissues examined so far. This isoform contains a 5' extension including a 75 bp extended open reading frame with two coupled SNPs leading to amino acid substitutions Q16R and R18S. About 1% of all Parvulin mRNAs include the novel extension as quantified by real-time PCR. The human Parvulin promoter is TATA-less and situated in a CpG island typical for house keeping genes. Thus, different Parvulin mRNAs seem to arise by alternative transcription initiation. N-terminally extended Parvulin is protected from rapid proteinaseK degradation. In HeLa and HepG2 cell lysates two protein species of about 17 and 28 KDa are detected by an antibody against an epitope within the N-terminal extension. These two bands are also recognized by an antibody towards the PPIase domain of Parvulin. The longer Parvulin protein is encoded by the human genome but absent from rodent, bovine and non-mammalian genomes.
Due to its molecular weight of 16.6 KDa we denote the novel Parvulin isoform as Par17 following the E. coli Par10 and human Par14 nomenclature. The N-terminal elongation of Par17-QR and Par17-RS suggests these isoforms to perform divergent functions within the eukaryotic cell than the well characterized Par14.