Nkx2.2 and Arx genetically interact to regulate pancreatic endocrine cell development and endocrine hormone expression

AbstractNkx2.2 and Arx are essential pancreatic transcription factors. Nkx2.2 is necessary for the appropriate specification of the islet alpha, beta, PP and epsilon cell lineages, whereas Arx is required to form the correct ratio of alpha, beta, delta and PP cells. To begin to understand the cooperative functions of Nkx2.2 and Arx in the development of endocrine cell lineages, we generated progenitor cell-specific deletions of Arx on the Nkx2.2 null background. The analysis of these mutants demonstrates that expansion of the ghrelin cell population in the Nkx2.2 null pancreas is not dependent on Arx; however, Arx is necessary for the upregulation of ghrelin mRNA levels in Nkx2.2 mutant epsilon cells. Alternatively, in the absence of Arx, delta cell numbers are increased and Nkx2.2 becomes essential for the repression of somatostatin gene expression. Interestingly, the dysregulation of ghrelin and somatostatin expression in the Nkx2.2/Arx compound mutant (Nkx2.2null;ArxΔpanc) results in the appearance of ghrelin+/somatostatin+ co-expressing cells. These compound mutants also revealed a genetic interaction between Nkx2.2 and Arx in the regulation of the PP cell lineage; the PP cell population is reduced when Nkx2.2 is deleted but is restored back to wildtype numbers in the Nkx2.2null;ArxΔpanc mutant. Moreover, conditional deletion of Arx in specific pancreatic cell populations established that the functions of Arx are necessary in the Neurog3+ endocrine progenitors. Together, these experiments identify novel genetic interactions between Nkx2.2 and Arx within the endocrine progenitor cells that ensure the correct specification and regulation of endocrine hormone-producing cells

The heart healthy lenoir project-an intervention to reduce disparities in hypertension control: study protocol

Background Racial disparities in blood pressure control are well established; however the impact of low health literacy (LHL) on blood pressure has garnered less attention. Office based interventions that are created with iterative patient, practice and community stakeholder input and are rolled out incrementally, may help address these disparities in hypertension control. This paper describes our study protocol. Methods/design Using a community based participatory research (CBPR) approach, we designed and implemented a cohort study that includes both a practice level and patient level intervention to enhance the care and support of patients with hypertension in primary care practices in a rural region of eastern North Carolina. The study is divided into a formative phase and an ongoing 2.5 year implementation phase. Our main care enhancement activities include the integration of a community health coach, using home blood pressure monitoring in clinical decision making, standardizing care delivery processes, and working to improve medication adherence. Main outcomes include overall blood pressure change, the differential change in blood pressure by race (African American vs. White) and health literacy level (low vs. higher health literacy). Discussion Using a community based participatory approach in primary care practice settings has helped to engage patients and practice staff and providers in the research effort and in making practice changes to support hypertension care. Practices have engaged at varying levels, but progress has been made in implementing and iteratively improving upon the interventions to date

Computational Treatment of Metalloproteins

Metalloproteins present a considerable challenge for modeling, especially when the starting point is far from thermodynamic equilibrium. Examples include formidable problems such as metalloprotein folding and structure prediction upon metal addition, removal, or even just replacement; metalloenzyme design, where stabilization of a transition state of the catalyzed reaction in the specific binding pocket around the metal needs to be achieved; docking to metal-containing sites and design of metalloenzyme inhibitors. Even more conservative computations, such as elucidations of the mechanisms and energetics of the reaction catalyzed by natural metalloenzymes, are often nontrivial. The reason is the vast span of time and length scales over which these proteins operate, and thus the resultant difficulties in estimating their energies and free energies. It is required to perform extensive sampling, properly treat the electronic structure of the bound metal or metals, and seamlessly merge the required techniques to assess energies and entropies, or their changes, for the entire system. Additionally, the machinery needs to be computationally affordable. Although a great advancement has been made over the years, including some of the seminal works resulting in the 2013 Nobel Prize in chemistry, many aforementioned exciting applications remain far from reach. We review the methodology on the forefront of the field, including several promising methods developed in our lab that bring us closer to the desired modern goals. We further highlight their performance by a few examples of applications

Arx polyalanine expansion in mice leads to reduced pancreatic α-cell specification and increased α-cell death.

ARX/Arx is a homeodomain-containing transcription factor necessary for the specification and early maintenance of pancreatic endocrine α-cells. Many transcription factors important to pancreas development, including ARX/Arx, are also crucial for proper brain development. Although null mutations of ARX in human patients result in the severe neurologic syndrome XLAG (X-linked lissencephaly associated with abnormal genitalia), the most common mutation is the expansion of the first polyalanine tract of ARX, which results primarily in the clinical syndrome ISSX (infantile spasms). Mouse models of XLAG, ISSX and other human ARX mutations demonstrate a direct genotype-phenotype correlation in ARX-related neurologic disorders. Furthermore, mouse models utilizing a polyalanine tract expansion mutation have illustrated critical developmental differences between null mutations and expansion mutations in the brain, revealing context-specific defects. Although Arx is known to be required for the specification and early maintenance of pancreatic glucagon-producing α-cells, the consequences of the Arx polyalanine expansion on pancreas development remain unknown. Here we report that mice with an expansion mutation in the first polyalanine tract of Arx exhibit impaired α-cell specification and maintenance, with gradual α-cell loss due to apoptosis. This is in contrast to the re-specification of α-cells into β- and δ-cells that occurs in mice null for Arx. Overall, our analysis of an Arx polyalanine expansion mutation on pancreatic development suggests that impaired α-cell function might also occur in ISSX patients

ArxE mice have almost complete loss of α-cell fate by P14 with a concomitant decrease in total endocrine mass, but no change in β- and δ-cell mass.

<p>(<b>A–H</b>): P14 pancreatic sections were stained for glucagon (green) and insulin (red; A–B), somatostatin (Sst; red; C–D), PP (red; E–F), and ghrelin (Ghr; red; G–H). Scale bar denotes 50 µm. (<b>I</b>): Quantification of endocrine hormone mass including total endocrine mass (ChrgA), insulin, somatostatin, PP, and ghrelin displayed as fold change in ArxE mice (white bar) relative to control (black bar). (<b>J</b>): Analysis of glucagon mass over time starting at E15.5 and ending at P14 in control (black bar) and ArxE (white bar) pancreata. Resulting p value is listed. (*) denotes significance where p<0.05. Error bars represent standard error of the mean (I, J). For all analysis 4–5 animals per group were analyzed with all ArxE mice being males and control mice consisting of male and female mice.</p

ArxE mice are able to specify a subset of α-cells at E15.5.

<p>(<b>A–F</b>): Control and ArxE E15.5 pancreatic sections were stained for glucagon (green) and insulin (red; A–B), somatostatin (Sst; red; C–D), and ghrelin (Ghr; red; E–F). Scale bar denotes 50 µm. (<b>G</b>): Quantification of total endocrine (ChrgA) and β-, δ-, and α-cell mass in control (black bar) and ArxE (white bar) pancreata. (<b>H</b>): Quantification of transcript levels for each endocrine hormone in control (black bar) and ArxE pancreata (white bar) at E15.5 using qRT-PCR. All results are graphed as fold change relative to littermate controls ± standard error of the mean. Significance is denoted with (*) when p≤0.05. All analysis consists of 4–5 animals per group.</p

ArxE mice are able to correctly specify a subset of α-cells, but α-cells are gradually lost through apoptosis.

<p>Model demonstrating normal proliferation, but increased apoptosis in ArxE mice. Normal proliferation during embryonic time points maintains the α-cell lineage by replacing cells lost to apoptosis. However, proliferation slows during the neonatal stage leading to loss of the α-cell lineage.</p

Pancreatic α-Cell Specific Deletion of Mouse Arx Leads to α-Cell Identity Loss

<div><p>The specification and differentiation of pancreatic endocrine cell populations (α-, β-, δ, PP- and ε-cells) is orchestrated by a combination of transcriptional regulators. In the pancreas, <i>Aristaless-related homeobox</i> gene (<i>Arx</i>) is expressed first in the endocrine progenitors and then restricted to glucagon-producing α-cells. While the functional requirement of <i>Arx</i> in early α-cell specification has been investigated, its role in maintaining α-cell identity has yet to be explored. To study this later role of <i>Arx</i>, we have generated mice in which the <i>Arx</i> gene has been ablated specifically in glucagon-producing α-cells. Lineage-tracing studies and immunostaining analysis for endocrine hormones demonstrate that ablation of <i>Arx</i> in neonatal α-cells results in an α-to-β-like conversion through an intermediate bihormonal state. Furthermore, these <i>Arx</i>-deficient converted cells express β-cell markers including <i>Pdx1, MafA,</i> and <i>Glut2</i>. Surprisingly, short-term ablation of <i>Arx</i> in adult mice does not result in a similar α-to-β-like conversion. Taken together, these findings reveal a potential temporal requirement for <i>Arx</i> in maintaining α-cell identity.</p></div

Arx is specifically ablated in YFP⁺ α-cells of GKO;Rosa-YFP mice.

<p>P5 pancreatic sections were stained for glucagon (blue), Arx (red), and YFP (green). (<b>A</b>): Arx is expressed in all glucagon⁺ cells in control;Rosa-YFP pancreata. A subset of glucagon⁺Arx⁺ cells is YFP⁺. (<b>B</b>): In GKO;Rosa-YFP animals, there is a subset of glucagon⁺ cells that express YFP. These YFP⁺ cells have lost Arx expression. Scale bar represents 25 µm. (<b>C</b>): Quantitative analysis of Arx and YFP expressing cells within glucagon⁺ population in P5 animals. Over 500 total glucagon⁺ cells were counted with three mice per group used. Error bars represent standard error of the mean with <i>p-value</i> indicated. N.S: not significant. (<b>D</b>): Quantitative PCR analysis for <i>Arx</i> mRNA in total pancreata at P5 and islets from P21 control and GKO animals. Control mRNA level was set at one fold ± standard error of the mean. Male and female control and GKO animals (n≥3) were sex-matched for all analyses.</p