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1.  ATP-induced cellular stress and mitochondrial toxicity in cells expressing purinergic P2X7 receptor 
Under pathological conditions, the purinergic P2X7 receptor is activated by elevated concentrations of extracellular ATP. Thereby, the receptor forms a slowly dilating pore, allowing cations and, upon prolonged stimulation, large molecules to enter the cell. This process has a strong impact on cell signaling, metabolism, and viability. This study aimed to establish a link between gradual P2X7 activation and pharmacological endpoints including oxidative stress, hydrogen peroxide generation, and cytotoxicity. Mechanisms of cellular stress and cytotoxicity were studied in P2X7-transfected HEK293 cells. We performed real-time monitoring of metabolic and respiratory activity of cells expressing the P2X7-receptor protein using a cytosensor system. Agonistic effects were monitored using exogenously applied ATP or the stable analogue BzATP. Oxidative stress induced by ATP or BzATP in target cells was monitored by hydrogen peroxide release in human mononuclear blood cells. P2X7-receptor activation was studied by patch-clamp experiments using a primary mouse microglia cell line. Stimulation of the P2X7 receptor leads to ion influx, metabolic activation of target cells, and ultimately cytotoxicity. Conversion of the P2X7 receptor from a small cation channel to a large pore occurring under prolonged stimulation can be monitored in real time covering a time frame of milliseconds to hours. Selectivity of the effects can be demonstrated using the selective P2X7-receptor antagonist AZD9056. Our findings established a direct link between P2X7-receptor activation by extracellular ATP or BzATP and cellular events culminating in cytotoxicity. Mechanisms of toxicity include metabolic and oxidative stress, increase in intracellular calcium concentration and disturbance of mitochondrial membrane potential. Mitochondrial toxicity is suggested to be a key event leading to cell death.
PMCID: PMC4448979  PMID: 26038699
Cytosensor; extracellular adenosine triphosphate (ATP); metabolic stress; mitochondrial toxicity; P2X7; purinergic receptor
2.  ESPIRiT — An Eigenvalue Approach to Autocalibrating Parallel MRI: Where SENSE meets GRAPPA 
Parallel imaging allows the reconstruction of images from undersampled multi-coil data. The two main approaches are: SENSE, which explicitly uses coil sensitivities, and GRAPPA, which makes use of learned correlations in k-space. The purpose of this work is to clarify their relationship and to develop and evaluate an improved algorithm
Theory and Methods
A theoretical analysis shows: 1. The correlations in k-space are encoded in the null space of a calibration matrix. 2. Both approaches restrict the solution to a subspace spanned by the sensitivities. 3. The sensitivities appear as the main eigenvector of a reconstruction operator computed from the null space. The basic assumptions and the quality of the sensitivity maps are evaluated in experimental examples. The appearance of additional eigenvectors motivates an extended SENSE reconstruction with multiple maps, which is compared to existing methods
The existence of a null space and the high quality of the extracted sensitivities are confirmed. The extended reconstruction combines all advantages of SENSE with robustness to certain errors similar to GRAPPA.
In this paper the gap between both approaches is finally bridged. A new autocalibration technique combines the benefits of both.
PMCID: PMC4142121  PMID: 23649942
Autocalibration; Parallel Imaging; GRAPPA; SENSE; Compressed Sensing
3.  Detecting Radiation-Induced Injury Using Rapid 3D Variogram Analysis of CT Images of Rat Lungs 
Academic radiology  2013;20(10):1264-1271.
Rationale and Objectives
To investigate the ability of variogram analysis of octree-decomposed CT images and volume change maps to detect radiation-induced damage in rat lungs.
Materials and Methods
The lungs of female Sprague-Dawley rats were exposed to one of five absorbed doses (0, 6, 9, 12, or 15 Gy) of gamma radiation from a Co-60 source. At 6 months post-exposure, pulmonary function tests were performed and 4DCT images were acquired using a respiratory-gated microCT scanner. Volume change maps were then calculated from the 4DCT images. Octree decomposition was performed on CT images and volume change maps, and variogram analysis was applied to the decomposed images. Correlations of measured parameters with dose were evaluated.
The effects of irradiation were not detectable from measured parameters, indicating only mild lung damage. Additionally, there were no significant correlations of pulmonary function results or CT densitometry with radiation dose. However, the variogram analysis did detect a significant correlation with dose in both the CT images (r=−0.57, p=0.003) and the volume change maps (r=−0.53, p=0.008).
This is the first study to utilize variogram analysis of lung images to assess pulmonary damage in a model of radiation injury. Results show that this approach is more sensitive to detecting radiation damage than conventional measures such as pulmonary function tests or CT densitometry.
PMCID: PMC3928809  PMID: 24029058
octree; variogram; lung; irradiation; CT imaging
4.  Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy 
Nature  2008;455(7217):1205-1209.
Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27Kip1. Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.
PMCID: PMC4084721  PMID: 18806775
5.  Interaction between DMRT1 function and genetic background modulates signaling and pluripotency to control tumor susceptibility in the fetal germ line 
Developmental biology  2013;377(1):67-78.
Dmrt1(doublesex and mab-3 related transcription factor 1) is a regulator of testis development in vertebrates that has been implicated in testicular germ cell tumors of mouse and human. In the fetal mouse testis Dmrt1 regulates germ cell pluripotency in a strain-dependent manner. Loss of Dmrt1 in 129Sv strain mice results in a >90% incidence of testicular teratomas, tumors consisting cells of multiple germ layers; by contrast, these tumors have never been observed in Dmrt1 mutants of C57BL/6J (B6) or mixed genetic backgrounds. To further investigate the interaction between Dmrt1 and genetic background we compared mRNA expression in wild type and Dmrt1 mutant fetal testes of 129Sv and B6 mice at embryonic day 15.5 (E15.5), prior to overt tumorigenesis. Loss of Dmrt1 caused misexpression of overlapping but distinct sets of mRNAs in the two strains. The mRNAs that were selectively affected included some that changed expression only in one strain or the other and some that changed in both strains but to a greater degree in one versus the other. In particular, loss of Dmrt1 in 129Sv testes caused a more severe failure to silence regulators of pluripotency than in B6 testes. A number of genes misregulated in 129Sv mutant testes also are misregulated in human testicular germ cell tumors (TGCTs), suggesting similar etiology between germ cell tumors in mouse and man. Expression profiling showed that DMRT1 also regulates pluripotency genes in the fetal ovary, although Dmrt1 mutant females do not develop teratomas. Pathway analysis indicated disruption of several signaling pathways in Dmrt1 mutant fetal testes, including Nodal, Notch, and GDNF. We used a Nanos3-cre knock-in allele to perform conditional gene targeting, testing the GDNF coreceptors Gfra1 and Ret for effects on teratoma susceptibility. Conditional deletion of Gfra1 but not Ret in fetal germ cells of animals outcrossed to 129Sv caused a modest but significant elevation in tumor incidence. Despite some variability in genetic background in these crosses, this result is consistent with previous genetic mapping of teratoma susceptibility loci to the region containing Gfra1. Using Nanos3-cre we also uncovered a strong genetic interaction between Dmrt1 and Nanos3, suggesting parallel functions for these two genes in fetal germ cells. Finally, we used chromatin immunoprecipitation (ChIP-seq) analysis to identify a number of potentially direct DMRT1 targets. This analysis suggested that DMRT1 controls pluripotency via transcriptional repression of Esrrb, Nr5a2/Lrh1, and Sox2. Given the strong evidence for involvement of DMRT1 in human TGCT, the downstream genes and pathways identified in this study provide potentially useful candidates for roles in the human disease.
PMCID: PMC3630265  PMID: 23473982
Dmrt1; Nanos3; teratoma; TGCT; pluripotency; germ cell
6.  The Importance of Dosimetry Standardization in Radiobiology 
Radiation dose is central to much of radiobiological research. Precision and accuracy of dose measurements and reporting of the measurement details should be sufficient to allow the work to be interpreted and repeated and to allow valid comparisons to be made, both in the same laboratory and by other laboratories. Despite this, a careful reading of published manuscripts suggests that measurement and reporting of radiation dosimetry and setup for radiobiology research is frequently inadequate, thus undermining the reliability and reproducibility of the findings. To address these problems and propose a course of action, the National Cancer Institute (NCI), the National Institute of Allergy and Infectious Diseases (NIAID), and the National Institute of Standards and Technology (NIST) brought together representatives of the radiobiology and radiation physics communities in a workshop in September, 2011. The workshop participants arrived at a number of specific recommendations as enumerated in this paper and they expressed the desirability of creating dosimetry standard operating procedures (SOPs) for cell culture and for small and large animal experiments. It was also felt that these SOPs would be most useful if they are made widely available through mechanism(s) such as the web, where they can provide guidance to both radiobiologists and radiation physicists, be cited in publications, and be updated as the field and needs evolve. Other broad areas covered were the need for continuing education through tutorials at national conferences, and for journals to establish standards for reporting dosimetry. This workshop did not address issues of dosimetry for studies involving radiation focused at the sub-cellular level, internally-administered radionuclides, biodosimetry based on biological markers of radiation exposure, or dose reconstruction for epidemiological studies.
PMCID: PMC4487307  PMID: 26401441
dosimetry; dosimetry protocols; dosimetry standards; radiobiology; radiobiology protocols; radiobiology standards
7.  MLL Becomes Functional through Intra-Molecular Interaction Not by Proteolytic Processing 
PLoS ONE  2013;8(9):e73649.
The mixed lineage leukemia (MLL) protein is an epigenetic transcriptional regulator that controls proliferative expansion of immature hematopoietic progenitors, whose aberrant activation triggers leukemogenesis. A mature MLL protein is produced by formation of an intra-molecular complex and proteolytic cleavage. However the biological significance of these two post-transcriptional events remains unclear. To address their in vivo roles, mouse mutant alleles were created that exclusively express either a variant protein incapable of intra-molecular interaction (designated de) or an uncleavable mutant protein (designated uc). The de homozygous mice died during midgestation and manifested devastating failure in embryonic development and reduced numbers of hematopoietic progenitors, whereas uc homozygous mice displayed no apparent defects. Expression of MLL target genes was severely impaired in de homozygous fibroblasts but unaffected in uc homozygous fibroblasts. These results unequivocally demonstrate that intra-molecular complex formation is a crucial maturation step whereas proteolytic cleavage is dispensable for MLL-dependent gene activation and proliferation in vivo.
PMCID: PMC3769346  PMID: 24040009
8.  Fast ℓ1-SPIRiT Compressed Sensing Parallel Imaging MRI: Scalable Parallel Implementation and Clinically Feasible Runtime 
IEEE transactions on medical imaging  2012;31(6):1250-1262.
We present ℓ1-SPIRiT, a simple algorithm for auto calibrating parallel imaging (acPI) and compressed sensing (CS) that permits an efficient implementation with clinically-feasible runtimes. We propose a CS objective function that minimizes cross-channel joint sparsity in the Wavelet domain. Our reconstruction minimizes this objective via iterative soft-thresholding, and integrates naturally with iterative Self-Consistent Parallel Imaging (SPIRiT). Like many iterative MRI reconstructions, ℓ1-SPIRiT’s image quality comes at a high computational cost. Excessively long runtimes are a barrier to the clinical use of any reconstruction approach, and thus we discuss our approach to efficiently parallelizing ℓ1-SPIRiT and to achieving clinically-feasible runtimes. We present parallelizations of ℓ1-SPIRiT for both multi-GPU systems and multi-core CPUs, and discuss the software optimization and parallelization decisions made in our implementation. The performance of these alternatives depends on the processor architecture, the size of the image matrix, and the number of parallel imaging channels. Fundamentally, achieving fast runtime requires the correct trade-off between cache usage and parallelization overheads. We demonstrate image quality via a case from our clinical experimentation, using a custom 3DFT Spoiled Gradient Echo (SPGR) sequence with up to 8× acceleration via poisson-disc undersampling in the two phase-encoded directions.
PMCID: PMC3522122  PMID: 22345529
Autocalibrating Parallel Imaging; SPIRiT; Compressed Sensing; GPGPU; Parallel Computing
9.  Rapid pediatric cardiac assessment of flow and ventricular volume with compressed sensing parallel imaging volumetric cine phase-contrast MRI 
AJR. American journal of roentgenology  2012;198(3):W250-W259.
Quantification of cardiac flow and ventricular volumes comprise essential goals of many congenital heart MRI examinations, often requiring acquisition of multiple two-dimensional phase contrast (2DPC) and bright blood cine SSFP planes. Scan prescription however, is lengthy and highly reliant on an imager well-versed in structural heart disease. Though also lengthy, 3D time-resolved phase-contrast (4DPC) MRI yields global flow patterns and is simpler to prescribe. We therefore sought to accelerate 4DPC and determine whether equivalent flow and volume measurements could be extracted.
Materials and Methods
4DPC was modified for higher acceleration with compressed-sensing. Custom software was developed to process 4DPC images. With IRB-approval and HIPAA-compliance, we studied 29 patients referred for congenital cardiac MRI, who underwent a routine clinical protocol including cine short-axis stack SSFP and 2DPC, followed by contrast-enhanced 4DPC. To compare quantitative measurements, Bland-Altman analysis, paired t-tests, and F-tests were used.
Ventricular end-diastolic, end-systolic and stroke volumes obtained from 4DPC and SSFP were well-correlated (ρ=0.91–0.95, r2=0.83–0.90), with no statistically significant difference. Ejection fractions were well-correlated in a subpopulation that underwent higher-resolution compressed-sensing 4DPC (ρ=0.88, r2=0.77). 4DPC and 2DPC flow rates were also well-correlated (ρ=0.90, r2=0.82). Excluding ventricles with valvular insufficiency, cardiac outputs derived from outlet valve flow and stroke volumes were more consistent by 4DPC than by 2DPC and SSFP.
Combined parallel imaging and compressed sensing can be applied to 4DPC. With custom software, flow and ventricular volumes may be extracted with comparable accuracy to SSFP and 2DPC. Further, cardiac outputs were more consistent by 4DPC.
PMCID: PMC3515670  PMID: 22358022
10.  Enrichment-based DNA methylation analysis using next-generation sequencing: sample exclusion, estimating changes in global methylation, and the contribution of replicate lanes 
BMC Genomics  2012;13(Suppl 8):S6.
DNA methylation is an important epigenetic mark and dysregulation of DNA methylation is associated with many diseases including cancer. Advances in next-generation sequencing now allow unbiased methylome profiling of entire patient cohorts, greatly facilitating biomarker discovery and presenting new opportunities to understand the biological mechanisms by which changes in methylation contribute to disease. Enrichment-based sequencing assays such as MethylCap-seq are a cost effective solution for genome-wide determination of methylation status, but the technical reliability of methylation reconstruction from raw sequencing data has not been well characterized.
We analyze three MethylCap-seq data sets and perform two different analyses to assess data quality. First, we investigate how data quality is affected by excluding samples that do not meet quality control cutoff requirements. Second, we consider the effect of additional reads on enrichment score, saturation, and coverage. Lastly, we verify a method for the determination of the global amount of methylation from MethylCap-seq data by comparing to a spiked-in control DNA of known methylation status.
We show that rejection of samples based on our quality control parameters leads to a significant improvement of methylation calling. Additional reads beyond ~13 million unique aligned reads improved coverage, modestly improved saturation, and did not impact enrichment score. Lastly, we find that a global methylation indicator calculated from MethylCap-seq data correlates well with the global methylation level of a sample as obtained from a spike-in DNA of known methylation level.
We show that with appropriate quality control MethylCap-seq is a reliable tool, suitable for cohorts of hundreds of patients, that provides reproducible methylation information on a feature by feature basis as well as information about the global level of methylation.
PMCID: PMC3535705  PMID: 23281662
11.  Methods for high-throughput MethylCap-Seq data analysis 
BMC Genomics  2012;13(Suppl 6):S14.
Advances in whole genome profiling have revolutionized the cancer research field, but at the same time have raised new bioinformatics challenges. For next generation sequencing (NGS), these include data storage, computational costs, sequence processing and alignment, delineating appropriate statistical measures, and data visualization. Currently there is a lack of workflows for efficient analysis of large, MethylCap-seq datasets containing multiple sample groups.
The NGS application MethylCap-seq involves the in vitro capture of methylated DNA and subsequent analysis of enriched fragments by massively parallel sequencing. The workflow we describe performs MethylCap-seq experimental Quality Control (QC), sequence file processing and alignment, differential methylation analysis of multiple biological groups, hierarchical clustering, assessment of genome-wide methylation patterns, and preparation of files for data visualization.
Here, we present a scalable, flexible workflow for MethylCap-seq QC, secondary data analysis, tertiary analysis of multiple experimental groups, and data visualization. We demonstrate the experimental QC procedure with results from a large ovarian cancer study dataset and propose parameters which can identify problematic experiments. Promoter methylation profiling and hierarchical clustering analyses are demonstrated for four groups of acute myeloid leukemia (AML) patients. We propose a Global Methylation Indicator (GMI) function to assess genome-wide changes in methylation patterns between experimental groups. We also show how the workflow facilitates data visualization in a web browser with the application Anno-J.
This workflow and its suite of features will assist biologists in conducting methylation profiling projects and facilitate meaningful biological interpretation.
PMCID: PMC3481483  PMID: 23134780
12.  DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary 
Developmental biology  2011;356(1):63-70.
Dmrt1 belongs to the DM domain gene family of conserved sexual regulators. In the mouse Dmrt1 is expressed in the genital ridge (the gonadal primordium) in both sexes and then becomes testis-specific shortly after sex determination. The essential role of DMRT1 in testicular differentiation is well established, and includes transcriptional repression of the meiotic inducer Stra8. However Dmrt1 mutant females are fertile and the role of Dmrt1 in the ovary has not been studied. Here we show in the mouse that most Dmrt1 mutant germ cells in the fetal ovary have greatly reduced expression of STRA8, and fail to properly localize SYCP3 and γH2AX during meiotic prophase. Lack of DMRT1 in the fetal ovary results in the formation of many fewer primordial follicles in the juvenile ovary, although these are sufficient for fertility. Genome-wide chromatin immunoprecipitiation (ChIP-chip) and quantitative ChIP (qChIP) combined with mRNA expression profiling suggests that transcriptional activation of Stra8 in fetal germ cells is the main function of DMRT1 in females, and that this regulation likely is direct. Thus DMRT1 controls Stra8 sex-specifically, activating it in the fetal ovary and repressing it in the adult testis.
PMCID: PMC3131262  PMID: 21621532
Dmrt1; DM domain; meiosis; Stra8; ovary
13.  A Scalable, Flexible Workflow for MethylCap-Seq Data Analysis 
Advances in whole genome profiling have revolutionized the cancer research field, but at the same time have raised new bioinformatics challenges. For next generation sequencing (NGS), these include data storage, computational costs, sequence processing and alignment, delineating appropriate statistical measures, and data visualization. The NGS application MethylCap-seq involves the in vitro capture of methylated DNA and subsequent analysis of enriched fragments by massively parallel sequencing. Here, we present a scalable, flexible workflow for MethylCap-seq Quality Control, secondary data analysis, tertiary analysis of multiple experimental groups, and data visualization. This workflow and its suite of features will assist biologists in conducting methylation profiling projects and facilitate meaningful biological interpretation.
PMCID: PMC3320741  PMID: 22484542
next generation sequencing; DNA methylation; epigenetics; cancer; data analysis; data visualization
14.  In-vivo delivery of FTY720 prevents radiation-induced ovarian failure and infertility in adult female non-human primates 
Fertility and sterility  2011;95(4):1440-1445.e7.
To determine if sphingosine-1-phosphate (S1P), or the S1P mimetic FTY720 shields ovaries of adult female rhesus monkeys from damage caused by 15 Gy of targeted radiotherapy, allowing for retention of long-term fertility; and, to evaluate if S1P protects human ovarian tissue xenografted into mice from radiation-induced damage.
Research animal study.
Research laboratory and teaching hospital.
Adult female rhesus macaques (8–14 years of age).
Two women (24 and 27 years of age) undergoing gynecologic surgery for benign reasons, after informed consent and approval.
Main Outcome Measure(s)
Ovarian histology, ovarian reserve measurements and fertility in mating trials.
Rapid ovarian failure was induced in female macaques by ovarian application of 15 Gy of radiation. Females given S1P or FTY720 by direct intraovarian cannulation for 1 week prior to ovarian irradiation rapidly resumed menstrual cycles due to maintenance of follicles, with greater beneficial effects achieved using FTY720. Monkeys given the S1P mimetic prior to ovarian irradiation also became pregnant in mating trials. Offspring conceived and delivered by radioprotected females developed normally, and showed no evidence of genomic instability as measured by micronucleus frequency in reticulocytes. Adult human ovarian cortical tissue xenografted into mice also exhibited a reduction in radiation-induced primordial oocyte depletion when pre-exposed to S1P.
S1P and its analogs hold clinical promise as therapeutic agents to preserve both ovarian function and fertility in female cancer patients exposed to cytotoxic treatments.
PMCID: PMC3063448  PMID: 21316047
Fertility preservation; cancer; ovary; oocyte; monkey; human; phingosine-1-phosphate; FTY720
15.  DMRT1 prevents female reprogramming in the postnatal mammalian testis 
Nature  2011;476(7358):101-104.
Sex in mammals is determined in the foetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells1. This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the foetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving Wnt/β-catenin signaling (Supplemental Fig. 1)2. In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogramme granulosa cells into Sertoli cells2. Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor3 in mouse Sertoli cells, even in adults, activates Foxl2 and reprogrammes Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced, and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the foetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates4,5 and Dmrt1-related sexual regulators are conserved throughout metazoans3. Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the etiology of human syndromes linked to DMRT1, including disorders of sexual differentiation6 and testicular cancer7.
PMCID: PMC3150961  PMID: 21775990
16.  The mammalian Doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells 
Developmental cell  2010;19(4):612-624.
The switch from mitosis to meiosis is a unique feature of germ cell development. In mammals, meiotic initiation requires retinoic acid (RA), which activates meiotic inducers including Stra8, but how the switch to meiosis is controlled in male germ cells (spermatogonia) remains poorly understood. Here we examine the role of the Doublesex-related transcription factor DMRT1 in adult spermatogenesis using conditional gene targeting in the mouse. Loss of Dmrt1 causes spermatogonia to precociously exit the spermatogonial program and enter meiosis. Dmrt1 therefore determines whether male germ cells undergo mitosis and spermatogonial differentiation or meiosis. Loss of Dmrt1 in spermatogonia also disrupts cyclical gene expression in Sertoli cells. DMRT1 acts in spermatogonia to restrict RA responsiveness, directly repress Stra8 transcription, and activate transcription of the spermatogonial differentiation factor Sohlh1, thereby preventing meiosis and promoting spermatogonial development. By coordinating spermatogonial development and mitotic amplification with meiosis, DMRT1 allows abundant, continuous production of sperm.
PMCID: PMC2996490  PMID: 20951351
17.  EGL-5/ABD-B plays an instructive role in male cell fate determination in the C. elegans somatic gonad 
Developmental biology  2010;344(2):827-835.
Hox genes of the Abdominal-B (Abd-B) class regulate gonadal development in diverse metazoans. Here we have investigated the role of the Abd-B homolog egl-5 in C. elegans gonadal development. Previous work showed that egl-5 is required male-specifically in the gonad and that mutant gonads are highly dysgenic and possibly feminized. We have used sex-specific gonadal reporter genes to confirm that the gonads of egl-5 males are extensively feminized. Sex-specific expression of egl-5 requires the global sex determination gene tra-1 and the gonadal masculinizing gene fkh-6, but mutagenesis of a short male gonadal enhancer element in egl-5 suggested that this regulation is indirect. Ectopic expression of EGL-5 in hermaphrodites is sufficient to induce male gonadal gene expression, indicating that EGL-5 plays an instructive role in male gonadal fate determination. EGL-5 acts in parallel with a Wnt/β -catenin pathway to regulate male gonadal fates and can physically interact with the Wnt pathway transcription factor POP-1 and modulate activity of a POP-1 dependent reporter gene. We propose that EGL-5 imparts sex-specific function on POP-1 by recruiting it to male-specific gonadal target genes.
PMCID: PMC2921588  PMID: 20553900
gonad; Wnt; Hox; sexual differentiation
18.  Inpatient hospital complications and lengths of stay: a short report 
BMC Research Notes  2011;4:135.
Increasingly, efforts are being made to link health care outcomes with more efficient use of resources. The current difficult economic times and health care reform efforts provide incentives for specific efforts in this area.
This study defined relationships between inpatient complications for urinary tract infection and pneumonia and hospital lengths of stay in three general hospitals in the metropolitan area of Syracuse, New York. It employed the Potentially Preventable Complications (PPC) software developed by 3M™ Health Information Services to identify lengths of stay for patients with and without urinary tract infection and pneumonia. The patient populations included individuals assigned to the same All Patients Refined Diagnosis Related Groups and severity of illness. The comparisons involved two nine month periods in 2008 and 2009.
The study demonstrated that patients who experienced the complications had substantially longer inpatient hospital stays than those who did not. Patients with a PPC of urinary tract infection stayed a mean of 8.9 - 11.9 days or 161 - 216 percent longer than those who did not for the two time periods. This increased stay produced 2,020 - 2,427 additional patient days.
The study demonstrated that patients who experienced the complications had substantially longer inpatient hospital stays than those who did not. Patients with a PPC of pneumonia stayed a mean of 13.0 - 16.3 days or 232 - 281 percent longer than those who did not for the two time periods. This increased stay produced 2,626 - 3,456 additional patient days. Similar differences were generated for median lengths of stay.
The differences in hospital stays for patients in the same APR DRGs and severity of illness with and without urinary tract infection and pneumonia in the Syracuse hospitals were substantial. The additional utilization for these complications was valued at between $2,000,000 - $3,000,000 for a three month period. These differences in the use of hospital resources have important implications for reduction of health care costs among providers and payors of care.
PMCID: PMC3098808  PMID: 21545741
19.  Fos-Tau-LacZ Mice Reveal Sex Differences in Brainstem c-fos Activation in Response to Mild Carbon Dioxide Exposure 
Brain research  2009;1311C:51-63.
There are sex differences in the neurochemistry of brainstem nuclei that participate in the control of breathing, as well as sex differences in respiratory responses to hypoxia. Central chemoreception refers to the detection within the brain of minute changes in carbon dioxide (CO2) levels and the subsequent modulation of breathing. Putative central chemoreceptor sites are widespread and include cells located near the ventral surface of the brainstem in the retrotrapezoid nucleus (RTN), in the medullary midline raphe nuclei, and, more dorsally in the medulla, in the nucleus of the solitary tract and in the locus coeruleus at the pontomedullary junction, as well as in the fastigial nucleus of the cerebellum. In this study, we ask if the cells that respond to CO2 differ between the sexes. We used a transgenic mouse with a c-fos promoter driven tau-lacZ reporter construct (FTL) to map the locations of cells in the mouse brainstem and cerebellum that responded to exposure of mice of both sexes to 5% CO2 or room air (control). X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside) histochemical staining to detect the beta-galactosidase enzyme produced staining in the brains of mice of both sexes in all of the previously identified putative chemoreceptor sites, with the exception of the fastigial nucleus. Notably, the male RTN region contained significantly more x-gal labeled cells than the female RTN region. In addition to new observations regarding potential sex differences in the retrotrapezoid region, we found the FTL mouse to be a useful tool for identifying cells that respond to the exposure of the whole animal to relatively low concentrations of CO2.
PMCID: PMC2812580  PMID: 19932690
ventilation; hypercapnia; chemosensation; c-fos; retrotrapezoid nucleus; sudden infant death syndrome
20.  Essential Role for Pbx1 in Corneal Morphogenesis 
A conditional knockout mouse model was used to specifically inactivate the Pbx1 TALE homeodomain protein in corneal epithelial cells, resulting in an alteration of corneal cell fate, high epithelial cell turnover, and marked disruption of the corneal basement membrane, with deficient epithelial barrier function demonstrating an essential role for Pbx1 in morphogenesis and maintenance of self-renewing adult tissues of the cornea.
The Pbx TALE (three-amino-acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in the corneal epithelium of mice.
Pbx1f/f mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently, the eyes of these mice were dissected and prepared for histologic or molecular analysis.
Tissue-specific deletion of Pbx1 in the corneal epithelium of mice resulted in corneal dystrophy and clouding that was apparent in newborns and progressively worsened with age. Thickening of the cornea epithelium was accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover was associated with perturbed expression of developmental regulators and aberrant differentiation, suggesting an important function for Pbx1 in determining corneal identity.
These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.
PMCID: PMC2821029  PMID: 19797217
21.  Pbx1 is essential for corneal morphogenesis 
The Pbx TALE (three amino acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in corneal epithelium of mice.
Pbx1f/f mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently the eyes from these mice were dissected and prepared for histological or molecular analysis.
Tissue-specific deletion of Pbx1 in the corneal epithelium of mice results in corneal dystrophy and clouding that is apparent in newborns and progressively worsens with age. Thickening of the cornea epithelium is accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover is associated with perturbed expression of developmental regulators and aberrant differentiation suggesting an important function for Pbx1 in determining corneal identity.
These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.
PMCID: PMC2821029  PMID: 19797217
22.  Application of High-Resolution 1H MAS NMR Spectroscopy to the Analysis of Intact Bones from Mice Exposed to Gamma Radiation 
Radiation research  2009;172(5):607-616.
Herein we demonstrate that high-resolution magic angle spinning (MAS) 1H NMR can be used to profile the pathology of bone marrow rapidly and with minimal sample preparation. The spectral resolution obtained allows several metabolites to be analyzed quantitatively. The level of NMR-detectable metabolites in the epiphysis + metaphysis sections of mouse femur were significantly higher than that observed in the diaphysis of the same femur. The major metabolite damage to bone marrow resulting from either 3.0 Gy or 7.8 Gy of whole-body γ radiation 4 days after exposure were (1) decreased total choline content, (2) increased fatty acids in bone marrow, and (3) decreased creatine content. These results suggest that the membrane choline phospholipid metabolism (MCPM) pathway and the fatty acid biosynthesis pathway were altered as a result of radiation exposure. We also found that the metabolic damage induced by radiation in the epiphysis + metaphysis sections of mouse femur was higher than that of the diaphysis of the same femur. Traditional histopathology analysis was also carried out to correlate radiation damage with changes in metabolites. Importantly, the molecular information gleaned from high-resolution MAS 1H NMR complements the pathology data.
PMCID: PMC2796857  PMID: 19883229
23.  Pbx1 regulates self-renewal of long-term hematopoietic stem cells by maintaining their quiescence 
Cell stem cell  2008;2(5):484-496.
Self-renewal is a defining characteristic of stem cells, however the molecular pathways underlying its regulation are poorly understood. Here we demonstrate that conditional inactivation of the Pbx1 proto-oncogene in the hematopoietic compartment results in a progressive loss of long-term hematopoietic stem cells (LT-HSCs) that is associated with concomitant reduction in their quiescence, leading to a defect in the maintenance of self-renewal as assessed by serial transplantation. Transcriptional profiling revealed that multiple stem cell maintenance factors are perturbed in Pbx1-deficient LT-HSCs, which prematurely express a large subset of genes, including cell cycle regulators, normally expressed in non-self-renewing multipotent progenitors. A significant proportion of Pbx1-dependent genes are associated with the Tgf-b pathway, which serves a major role in maintaining HSC quiescence. Prospectively isolated, Pbx1-deficient LT-HSCs display altered transcriptional responses to Tgf-b stimulation in vitro, suggesting a possible mechanism through which Pbx1 maintenance of stem cell quiescence may in part be achieved.
PMCID: PMC2416441  PMID: 18462698
24.  Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA 
The DM domain is a zinc finger-like DNA binding motif first identified in the sexual regulatory proteins Doublesex (DSX) and MAB-3, and is widely conserved among metazoans. DM domain proteins regulate sexual differentiation in at least three phyla and also control other aspects of development, including vertebrate segmentation. Most DM domain proteins share little similarity outside the DM domain. DSX and MAB-3 bind partially overlapping DNA sequences, and DSX has been shown to interact with DNA via the minor groove without inducing DNA bending. DSX and MAB-3 exhibit unusually high DNA sequence specificity relative to other minor groove binding proteins. No detailed analysis of DNA binding by the seven vertebrate DM domain proteins, DMRT1-DMRT7 has been reported, and thus it is unknown whether they recognize similar or diverse DNA sequences.
We used a random oligonucleotide in vitro selection method to determine DNA binding sites for six of the seven proteins. These proteins selected sites resembling that of DSX despite differences in the sequence of the DM domain recognition helix, but they varied in binding efficiency and in preferences for particular nucleotides, and some behaved anomalously in gel mobility shift assays. DMRT1 protein from mouse testis extracts binds the sequence we determined, and the DMRT proteins can bind their in vitro-defined sites in transfected cells. We also find that some DMRT proteins can bind DNA as heterodimers.
Our results suggest that target gene specificity of the DMRT proteins does not derive exclusively from major differences in DNA binding specificity. Instead target specificity may come from more subtle differences in DNA binding preference between different homodimers, together with differences in binding specificity between homodimers versus heterodimers.
PMCID: PMC1931443  PMID: 17605809
25.  A method for detecting functional activity related expression in gross brain regions, specific brain nuclei and individual neuronal cell bodies and their projections 
We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of β-galactosidase (βgal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of βgal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain.
PMCID: PMC1821346  PMID: 17364022
Mice, Transgenic; Brain Chemistry; beta-Galactosidase

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