Publications by authors named "James A Richardson"

205 Publications

A bedside rescue method for retrieving retained guidewires: The 'Suck Out' technique.

J Vasc Access 2020 Jul 25:1129729820943457. Epub 2020 Jul 25.

Critical Care Department, The Queen Elizabeth Hospital, King's Lynn, UK.

Background: Central venous catheter guidewire retention is classed as a 'never event' in the United Kingdom, with the potential for significant patient harm. If the retained guidewire remains within the central venous catheter lumen, bedside techniques may facilitate guidewire retrieval. However, these techniques may be ineffective if the guidewire has already passed below skin level. We investigated a novel 'suck out' technique for bedside guidewire retrieval and compared this against traditional retrieval methods.

Methods: Simulation 1: in a benchtop model, seven different central venous catheters had their corresponding guidewire placed in the last 2 cm of the catheter tip which was immersed horizontally in fluid. A 50-mL syringe was attached to the distal lumen central venous catheter hub and suction applied for 5 s, and the distance of guidewire retraction was recorded. Simulation 2: a central venous catheter guidewire was intentionally retained within the catheter at either 5 cm above or below skin level in a pigskin model. Simple catheter withdrawal, catheter clamping withdrawal and the 'suck out' method were compared for efficacy using Fisher's exact test.

Results: Simulation 1: retained guidewires were retracted by 13 cm on average. Simulation 2: when guidewires were retained 5 cm above skin level, all retrieval methods were 100% effective; however, when retained 5 cm below skin level, simple catheter withdrawal was ineffective, clamping and withdrawal was only 10% effective and the 'suck out' technique was 90% effective (p < 0.001).

Conclusion: The 'suck out' technique can effectively retract guidewires retained within central venous catheter lumens and demonstrates superiority over traditional methods of retained guidewire extraction in simulated models.
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http://dx.doi.org/10.1177/1129729820943457DOI Listing
July 2020

Lung cancer progression using fast switching multiple ion beam radiation and countermeasure prevention.

Life Sci Space Res (Amst) 2020 Feb 1;24:108-115. Epub 2019 Aug 1.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

Most of the research in understanding space radiation-induced cancer progression and risk assessment has been performed using mono-energetic single-ion beams. However, the space radiation environment consists of a wide variety of ion species with a various range of energies. Using the fast beam switching technology developed at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), ion species can be switched rapidly allowing investigators to use multiple ions with different energies to simulate more closely the radiation environment found in space. Here, we exposed a lung cancer susceptible mouse model (K-ras) to three sequential ion beams: Proton (H) (120 MeV/n) 20 cGy, Helium (He) (250 MeV/n) 5.0 cGy, and Silicon (Si) (300 MeV/n) 5.0 cGy with a dose rate of 0.5 cGy/min. Using three ion beams we performed whole body irradiation with a total dose of 30 cGy in two different orders: 3B-1 (H→He→Si) and 3B-2 (Si→He→H) and used 30 cGy H single-ion beam as a reference. In this study we show that whole-body irradiation with H→He→Si increases the incidence of premalignant lesions and systemic oxidative stress in mice 100 days post-irradiation more than (Si→He→H) and H only irradiation. Additionally, we observed an increase in adenomas with atypia and adenocarcinomas in H→He→Si irradiated mice but not in (Si→He→H) or H (30 cGy) only irradiated mice. When we used the H→He→Si irradiation sequence but skipped a day before exposing the mice to Si, we did not observe the increased incidence of cancer initiation and progression. We also found that a non-toxic anti-inflammatory, anti-oxidative radioprotector (CDDO-EA) reduced H→He→Si induced oxidative stress and cancer initiation almost back to baseline. Thus, exposure to H→He→Si elicits significant changes in lung cancer initiation that can be mitigated using CDDO-EA.
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http://dx.doi.org/10.1016/j.lssr.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6991460PMC
February 2020

The effect of flow on blood oxygen level dependent (R ) MRI of orthotopic lung tumors.

Magn Reson Med 2019 06 30;81(6):3787-3797. Epub 2019 Jan 30.

Department of Radiology, University of Texas Southwestern Medical Center, Dallas, Texas.

Purpose: Blood oxygen level dependent (BOLD) MRI based on measurements can provide insights into tumor vascular oxygenation. However, measurements are susceptible to blood flow, which may vary accompanying a hyperoxic gas challenge. We investigated flow sensitivity by comparing measurements with and without flow suppression (fs) in 2 orthotopic lung xenograft tumor models.

Methods: H460 (n = 20) and A549 (n = 20) human lung tumor xenografts were induced by surgical implantation of cancer cells in the right lung of nude rats. MRI was performed at 4.7T after tumors reached 5 to 8 mm in diameter. A multiecho gradient echo MRI sequence was acquired with and without spatial saturation bands on each side of the imaging plane to evaluate the effect of flow on . fs and non-fs MRI measurements were interleaved during an oxygen breathing challenge (from air to 100% O ). -weighted signal intensity changes (ΔSI(%)) and measurements were obtained for regions of interest and on a voxel-by-voxel basis and discrepancies quantified with Bland-Altman analysis.

Results: Flow suppression affected ΔSI(%) and measurements in each tumor model. Average discrepancy and limits of agreement from Bland-Altman analyses revealed greater flow-related bias in A549 than H460.

Conclusion: The effect of flow on , and hence BOLD, was tumor model dependent with measurements being more sensitive in well-perfused A549 tumors.
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http://dx.doi.org/10.1002/mrm.27661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527131PMC
June 2019

Proton radiation-induced cancer progression.

Life Sci Space Res (Amst) 2018 Nov 18;19:31-42. Epub 2018 Aug 18.

Department of Cell Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390, USA. Electronic address:

There are considerable health risks related to ionizing and proton radiation exposure. While there is a long history of health risks associated with ionizing (photon) radiation exposure, there is a limited understanding of the long-term health risks associated with proton radiation exposure. Since proton radiation is becoming more common in cancer therapy, the long-term biological effects of proton radiation remain less well characterized in terms of radiotherapy and well as for astronauts during deep space explorations. In this study, we compared the long-term side effects of proton radiation to equivalent doses of X-rays in the initiation and progression of premalignant lesions in a lung cancer susceptible mouse model (K-ras). We show proton irradiation causes more complex DNA damage that is not completely repaired resulting in increased oxidative stress in the lungs both acutely and persistently. We further observed K-ras mice irradiated with protons had an increased number and size of initiated and premalignant lesions and adenomas that were often infiltrated with inflammatory cells. Proton irradiated mice had a lower median survival and increased carcinoma incidence as compared to unirradiated controls and X-rays exposed mice. Our conclusion is that exposure to proton irradiation enhances the progression of premalignant lesions to invasive carcinomas through persistent DNA damage, chronic oxidative stress, and immunosuppression.
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http://dx.doi.org/10.1016/j.lssr.2018.08.002DOI Listing
November 2018

Modulation of Mutant -Driven Lung Tumorigenesis by Gain or Loss of PCDH7 Function.

Mol Cancer Res 2019 02 8;17(2):594-603. Epub 2018 Nov 8.

Department of Molecular Biology, UT Southwestern Medical Center, Dallas, Texas.

PROTOCADHERIN 7 (PCDH7), a transmembrane receptor and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinoma and is associated with poor clinical outcome. Although PCDH7 was recently shown to promote transformation and facilitate brain metastasis in lung and breast cancers, decreased PCDH7 expression has also been documented in colorectal, gastric, and invasive bladder cancers. These data suggest context-dependent functions for PCDH7 in distinct tumor types. Given that PCDH7 is a potentially targetable molecule on the surface of cancer cells, further investigation of its role in tumorigenesis is needed to evaluate the therapeutic potential of its inhibition. Here, we report the analysis of novel PCDH7 gain- and loss-of-function mouse models and provide compelling evidence that this cell-surface protein acts as a potent lung cancer driver. Employing a Cre-inducible transgenic allele, we demonstrated that enforced PCDH7 expression significantly accelerates -driven lung tumorigenesis and potentiates MAPK pathway activation. Furthermore, we performed somatic genome editing with CRISPR/Cas9 in ; (KP) mice to assess the consequences of PCDH7 loss of function. Inactivation of PCDH7 in KP mice significantly reduced lung tumor development, prolonged survival, and diminished phospho-activation of ERK1/2. Together, these findings establish a critical oncogenic function for PCDH7 and highlight the therapeutic potential of PCDH7 inhibition for lung cancer. Moreover, given recent reports of elevated or reduced PCDH7 in distinct tumor types, the new inducible transgenic model described here provides a robust experimental system for broadly elucidating the effects of PCDH7 overexpression . IMPLICATIONS: In this study, we establish a critical oncogenic function for PCDH7 using novel mouse models and CRISPR/Cas9 genome editing, and we validate the therapeutic potential of PCDH7 inhibition for lung cancer.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359939PMC
February 2019

Loss of partially phenocopies Perlman syndrome in mice and results in up-regulation of in nephron progenitor cells.

Genes Dev 2018 07 27;32(13-14):903-908. Epub 2018 Jun 27.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Loss of function of the DIS3L2 exoribonuclease is associated with Wilms tumor and the Perlman congenital overgrowth syndrome. LIN28, a Wilms tumor oncoprotein, triggers the DIS3L2-mediated degradation of the precursor of let-7, a microRNA that inhibits Wilms tumor development. These observations have led to speculation that DIS3L2-mediated tumor suppression is attributable to let-7 regulation. Here we examine new DIS3L2-deficient cell lines and mouse models, demonstrating that DIS3L2 loss has no effect on mature let-7 levels. Rather, analysis of -null nephron progenitor cells, a potential cell of origin of Wilms tumors, reveals up-regulation of , a growth-promoting gene strongly associated with Wilms tumorigenesis. These findings nominate a new potential mechanism underlying the pathology associated with DIS3L2 deficiency.
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http://dx.doi.org/10.1101/gad.315804.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075040PMC
July 2018

Remote acoustic sensing as a safety mechanism during exposure of metal implants to alternating magnetic fields.

PLoS One 2018 10;13(5):e0197380. Epub 2018 May 10.

Department of Radiology, UT Southwestern Medical Center, Dallas TX, United States of America.

Treatment of prosthetic joint infections often involves multiple surgeries and prolonged antibiotic administration, resulting in a significant burden to patients and the healthcare system. We are exploring a non-invasive method to eradicate biofilm on metal implants utilizing high-frequency alternating magnetic fields (AMF) which can achieve surface induction heating. Although proof-of-concept studies demonstrate the ability of AMF to eradicate biofilm in vitro, there is a legitimate safety concern related to the potential for thermal damage to surrounding tissues when considering heating implanted metal objects. The goal of this study was to explore the feasibility of detecting acoustic emissions associated with boiling at the interface between a metal implant and surrounding soft tissue as a wireless safety sensing mechanism. Acoustic emissions generated during in vitro and in vivo AMF exposures were captured with a hydrophone, and the relationship with surface temperature analyzed. The effect of AMF exposure power, surrounding media composition, implant location within the AMF transmitter, and implant geometry on acoustic detection during AMF therapy was also evaluated. Acoustic emissions were reliably identified in both tissue-mimicking phantom and mouse studies, and their onset coincided with the implant temperature reaching the boiling threshold. The viscosity of the surrounding medium did not impact the production of acoustic emissions; however, emissions were not present when the medium was oil due to the higher boiling point. Results of simulations and in vivo studies suggest that short-duration, high-power AMF exposures combined with acoustic sensing can be used to minimize the amount of thermal damage in surrounding tissues. These studies support the hypothesis that detection of boiling associated acoustic emissions at a metal/tissue interface could serve as a real-time, wireless safety indicator during AMF treatment of biofilm on metallic implants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197380PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944992PMC
December 2018

The G protein-coupled receptor Gpr161 regulates forelimb formation, limb patterning and skeletal morphogenesis in a primary cilium-dependent manner.

Development 2018 01 8;145(1). Epub 2018 Jan 8.

Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas, USA

The role of basal suppression of the sonic hedgehog (Shh) pathway and its interaction with Indian hedgehog (Ihh) signaling during limb/skeletal morphogenesis is not well understood. The orphan G protein-coupled receptor Gpr161 localizes to primary cilia and functions as a negative regulator of Shh signaling by promoting Gli transcriptional repressor versus activator formation. Here, we show that forelimb buds are not formed in knockout mouse embryos despite establishment of prospective limb fields. Limb-specific deletion of resulted in prematurely expanded Shh signaling and ectopic Shh-dependent patterning defects resulting in polysyndactyly. In addition, endochondral bone formation in forearms, including formation of both trabecular bone and bone collar was prevented. Endochondral bone formation defects resulted from accumulation of proliferating round/periarticular-like chondrocytes, lack of differentiation into columnar chondrocytes, and corresponding absence of Ihh signaling. deficiency in craniofacial mesenchyme also prevented intramembranous bone formation in calvarium. Defects in limb patterning, endochondral and intramembranous skeletal morphogenesis were suppressed in the absence of cilia. Overall, Gpr161 promotes forelimb formation, regulates limb patterning, prevents periarticular chondrocyte proliferation and drives osteoblastogenesis in intramembranous bones in a cilium-dependent manner.
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http://dx.doi.org/10.1242/dev.154054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825871PMC
January 2018

Local Hypothermia as a Radioprotector of the Rectal Wall During Prostate Stereotactic Body Radiation Therapy.

Int J Radiat Oncol Biol Phys 2017 05 11;98(1):75-82. Epub 2017 Jan 11.

Department of Radiation Oncology, UT Southwestern Medical Center, Dallas, Texas.

Purpose: To compare the single-fraction dose-related incidence of rectal obstruction and/or bleeding in normothermic and hypothermic rectums of a rat model.

Methods And Materials: A 1.9-cm length of rectum was irradiated with a single fraction in 57 Sprague-Dawley rats using a dedicated image-guided small animal irradiator and Monte Carlo-based treatment planning system. All rats had a rectal temperature control apparatus placed during irradiation and were stratified to achieve either a normothermic (37°C) or hypothermic (15°C) rectal wall temperature. Radiation was delivered to a 1-cm-diameter cylindrical volume about the cooling device and rectal wall. The radiation dose was escalated from 16 Gy up to 37 Gy to assess the dose response in each arm. The primary endpoint of this study was rectal obstruction and/or bleeding during a follow-up of 180 to 186 days. Histologic scoring was performed on all study rats.

Results: Probit analysis showed a dose associated with a 50% incidence of rectal obstruction of 24.6 Gy and 40.8 Gy for normothermic and hypothermic arms, respectively. The occurrence of obstruction and/or bleeding correlated with the posttreatment histologic score for normothermic rats; however, there was no difference in histologic score between normothermic and hypothermic rats at the highest dose levels evaluated.

Conclusions: A significant radioprotective effect was observed using local hypothermia during a single large dose of radiation for the functional endpoint of rectal obstruction and/or bleeding. A confirmatory study in a large animal model with anatomic and physiologic similarities to humans is suggested.
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http://dx.doi.org/10.1016/j.ijrobp.2017.01.017DOI Listing
May 2017

Bench and initial preclinical results of a novel 8 mm diameter double opposed helical biodegradable stent.

Catheter Cardiovasc Interv 2016 Nov 29;88(6):902-911. Epub 2016 Jul 29.

Division of Cardiology, Department of Pediatrics, UT Southwestern Medical Center, Dallas, Texas.

Background: Metallic endovascular stents are utilized off-label in congenital heart disease. Biodegradable stents (BDS) offer potential advantages in a growing child. We have previously reported double opposed helical (DH) BDS up to 6 mm diameter (DH-6). The objectives are to investigate the bench characteristics of larger 8 mm diameter BDS (DH-8) manufactured with increasing strut thicknesses and the inflammatory profile in a porcine model.

Methods: DH-8 were manufactured with strut thicknesses 0.10, 0.12, and 0.18 mm and mechanical testing performed. Stents were deployed into the infrarenal descending aorta (DAO) of nine minipigs. At insertion (nonsurvival = 2), 1 week (n = 2), 1 month (n = 2), and 9 months (n = 3) follow-up angiography, intravascular ultrasound and histopathology were performed.

Results: There was superior recoil and collapse pressure with increasing strut thickness, with 0.18 mm having 1.0% elastic recoil and collapse pressure 0.75 Atmospheres. There was good wall apposition at insertion with 5 BDS (4 DH-8 and 1 DH-6) but suboptimal in 4 as the minipigs infrarenal DAO were >8 mm (deployed at iliac bifurcation). Structural integrity was maintained in 8 BDS with 1 DH-8 collapsed at 9 months, secondary to strut damage at insertion. No thrombosis was seen. There was mild inflammation and neointimal proliferation at 1 week and 1 month, but a moderate inflammatory response at 9 months.

Conclusions: DH-8 with increased strut thickness had acceptable mechanical properties at the cost of an increased inflammatory response. Miniaturization to improve delivery and further investigation on the long-term inflammatory profile of thicker struts, including through degradation, is needed. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ccd.26647DOI Listing
November 2016

Hypoxia fate mapping identifies cycling cardiomyocytes in the adult heart.

Nature 2015 Jul 22;523(7559):226-30. Epub 2015 Jun 22.

1] Department of Internal Medicine, Division of Cardiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA [2] Hamon Center for Regenerative Science and Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts, mediated primarily by proliferation of pre-existing cardiomyocytes. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.
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http://dx.doi.org/10.1038/nature14582DOI Listing
July 2015

Synthesis and use of universal sequence probes in fluorogenic multi-strand hybridisation complexes for economical nucleic acid testing.

Mol Cell Probes 2015 Aug 14;29(4):228-36. Epub 2015 May 14.

LGC, Queens Road, Teddington TW11 0LY, UK. Electronic address:

Analysis of nucleic acid amplification products has become the gold standard for applications such as pathogen detection and characterisation of single nucleotide polymorphisms and short tandem repeat sequences. The development of real-time PCR and melting curve analysis using fluorescent probes has simplified nucleic acid analyses. However, the cost of probe synthesis can be prohibitive when developing large panels of tests. We describe an economic two-stage method for probe synthesis, and a new method for nucleic acid sequence analysis which together considerably reduce costs. The analysis method utilises three-strand and four-strand hybridisation complexes for the detection and identification of nucleic acid target sequences by real-time PCR and fluorescence melting.
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http://dx.doi.org/10.1016/j.mcp.2015.05.007DOI Listing
August 2015

Severe myopathy in mice lacking the MEF2/SRF-dependent gene leiomodin-3.

J Clin Invest 2015 Apr 16;125(4):1569-78. Epub 2015 Mar 16.

Maintenance of skeletal muscle structure and function requires a precise stoichiometry of sarcomeric proteins for proper assembly of the contractile apparatus. Absence of components of the sarcomeric thin filaments causes nemaline myopathy, a lethal congenital muscle disorder associated with aberrant myofiber structure and contractility. Previously, we reported that deficiency of the kelch-like family member 40 (KLHL40) in mice results in nemaline myopathy and destabilization of leiomodin-3 (LMOD3). LMOD3 belongs to a family of tropomodulin-related proteins that promote actin nucleation. Here, we show that deficiency of LMOD3 in mice causes nemaline myopathy. In skeletal muscle, transcription of Lmod3 was controlled by the transcription factors SRF and MEF2. Myocardin-related transcription factors (MRTFs), which function as SRF coactivators, serve as sensors of actin polymerization and are sequestered in the cytoplasm by actin monomers. Conversely, conditions that favor actin polymerization de-repress MRTFs and activate SRF-dependent genes. We demonstrated that the actin nucleator LMOD3, together with its stabilizing partner KLHL40, enhances MRTF-SRF activity. In turn, SRF cooperated with MEF2 to sustain the expression of LMOD3 and other components of the contractile apparatus, thereby establishing a regulatory circuit to maintain skeletal muscle function. These findings provide insight into the molecular basis of the sarcomere assembly and muscle dysfunction associated with nemaline myopathy.
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http://dx.doi.org/10.1172/JCI80115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396495PMC
April 2015

Implementation of NICE Clinical Guideline 95 for assessment of stable chest pain in a rapid access chest pain clinic reduces the mean number of investigations and cost per patient.

Open Heart 2015;2(1):e000151. Epub 2015 Feb 19.

The Heart Hospital, University College London Hospitals NHS Foundation Trust , London , UK.

Objective: In 2010, the National Institute for Health and Care Excellence (NICE) in the UK published Clinical Guideline 95 (CG95) advocating risk stratification of patients using 'CADScore' to guide appropriate cardiac investigations for chest pain of recent onset. Implementation of the guideline in the University College London Hospitals NHS Foundation Trust was evaluated to see if it led to a reduction in the average cost of the diagnostic journey per patient and fewer investigations per patient in order to confirm a diagnosis.

Methods: This was a single centre study at a Tertiary Centre in Central London. The investigative journey for each patient presenting to the Rapid Access Chest Pain Clinic (RACPC) at University College London Hospitals NHS Foundation Trust was recorded. Retrospective analysis on this data was performed.

Results: Data for 4968 patients presenting to the RACPC from 2004 to 2012 was analysed and a size-matched cohort of 1503 patients preimplementation and postimplementation of the guidelines was compared. The mean cost of investigations postimplementation was £291.83 as compared to £319.54 preimplementation of the guidelines despite higher costs associated with some of the recommended initial investigations. The mean number of tests per patient postguidelines was 0.78 compared to 0.97 for preguidelines. An approximate twofold increase in patients not requiring tests was seen post-CG95 implementation (245 pre-CG95 vs 476 post-CG95).

Conclusions: The implementation of the NICE guidelines in our trust has reduced the average cost of the investigative journey and the number of investigations required per patient.
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http://dx.doi.org/10.1136/openhrt-2014-000151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336864PMC
February 2015

Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes--a homogenous system for sequence-specific detection.

Mol Cell Probes 2015 Apr 24;29(2):92-8. Epub 2014 Dec 24.

LGC, Queens Road, Teddington, TW11 0LY, UK. Electronic address:

Isothermal amplification is a rapid, simple alternative to PCR, with amplification commonly detected using fluorescently labelled oligonucleotide probes, intercalating dyes or increased turbidity as a result of magnesium pyrophosphate generation. SNP identification is possible but requires either allele-specific primers or multiple dye-labelled probes, but further downstream processing is often required for allelic identification. Here we demonstrate that modification of common isothermal amplification methods by the addition of HyBeacon probes permits homogeneous sequence detection and discrimination by melting or annealing curve analysis. Furthermore, we demonstrate that isothermal amplification and sequence discrimination is possible directly from a crude sample such as an expressed buccal swab.
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http://dx.doi.org/10.1016/j.mcp.2014.12.001DOI Listing
April 2015

Tumor suppression by miR-26 overrides potential oncogenic activity in intestinal tumorigenesis.

Genes Dev 2014 Dec 13;28(23):2585-90. Epub 2014 Nov 13.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; Simmons Cancer Center, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

Down-regulation of miR-26 family members has been implicated in the pathogenesis of multiple malignancies. In some settings, including glioma, however, miR-26-mediated repression of PTEN promotes tumorigenesis. To investigate the contexts in which the tumor suppressor versus oncogenic activity of miR-26 predominates in vivo, we generated miR-26a transgenic mice. Despite measureable repression of Pten, elevated miR-26a levels were not associated with malignancy in transgenic animals. We documented reduced miR-26 expression in human colorectal cancer and, accordingly, showed that miR-26a expression potently suppressed intestinal adenoma formation in Apc(min/+) mice, a model known to be sensitive to Pten dosage. These studies reveal a tumor suppressor role for miR-26 in intestinal cancer that overrides putative oncogenic activity, highlighting the therapeutic potential of miR-26 delivery to this tumor type.
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http://dx.doi.org/10.1101/gad.250951.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248289PMC
December 2014

Trans-cranial opening of the blood-brain barrier in targeted regions using a stereotaxic brain atlas and focused ultrasound energy.

J Ther Ultrasound 2014 4;2:13. Epub 2014 Aug 4.

Department of Radiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9061, USA.

Objective: The blood-brain barrier (BBB) protects the brain by preventing the entry of large molecules; this poses a major obstacle for the delivery of drugs to the brain. A novel technique using focused ultrasound (FUS) energy combined with microbubble contrast agents has been widely used for non-invasive trans-cranial BBB opening. Traditionally, FUS research is conducted with magnetic resonance imaging (MRI) guidance, which is expensive and poses physical limitations due to the magnetic field. A system that could allow researchers to test brain therapies without MR intervention could facilitate and accelerate translational research.

Methods: In this study, we present a novel FUS system that uses a custom-built FUS generator mounted on a motorized stereotaxic apparatus with embedded brain atlas to locally open the BBB in rodents. The system was initially characterized using a tissue-mimicking phantom. Rodent studies were also performed to evaluate whether non-invasive, localized BBB opening could be achieved using brain atlas-based targeting. Brains were exposed to pulsed focused ultrasound energy at 1.06 MHz in rats and 3.23 MHz in mice, with the focal pressure estimated to be 0.5-0.6 MPa through the skull. BBB opening was confirmed in gross tissue sections by the presence of Evans blue leakage in the exposed region of the brain and by histological assessment.

Results: The targeting accuracy of the stereotaxic system was better than 0.5 mm in the tissue-mimicking phantom. Reproducible localized BBB opening was verified with Evans blue dye leakage in 32/33 rats and had a targeting accuracy of ±0.3 mm. The use of higher frequency exposures in mice enabled a similar precision of localized BBB opening as was observed with the low frequency in the rat model.

Conclusions: With this dedicated small-animal motorized stereotaxic-FUS system, we achieved accurate targeting of focused ultrasound exposures in the brain for non-invasive opening of the BBB. This system can be used as an alternative to MR-guided FUS and offers researchers the ability to perform efficient studies (30 min per experiment including preparation) at a reduced cost in a conventional laboratory environment.
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http://dx.doi.org/10.1186/2050-5736-2-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4160001PMC
September 2014

Loss of Tbx1 induces bone phenotypes similar to cleidocranial dysplasia.

Hum Mol Genet 2015 Jan 10;24(2):424-35. Epub 2014 Sep 10.

Department of Molecular Biology and

T-box transcription factor, TBX1, is the major candidate gene for 22q11.2 deletion syndrome (DiGeorge/ Velo-cardio-facial syndrome) characterized by facial defects, thymus hypoplasia, cardiovascular anomalies and cleft palates. Here, we report that the loss of Tbx1 in mouse (Tbx1(-/-)) results in skeletal abnormalities similar to those of cleidocranial dysplasia (CCD) in humans, which is an autosomal-dominant skeletal disease caused by mutations in RUNX2. Tbx1(-/-) mice display short stature, absence of hyoid bone, failed closure of fontanelle, bifid xiphoid process and hypoplasia of clavicle and zygomatic arch. A cell-type-specific deletion of Tbx1 in osteochondro-progenitor (Tbx1(OPKO)) or mesodermal (Tbx1(MKO)) lineage partially recapitulates the Tbx1(-/-) bone phenotypes. Although Tbx1 expression has not been previously reported in neural crest, inactivation of Tbx1 in the neural crest lineage (Tbx1(NCKO)) leads to an absence of the body of hyoid bone and postnatal lethality, indicating an unanticipated role of Tbx1 in neural crest development. Indeed, Tbx1 is expressed in the neural crest-derived hyoid bone primordium, in addition to mesoderm-derived osteochondral progenitors. Ablation of Tbx1 affected Runx2 expression in calvarial bones and overexpression of Tbx1 induced Runx2 expression in vitro. Taken together, our current studies reveal that Tbx1 is required for mesoderm- and neural crest-derived osteoblast differentiation and normal skeletal development. TBX1 mutation could lead to CCD-like bone phenotypes in human.
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http://dx.doi.org/10.1093/hmg/ddu458DOI Listing
January 2015

A novel design biodegradable stent for use in congenital heart disease: mid-term results in rabbit descending aorta.

Catheter Cardiovasc Interv 2015 Mar 3;85(4):629-39. Epub 2014 Sep 3.

Departments of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.

Objectives: This study evaluates the feasibility of delivery and deployment of low and medium molecular weight (LMW and MMW, respectively) double-opposing helical (DH) poly-l-lactic acid biodegradable stent (BDS) in rabbit descending aorta (DAO). Secondary objectives were to assess patency and inflammation of stented vessels at 9 months and to investigate safety following intentional embolization of stent fragments in DAO.

Background: A BDS that will relieve aortic obstruction and disappears as the child grows older allowing for preservation of aortic wall elasticity and natural growth of aorta will be ideal to treat Coarctation (CoA). BDS have never been evaluated in the DAO.

Methods: Seven New Zealand white rabbits underwent implantation of DH-LMW (n = 7), DH-MMW (n = 3), and metal stents (n = 7) in DAO. BDS fragments were intentionally embolized into DAO in two rabbits.

Results: All stents were deployed via a 6-French sheath. Five BDS covered the origin of major DAO side branches. Angiography and intravascular ultrasound showed good stent apposition to the wall of DAO with minimal luminal loss at 9 months follow-up. All stents had minimal neointimal hyperplasia on histopathology. Adverse events included 1 death, 1 aortic aneurysm, and lower extremity ulceration due to self-mutilation in an embolization rabbit.

Conclusions: Pilot study confirms the feasibility of delivery and deployment of up to 6-millimeter diameter DH BDS in rabbit DAO. Stent integrity with DH design was maintained at 9 months with minimal vessel inflammation. Potential morbidity due to embolized BD fragments cannot be ruled out and needs further evaluation.
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http://dx.doi.org/10.1002/ccd.25648DOI Listing
March 2015

Ataxia and Purkinje cell degeneration in mice lacking the CAMTA1 transcription factor.

Proc Natl Acad Sci U S A 2014 Aug 21;111(31):11521-6. Epub 2014 Jul 21.

Departments of Molecular Biology.

Members of the calmodulin-binding transcription activator (CAMTA) family of proteins function as calcium-sensitive regulators of gene expression in multicellular organisms ranging from plants to humans. Here, we show that global or nervous system deletion of CAMTA1 in mice causes severe ataxia with Purkinje cell degeneration and cerebellar atrophy, partially resembling the consequences of haploinsufficiency of the human CAMTA1 locus. Gene-expression analysis identified a large collection of neuronal genes that were dysregulated in the brains of CAMTA1-mutant mice, and elucidation of a consensus sequence for binding of CAMTA proteins to DNA revealed the association of CAMTA-binding sites with many of these genes. We conclude that CAMTA1 plays an essential role in the control of Purkinje cell function and survival. CAMTA1-mutant mice provide a model to study the molecular mechanisms of neurodegenerative diseases and for screening potential therapeutic interventions for such disorders.
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http://dx.doi.org/10.1073/pnas.1411251111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128133PMC
August 2014

Tissue-specific regulation of the mouse Pkhd1 (ARPKD) gene promoter.

Am J Physiol Renal Physiol 2014 Aug 4;307(3):F356-68. Epub 2014 Jun 4.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas; Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas;

Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1β, which is required for activity in transfected cells. Mutation of the HNF-1β-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1β. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1β is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues.
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http://dx.doi.org/10.1152/ajprenal.00422.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121570PMC
August 2014

Requirement of MEF2A, C, and D for skeletal muscle regeneration.

Proc Natl Acad Sci U S A 2014 Mar 3;111(11):4109-14. Epub 2014 Mar 3.

Departments of Molecular Biology, Internal Medicine, and Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Regeneration of adult skeletal muscle following injury occurs through the activation of satellite cells, an injury-sensitive muscle stem cell population that proliferates, differentiates, and fuses with injured myofibers. Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors play essential roles in muscle differentiation during embryogenesis, but their potential contributions to adult muscle regeneration have not been systematically explored. To investigate the potential involvement of MEF2 factors in muscle regeneration, we conditionally deleted the Mef2a, c, and d genes, singly and in combination, within satellite cells in mice, using tamoxifen-inducible Cre recombinase under control of the satellite cell-specific Pax7 promoter. We show that deletion of individual Mef2 genes has no effect on muscle regeneration in response to cardiotoxin injury. However, combined deletion of the Mef2a, c, and d genes results in a blockade to regeneration. Satellite cell-derived myoblasts lacking MEF2A, C, and D proliferate normally in culture, but cannot differentiate. The absence of MEF2A, C, and D in satellite cells is associated with aberrant expression of a broad collection of known and unique protein-coding and long noncoding RNA genes. These findings reveal essential and redundant roles of MEF2A, C, and D in satellite cell differentiation and identify a MEF2-dependent transcriptome associated with skeletal muscle regeneration.
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http://dx.doi.org/10.1073/pnas.1401732111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964114PMC
March 2014

Radiation-enhanced lung cancer progression in a transgenic mouse model of lung cancer is predictive of outcomes in human lung and breast cancer.

Clin Cancer Res 2014 Mar 31;20(6):1610-22. Epub 2014 Jan 31.

Authors' Affiliations: Departments of Cell Biology; Pathology; Molecular Biology; Plastic Surgery; and Clinical Sciences; Simmons Comprehensive Cancer Center; Hamon Center for Therapeutic Oncology; Departments of Internal Medicine; Pharmacology; and Radiation Oncology, UT Southwestern Medical Center, Dallas; Departments of Thoracic/Head and Neck Medical Oncology; and Translational Molecular Pathology; The University of Texas MD Anderson Cancer Center, Houston, Texas; and Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia.

Purpose: Carcinogenesis is an adaptive process between nascent tumor cells and their microenvironment, including the modification of inflammatory responses from antitumorigenic to protumorigenic. Radiation exposure can stimulate inflammatory responses that inhibit or promote carcinogenesis. The purpose of this study is to determine the impact of radiation exposure on lung cancer progression in vivo and assess the relevance of this knowledge to human carcinogenesis.

Experimental Design: K-ras(LA1) mice were irradiated with various doses and dose regimens and then monitored until death. Microarray analyses were performed using Illumina BeadChips on whole lung tissue 70 days after irradiation with a fractionated or acute dose of radiation and compared with age-matched unirradiated controls. Unique group classifiers were derived by comparative genomic analysis of three experimental cohorts. Survival analyses were performed using principal component analysis and k-means clustering on three lung adenocarcinoma, three breast adenocarcinoma, and two lung squamous carcinoma annotated microarray datasets.

Results: Radiation exposure accelerates lung cancer progression in the K-ras(LA1) lung cancer mouse model with dose fractionation being more permissive for cancer progression. A nonrandom inflammatory signature associated with this progression was elicited from whole lung tissue containing only benign lesions and predicts human lung and breast cancer patient survival across multiple datasets. Immunohistochemical analyses suggest that tumor cells drive predictive signature.

Conclusions: These results demonstrate that radiation exposure can cooperate with benign lesions in a transgenic model of cancer by affecting inflammatory pathways, and that clinically relevant similarities exist between human lung and breast carcinogenesis.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-2589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961755PMC
March 2014

A reevaluation of CD22 expression in human lung cancer.

Cancer Res 2014 Jan;74(1):263-71

Authors' Affiliations: Cancer Immunobiology Center and Hamon Center for Therapeutic Oncology Research; Departments of Immunology, Internal Medicine, Microbiology, Pathology, and Pharmacology, University of Texas Southwestern Medical Center, Dallas; Departments of Thoracic/Head and Neck Medical Oncology and Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston; Bio-Synthesis Inc., Lewisville, Texas; and Department of Immunology, Duke University Medical Center, Durham, North Carolina.

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-1436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903042PMC
January 2014

Cytoglobin modulates myogenic progenitor cell viability and muscle regeneration.

Proc Natl Acad Sci U S A 2014 Jan 23;111(1):E129-38. Epub 2013 Dec 23.

Departments of Internal Medicine, Molecular Biology, and Pathology, the Donald W. Reynolds Cardiovascular Clinical Research Center, and the Heart Failure, Ventricular Assist Device and Heart Transplant Program, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Mammalian skeletal muscle can remodel, repair, and regenerate itself by mobilizing satellite cells, a resident population of myogenic progenitor cells. Muscle injury and subsequent activation of myogenic progenitor cells is associated with oxidative stress. Cytoglobin is a hemoprotein expressed in response to oxidative stress in a variety of tissues, including striated muscle. In this study, we demonstrate that cytoglobin is up-regulated in activated myogenic progenitor cells, where it localizes to the nucleus and contributes to cell viability. siRNA-mediated depletion of cytoglobin from C2C12 myoblasts increased levels of reactive oxygen species and apoptotic cell death both at baseline and in response to stress stimuli. Conversely, overexpression of cytoglobin reduced reactive oxygen species levels, caspase activity, and cell death. Mice in which cytoglobin was knocked out specifically in skeletal muscle were generated to examine the role of cytoglobin in vivo. Myogenic progenitor cells isolated from these mice were severely deficient in their ability to form myotubes as compared with myogenic progenitor cells from wild-type littermates. Consistent with this finding, the capacity for muscle regeneration was severely impaired in mice deficient for skeletal-muscle cytoglobin. Collectively, these data demonstrate that cytoglobin serves an important role in muscle repair and regeneration.
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http://dx.doi.org/10.1073/pnas.1314962111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890830PMC
January 2014

The role of Cdk5 in neuroendocrine thyroid cancer.

Cancer Cell 2013 Oct;24(4):499-511

Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.
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http://dx.doi.org/10.1016/j.ccr.2013.08.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849320PMC
October 2013

Linking spermatid ribonucleic acid (RNA) binding protein and retrogene diversity to reproductive success.

Mol Cell Proteomics 2013 Nov 12;12(11):3221-36. Epub 2013 Aug 12.

Departments of Pharmacology.

Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness.
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http://dx.doi.org/10.1074/mcp.M113.030585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820935PMC
November 2013

Hippo pathway effector Yap promotes cardiac regeneration.

Proc Natl Acad Sci U S A 2013 Aug 5;110(34):13839-44. Epub 2013 Aug 5.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process.
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http://dx.doi.org/10.1073/pnas.1313192110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3752208PMC
August 2013

Use of a large Stokes-shift fluorophore to increase the multiplexing capacity of a point-of-care DNA diagnostic device.

Analyst 2013 Jul;138(13):3626-8

Chemistry, University of Southampton, Southampton, Hampshire SO17 1BJ, UK.

The intense demand for fluorescence-based point of care (POC) DNA diagnostics is driving developments to reduce the size of instrumentation, imposing limitations on the optical hardware that can be included. Here we describe a combination of instrumentation and fluorogenic probes to detect three fluorophores using two excitation and two detection channels.
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http://dx.doi.org/10.1039/c3an00593cDOI Listing
July 2013