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1.  Considerations in the design of clinical trials for pediatric acute lymphoblastic leukemia 
Clinical investigation  2013;3(9):10.4155/cli.13.71.
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although outcomes for children with ALL have improved dramatically over the last 50 years, ALL remains the leading cause of childhood cancer death. In addition, high-risk patient subsets can be identified with significantly inferior survival. In the current era of therapies directed at specific molecular targets, the use of conventional randomized Phase III trials to show benefit from a new treatment regimen may not be feasible when these biologically defined subsets are small. This review presents the traditional approaches to designing trials for children with ALL, as well as innovative approaches attempting to study the benefit of new treatments as reliably as possible for patient subsets with distinctive biological characteristics.
doi:10.4155/cli.13.71
PMCID: PMC3834963  PMID: 24273641
acute lymphoblastic leukemia; clinical trials; historical controls
2.  Hexa-Acylated Lipid A Is Required for Host Inflammatory Response to Neisseria gonorrhoeae in Experimental Gonorrhea 
Infection and Immunity  2014;82(1):184-192.
Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients with N. gonorrhoeae remain asymptomatic, without evidence of localized inflammation. Inflammatory responses to N. gonorrhoeae are generated by host innate immune recognition of N. gonorrhoeae by several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1β (IL-1β) processing complex known as the inflammasome. The lipooligosaccharide of N. gonorrhoeae has a hexa-acylated lipid A. N. gonorrhoeae strains that carry an inactivated msbB (also known as lpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show that msbB-deficient N. gonorrhoeae induces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affect N. gonorrhoeae-mediated activation of the inflammasome. We demonstrate that N. gonorrhoeae msbB is dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection with msbB-deficient N. gonorrhoeae is associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition of N. gonorrhoeae LOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.
doi:10.1128/IAI.00890-13
PMCID: PMC3911856  PMID: 24126526
3.  2010 Panel on the Biomaterials Grand Challenges 
In 2009, the National Academy for Engineering issued the Grand Challenges for Engineering in the 21st Century comprised of 14 technical challenges that must be addressed to build a healthy, profitable, sustainable, and secure global community (http://www.engineeringchallenges.org). Although crucial, none of the NEA Grand Challenges adequately addressed the challenges that face the biomaterials community. In response to the NAE Grand Challenges, Monty Reichert of Duke University organized a panel entitled Grand Challenges in Biomaterials at the at the 2010 Society for Biomaterials Annual Meeting in Seattle. Six members of the National Academies—Buddy Ratner, James Anderson, Allan Hoffman, Art Coury, Cato Laurencin, and David Tirrell—were asked to propose a grand challenge to the audience that, if met, would significantly impact the future of biomaterials and medical devices. Successfully meeting these challenges will speed the 60-plus year transition from commodity, off-the-shelf biomaterials to bioengineered chemistries, and biomaterial devices that will significantly advance our ability to address patient needs and also to create new market opportunities.
doi:10.1002/jbm.a.32969
PMCID: PMC4076103  PMID: 21171147
biomaterial; grand challenges; biocompatibility testing; smart polymers; regenerative medicine
4.  Certain Adenylated Non-Coding RNAs, Including 5′ Leader Sequences of Primary MicroRNA Transcripts, Accumulate in Mouse Cells following Depletion of the RNA Helicase MTR4 
PLoS ONE  2014;9(6):e99430.
RNA surveillance plays an important role in posttranscriptional regulation. Seminal work in this field has largely focused on yeast as a model system, whereas exploration of RNA surveillance in mammals is only recently begun. The increased transcriptional complexity of mammalian systems provides a wider array of targets for RNA surveillance, and, while many questions remain unanswered, emerging data suggest the nuclear RNA surveillance machinery exhibits increased complexity as well. We have used a small interfering RNA in mouse N2A cells to target the homolog of a yeast protein that functions in RNA surveillance (Mtr4p). We used high-throughput sequencing of polyadenylated RNAs (PA-seq) to quantify the effects of the mMtr4 knockdown (KD) on RNA surveillance. We demonstrate that overall abundance of polyadenylated protein coding mRNAs is not affected, but several targets of RNA surveillance predicted from work in yeast accumulate as adenylated RNAs in the mMtr4KD. microRNAs are an added layer of transcriptional complexity not found in yeast. After Drosha cleavage separates the pre-miRNA from the microRNA's primary transcript, the byproducts of that transcript are generally thought to be degraded. We have identified the 5′ leading segments of pri-miRNAs as novel targets of mMtr4 dependent RNA surveillance.
doi:10.1371/journal.pone.0099430
PMCID: PMC4057207  PMID: 24926684
5.  Beta-propeller protein-associated neurodegeneration: a new X-linked dominant disorder with brain iron accumulation 
Brain  2013;136(6):1708-1717.
Neurodegenerative disorders with high iron in the basal ganglia encompass an expanding collection of single gene disorders collectively known as neurodegeneration with brain iron accumulation. These disorders can largely be distinguished from one another by their associated clinical and neuroimaging features. The aim of this study was to define the phenotype that is associated with mutations in WDR45, a new causative gene for neurodegeneration with brain iron accumulation located on the X chromosome. The study subjects consisted of WDR45 mutation-positive individuals identified after screening a large international cohort of patients with idiopathic neurodegeneration with brain iron accumulation. Their records were reviewed, including longitudinal clinical, laboratory and imaging data. Twenty-three mutation-positive subjects were identified (20 females). The natural history of their disease was remarkably uniform: global developmental delay in childhood and further regression in early adulthood with progressive dystonia, parkinsonism and dementia. Common early comorbidities included seizures, spasticity and disordered sleep. The symptoms of parkinsonism improved with l-DOPA; however, nearly all patients experienced early motor fluctuations that quickly progressed to disabling dyskinesias, warranting discontinuation of l-DOPA. Brain magnetic resonance imaging showed iron in the substantia nigra and globus pallidus, with a ‘halo’ of T1 hyperintense signal in the substantia nigra. All patients harboured de novo mutations in WDR45, encoding a beta-propeller protein postulated to play a role in autophagy. Beta-propeller protein-associated neurodegeneration, the only X-linked disorder of neurodegeneration with brain iron accumulation, is associated with de novo mutations in WDR45 and is recognizable by a unique combination of clinical, natural history and neuroimaging features.
doi:10.1093/brain/awt095
PMCID: PMC3673459  PMID: 23687123
iron; NBIA; autophagy; basal ganglia; Rett syndrome
6.  Improved synthesis of structural analogues of (−)-epicatechin gallate for modulation of staphylococcal β-lactam resistance☆ 
Tetrahedron  2014;70(21):3485-3490.
The high-yielding synthesis of enantiomerically pure epicatechin gallate analogues where the A and/or B-ring hydroxylation is reduced or altered has been achieved by optimising routes to the catechin stereochemistry. The B-ring analogues were synthesised by using an electrophilic ring closure onto an enantiomerically enriched epoxide as a key step. The A and B-ring hydroxyl-deleted analogues were synthesised through a Mitsunobu cyclisation. For the B-ring analogues, the anti- (catechin) stereochemistry was converted to the syn- (epicatechin) stereochemistry by a known oxidation/reduction protocol. Absolute stereochemistry was derived from either a Sharpless epoxidation or asymmetric dihydroxylation.
Graphical abstract
doi:10.1016/j.tet.2014.03.052
PMCID: PMC4006114  PMID: 24876661
(−)-Epicatechin gallate analogues; Asymmetric synthesis; Cyclisation; Mitsunobu; MRSA
7.  Mortality Increase in Late-Middle and Early-Old Age: Heterogeneity in Death Processes as a New Explanation 
Demography  2013;50(5):1563-1591.
Deviations from the Gompertz law of exponential mortality increases in late-middle and early-old age are commonly neglected in overall mortality analyses. In this study, we examined mortality increase patterns between ages 40 and 85 in 16 low-mortality countries and demonstrated sex differences in these patterns, which also changed across period and cohort. These results suggest that the interaction between aging and death is more complicated than what is usually assumed from the Gompertz law and also challenge existing biodemographic hypotheses about the origin and mechanisms of sex differences in mortality. We propose a two-mortality model that explains these patterns as the change in the composition of intrinsic and extrinsic death rates with age. We show that the age pattern of overall mortality and the population heterogeneity therein are possibly generated by multiple dynamics specified by a two-mortality model instead of a uniform process throughout most adult ages.
doi:10.1007/s13524-013-0222-4
PMCID: PMC4028711  PMID: 23743628
Mortality acceleration; Sex differences; Two-mortality processes; Vitality
8.  Instability of Self-Assembled Monolayers (SAM) as a Model Material System for Macrophage/FBGC Cellular Behavior 
Novel self-assembled monolayers (SAMs) designed to present homogenous surface chemistries were utilized to further investigate the material surface chemistry dependent macrophage and foreign body giant cell (FBGC) behaviors including macrophage adhesion, fusion, and apoptosis. Contact angle analysis revealed instabilities in the –CH3 and –COOH terminate SAM surfaces upon incubation in serum-free media at 37oC or under dry, room temperatureconditions. Further analysis indicated that the –CH3 terminated SAM surface degraded rapidly within 2 hours and loss of sufficient SAM units to be comparable to the gold (Au) control surface within 24 hours of incubation in serum-free media (SFM) at 37oC. After 5days of incubation in SFM at 37oC, the contact angles for the –COOH terminated SAMsurfaces increased markedly. AFM analysis confirmed the desorption of –CH3 terminated SAM molecules from the surface with increased roughness and marked appearance of peaks andvalleys within 2 hours. A decrease in the thickness of the –COOH terminated SAM surface also suggests molecular desorption over time. No significant changes in contact angle or AFM analyses were observed on the –OH terminated SAM surfaces. Cellular adhesion decreased morerapidly on the Au control and –CH3 terminated SAM surfaces in comparison to the other surfaces. However by day 10, cellular adhesion, fusion, and apoptosis were comparable on all SAM surfaces and the Au control . These studies suggest that SAM surfaces may not be suitable for long-term studies where material dependent properties are investigated.
doi:10.1002/jbm.a.31660
PMCID: PMC4017672  PMID: 18412136
self-assembled monolayers; instability; macrophage; foreign body giant cell; contact angles
9.  Patterns of Chemotherapy-Induced Toxicities in Younger Children and Adolescents with Rhabdomyosarcoma: A Report from the Children’s Oncology Group Soft Tissue Sarcoma Committee 
Cancer  2011;118(4):1130-1137.
Purpose
Patients aged older than 10 years with rhabdomyosarcoma (RMS) have an inferior outcome compared to patients aged 1–9 years which might be explained by toxicities (adverse events, AE) resulting in chemotherapy dose reductions.
Methods
AEs observed during one of 3 randomized chemotherapy regimens (VAC, VAI or VIE) in Intergroup Rhabdomyosarcoma Study (IRS)-IV were recorded. The incidence of toxicities by age and treatment regimen was determined. The odds of developing an AE in a particular age group (5–9, 10–14, and 15–20 years) were compared to the control age group, 1–4 years.
Results
657 patients were eligible for analysis. Estimated 5-year EFS were 78%, 83%, 67%, and 58% for age groups 1–4, 5–9, 10–14, and 15–20, respectively. Patients 15–20 years experienced less neutropenia (OR 0.43, p<0.0001), thrombocytopenia (OR 0.41, p<0.0001), anemia (OR 0.34, p<0.0001) and infection (OR 0.41, p<0.0001) compared to younger patients, despite receiving similar amounts of chemotherapy. In contrast, peripheral nervous system (PNS) toxicity was higher in adolescents older than 10 years (OR 4.18, p<0.0001). Females experienced more neutropenia (OR 1.28, p=0.05) and thrombocytopenia (OR 1.26, p=0.06) compared to males.
Conclusions
Adolescents receiving treatment for RMS experience significantly less hematologic and more PNS toxicity compared to younger children despite receiving similar amounts of chemotherapy. Although outcome is inferior in adolescents, it is unclear whether the differences in toxicity observed in our study impact on outcome. Future studies examining the age and gender-related differences in pharmacokinetics of chemotherapy are necessary.
doi:10.1002/cncr.26358
PMCID: PMC4008942  PMID: 21761400
adolescent; rhabdomyosarcoma; toxicity; chemotherapy; age; gender
10.  The immunophenotype of T-lymphoblastic lymphoma in children and adolescents: a Children’s Oncology Group report 
British journal of haematology  2012;159(4):454-461.
Summary
T-lymphoblastic leukaemia (T-ALL) and T-lymphoblastic lymphoma (T-LBL) are neoplasms derived from immature lymphoid cells of T-cell lineage. These neoplasms are biologically similar, but significant differences may exist between the two given their clinical differences. Although ample data regarding the immunophenotypic characterization T-ALL are available, there is a paucity of such data in children and adolescents with T-LBL. We used flow cytometry and/or immunohistochemistry to characterize the immunophenotypic profile of 180 children and adolescents with newly diagnosed T-LBL enrolled in the Children’s Oncology Group 5971 study. Multiple T-cell, B-cell, myeloid, and other markers were evaluated. We identified diagnostically useful immunophenotypic features of T-LBL as well as distinct immunophenotypic subgroups, although none of these was statistically related to event-free or overall survival in this retrospective analysis. Further studies of biologically and immunophenotypically distinct subgroups of T-LBL, such as the early T-cell precursor phenotype, are warranted.
doi:10.1111/bjh.12042
PMCID: PMC4008319  PMID: 22994934
T-lymphoblastic lymphoma; early T-cell precursor; immunophenotypic analysis; paediatric lymphoma; T-cell antigens
11.  Lymph node involvement in Wilms tumor: results from National Wilms Tumor Studies 4 and 5 
Journal of pediatric surgery  2012;47(4):700-706.
Purpose
The aim of the study was to determine the prognostic impact of lymph node (LN) involvement and sampling in patients with Wilms tumor (WT) and the minimum number of LNs needed for accurate staging.
Methods
We reviewed all patients with unilateral, nonmetastatic WT enrolled in the National Wilms Tumor Study 4 or 5. Data were abstracted on patient demographics, tumor histology, staging, number of LNs sampled, and disease-specific and overall patient outcomes.
Results
A total of 3409 patients had complete information on LN sampling. Five-year event-free survival (EFS) was lower in patients with nodal disease (P = .001); the effect of LN positivity was greater for patients with anaplastic (P = .047) than with favorable histology (P = .02). The likelihood of obtaining a positive LN was higher when sampling at least 7 LNs. However, after controlling for tumor histology and stage, the number of LNs sampled did not predict EFS variations (P = .75). Among patients with stage II disease, patients with LN sampling (P = .055) had improved EFS, largely reflecting poorer EFS in patients with anaplastic tumors (P = .03).
Conclusions
Lymph node sampling is particularly important for patients with stage II anaplastic WT. Although the likelihood of finding a positive LN was greater when more than 7 LNs were sampled, EFS was not impacted by the number of LNs sampled.
doi:10.1016/j.jpedsurg.2011.08.017
PMCID: PMC3976547  PMID: 22498384
Wilms tumor; Lymph node; Treatment outcome; Child
12.  Staphylococcal Phenotypes Induced by Naturally Occurring and Synthetic Membrane-Interactive Polyphenolic β-Lactam Resistance Modifiers 
PLoS ONE  2014;9(4):e93830.
Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation.
doi:10.1371/journal.pone.0093830
PMCID: PMC3974817  PMID: 24699700
13.  ON Cone Bipolar Cell Axonal Synapses in the OFF Inner Plexiform Layer of the Rabbit Retina 
The Journal of comparative neurology  2013;521(5):977-1000.
Analysis of the rabbit retinal connectome RC1 reveals that the division between the ON and OFF inner plexiform layer (IPL) is not structurally absolute. ON cone bipolar cells make non-canonical axonal synapses onto specific targets and receive amacrine cell synapses in the nominal OFF layer, creating novel motifs, including inhibitory crossover networks. Automated transmission electron microscope (ATEM) imaging, molecular tagging, tracing, and rendering of ≈ 400 bipolar cells reveals axonal ribbons in 36% of ON cone bipolar cells, throughout the OFF IPL. The targets include GABA-positive amacrine cells (γACs), glycine-positive amacrine cells (GACs) and ganglion cells. Most ON cone bipolar cell axonal contacts target GACs driven by OFF cone bipolar cells, forming new architectures for generating ON-OFF amacrine cells. Many of these ON-OFF GACs target ON cone bipolar cell axons, ON γACs and/or ON-OFF ganglion cells, representing widespread mechanisms for OFF to ON crossover inhibition. Other targets include OFF γACs presynaptic to OFF bipolar cells, forming γAC-mediated crossover motifs. ON cone bipolar cell axonal ribbons drive bistratified ON-OFF ganglion cells in the OFF layer and provide ON drive to polarity-appropriate targets such as bistratified diving ganglion cells (bsdGCs). The targeting precision of ON cone bipolar cell axonal synapses shows that this drive incidence is necessarily a joint distribution of cone bipolar cell axonal frequency and target cell trajectories through a given volume of the OFF layer. Such joint distribution sampling is likely common when targets are sparser than sources and when sources are coupled, as are ON cone bipolar cells.
doi:10.1002/cne.23244
PMCID: PMC3560306  PMID: 23042441
retina; inner plexiform layer; connectomics; circuitry; neural network; bipolar cell; axonal ribbon; axonal cistern; amacrine cell; bistratified diving ganglion cell; intrinsically photosensitive ganglion cell; crossover inhibition; within channel inhibition; ON-OFF crosstalk; functional network; structure-function; network topology
14.  A high-resolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans 
eLife  2014;3:e01948.
Many primary sensory cilia exhibit unique architectures that are critical for transduction of specific sensory stimuli. Although basic ciliogenic mechanisms are well described, how complex ciliary structures are generated remains unclear. Seminal work performed several decades ago provided an initial but incomplete description of diverse sensory cilia morphologies in C. elegans. To begin to explore the mechanisms that generate these remarkably complex structures, we have taken advantage of advances in electron microscopy and tomography, and reconstructed three-dimensional structures of fifty of sixty sensory cilia in the C. elegans adult hermaphrodite at high resolution. We characterize novel axonemal microtubule organization patterns, clarify structural features at the ciliary base, describe new aspects of cilia–glia interactions, and identify structures suggesting novel mechanisms of ciliary protein trafficking. This complete ultrastructural description of diverse cilia in C. elegans provides the foundation for investigations into underlying ciliogenic pathways, as well as contributions of defined ciliary structures to specific neuronal functions.
DOI: http://dx.doi.org/10.7554/eLife.01948.001
eLife digest
To survive, animals must constantly gather information about their surroundings and then decide how to respond. Animals rely on cells called sensory neurons to help them perceive and process this information, and these neurons in most animals have smaller structures called cilia that help them to gather this information. The structures of these cilia can range from simple hair-like rods to complex branched arbors. Defective cilia can lead to cell degeneration and death.
Scientists have identified and determined the functions of many of the 60 sensory neurons with cilia in C. elegans, a tiny roundworm with a simple nervous system. These experiments have revealed that the shapes of these cilia are quite diverse, and that the shape determines the type of information the neurons process. Learning more about how cilia are shaped, and how these shapes allow them to perform specific sensory functions, would give scientists a better understanding of how the brain processes sensory information.
Doroquez et al. have now taken advantage of advances in imaging technology to generate highly detailed three-dimensional reconstructions of the cilia on 50 neurons in the nose of C. elegans. The experiments involved rapidly freezing the worms, slowly replacing the frozen water molecules with a preservative solution, and then embedding in resin. This allowed Doroquez et al. to slice the samples into very thin sections—some 1400 times thinner than a sheet of paper—and then image them with transmission electron microscopy and electron tomography. Finally, all these images were combined in a computer to produce 3D models of the cilia.
The models reveal a wide range of cilia structures, including some that have never been examined in detail before. Doroquez et al. were also able to see detailed structures within the cilia, including compartments that determine which proteins should enter into, or be excluded from, an individual cilium. The models, along with the results of previous studies, suggest that cilia are shaped by genetic factors and also by interactions with the environment. This detailed description of diverse cilia structures should now allow researchers to identify the genes that determine their unique shapes, and explore how specific shapes contribute to specific sensory functions.
DOI: http://dx.doi.org/10.7554/eLife.01948.002
doi:10.7554/eLife.01948
PMCID: PMC3965213  PMID: 24668170
cilia; electron microscopy; electron tomography; C. elegans
15.  Clinicopathologic Findings Predictive of Relapse in Children With Stage III Favorable-Histology Wilms Tumor 
Journal of Clinical Oncology  2013;31(9):1196-1201.
Purpose
Stage III designation in NWTS-5 (National Wilms Tumor Study–5) was determined by four pathologic criteria: positive lymph nodes (LNs), peritoneal implants, residual disease, and tumor rupture. The objective of this study was to determine the prognostic significance of each of the stage III criteria.
Patients and Methods
Children with stage III Wilms tumor (WT) treated in NWTS-5 were assessed for event-free (EFS) and overall survival (OS). Sites of relapse and molecular status of tumors are reported. EFS and OS are reported 8 years after diagnosis.
Results
There were 569 patients with local stage III favorable-histology (FH) WT in this analysis, of whom 109 had overall stage IV disease. LN involvement alone was the most frequent criterion for stage III designation (38%), followed by microscopic residual disease alone (20%), microscopic residual disease and LN involvement (14%), and spill or soilage alone (9%). The 8-year EFS and OS estimates for all patients with local stage III FHWT were 82% and 91%, respectively. Multivariate analysis demonstrated that both LN involvement (relative risk, 1.89; P = .005) and microscopic residual disease (relative risk, 1.87; P = .007) were predictive of EFS, and OS results were similar. There was no apparent difference in pattern of relapse according to stage III subtype. The rate of loss of heterozygosity was higher (6%) for those with positive LNs than for those without (2%; P = .05).
Conclusion
LN involvement and microscopic residual are the stage III criteria highly predictive of EFS and OS for patients with stage III FHWT. It is possible that in future studies, patients with different stage III criteria may receive different therapies.
doi:10.1200/JCO.2011.41.1165
PMCID: PMC3595426  PMID: 23382471
16.  Aberrant Activation, Nuclear Localization, and Phosphorylation of Yes-Associated Protein-1 in the Embryonic Kidney and Wilms Tumor 
Pediatric blood & cancer  2013;61(2):198-205.
Background
The Yes-associated-protein-1 (YAP1) is a novel, direct regulator of stem cell genes both in development and cancer. FAT4 is an upstream regulator that induces YAP1 cytosolic sequestering by phosphorylation (p-Ser 127) and therefore inhibits YAP1-dependent cellular proliferation. We hypothesized that loss of FAT4 signaling would result in expansion of the nephron progenitor population in kidney development and that YAP1 subcellular localization would be dysregulated in Wilms tumor (WT), an embryonal malignancy that retains gene expression profiles and histologic features reminiscent of the embryonic kidney.
Methods
Fetal kidneys from Fat4−/− mice were harvested at e18.5 and markers of nephron progenitors were investigated using immunohistochemical analysis. To examine YAP1 subcellular localization in WT, a primary WT cell line (VUWT30) was analyzed by immunofluorescence. Forty WT specimens evenly distributed between favorable and unfavorable histology (n = 20 each), and treatment failure or success (n = 20 each) was analyzed for total and phosphorylated YAP1 using immunohistochemistry and Western blot.
Results
Fat4−/− mouse fetal kidneys exhibit nuclear YAP1 with increased proliferation and expansion of nephron progenitor cells. In contrast to kidney development, subcellular localization of YAP1 is dysregulated in WT, with a preponderance of nuclear p-YAP1. By Western blot, median p-YAP1 quantity was 5.2-fold greater in unfavorable histology WT (P = 0.05).
Conclusions
Fetal kidneys in Fat4−/− mice exhibit a phenotype reminiscent of nephrogenic rests, a WT precursor lesion. In WT, YAP1 subcellular localization is dysregulated and p-YAP1 accumulation is a novel biomarker of unfavorable histology.
doi:10.1002/pbc.24788
PMCID: PMC3955491  PMID: 24115727
anaplasia; biomarker; nephrogenic rests; Wilms tumor; YAP1
17.  Neurologically Intact Patient Following Bilateral Facet Dislocation: Case Report and Review of Literature 
The Ochsner Journal  2014;14(1):108-111.
Background
Complete spinal cord lesions and quadriplegia occur in 50%-84% of patients with bilateral facet dislocation. We present a patient who suffered both bilateral facet dislocation and bilateral pedicle fractures while remaining neurologically intact. Based on this case and our literature review, we hypothesize that bilateral facet dislocations without neurological deficits are accompanied by significant associated fractures that facilitate the maintenance of cervical spine canal patency.
Case Report
After a fall down a flight of stairs, an 86-year-old woman presented to the hospital complaining of neck pain. She denied numbness and weakness of her extremities. On physical examination she was neurologically intact without focal sensory or motor deficits and with normal reflexes throughout. Computed tomography (CT) of her neck demonstrated bilateral C5-C6 facet dislocation with locking of the C6 superior articular process dorsal to the C5 inferior articular process, as well as corresponding bilateral C6 pedicle fractures. Additional acute fractures were identified on the thoracic CT. Magnetic resonance imaging demonstrated no spinal cord compression, edema, or hemorrhage. The patient had a C6-C7 anterior cervical discectomy and allograft fusion and a C5-T1 anterior cervical plate with screw fixation.
Conclusion
Because bilateral facet dislocations without neurological deficits are rare, the most appropriate surgical intervention is not evident. We believe the best choice as a first step is an anterior cervical discectomy and allograft fusion with plating.
PMCID: PMC3963039  PMID: 24688342
Cervical vertebrae; decompression–surgical; spinal canal; spinal cord compression; traction
18.  Retinal Remodeling in the Tg P347L Rabbit, a Large-Eye Model of Retinal Degeneration 
The Journal of comparative neurology  2011;519(14):2713-2733.
Retinitis pigmentosa (RP) is an inherited blinding disease characterized by progressive loss of retinal photo-receptors. There are numerous rodent models of retinal degeneration, but most are poor platforms for interventions that will translate into clinical practice. The rabbit possesses a number of desirable qualities for a model of retinal disease including a large eye and an existing and substantial knowledge base in retinal circuitry, anatomy, and ophthalmology. We have analyzed degeneration, remodeling, and reprogramming in a rabbit model of retinal degeneration, expressing a rhodopsin proline 347 to leucine transgene in a TgP347L rabbit as a powerful model to study the pathophysiology and treatment of retinal degeneration. We show that disease progression in the TgP347L rabbit closely tracks human cone-sparing RP, including the cone-associated preservation of bipolar cell signaling and triggering of reprogramming. The relatively fast disease progression makes the TgP347L rabbit an excellent model for gene therapy, cell biological intervention, progenitor cell transplantation, surgical interventions, and bionic prosthetic studies.
doi:10.1002/cne.22703
PMCID: PMC3894993  PMID: 21681749
retinitis pigmentosa; retinal degeneration; retinal remodeling; neural remodeling; transgenic; electron microscopy; light microscopy; confocal microscopy; electroretinogram; computational molecular phenotyping; human; rabbit
19.  Growth and Maximum Size of Tiger Sharks (Galeocerdo cuvier) in Hawaii 
PLoS ONE  2014;9(1):e84799.
Tiger sharks (Galecerdo cuvier) are apex predators characterized by their broad diet, large size and rapid growth. Tiger shark maximum size is typically between 380 & 450 cm Total Length (TL), with a few individuals reaching 550 cm TL, but the maximum size of tiger sharks in Hawaii waters remains uncertain. A previous study suggested tiger sharks grow rather slowly in Hawaii compared to other regions, but this may have been an artifact of the method used to estimate growth (unvalidated vertebral ring counts) compounded by small sample size and narrow size range. Since 1993, the University of Hawaii has conducted a research program aimed at elucidating tiger shark biology, and to date 420 tiger sharks have been tagged and 50 recaptured. All recaptures were from Hawaii except a single shark recaptured off Isla Jacques Cousteau (24°13′17″N 109°52′14″W), in the southern Gulf of California (minimum distance between tag and recapture sites  =  approximately 5,000 km), after 366 days at liberty (DAL). We used these empirical mark-recapture data to estimate growth rates and maximum size for tiger sharks in Hawaii. We found that tiger sharks in Hawaii grow twice as fast as previously thought, on average reaching 340 cm TL by age 5, and attaining a maximum size of 403 cm TL. Our model indicates the fastest growing individuals attain 400 cm TL by age 5, and the largest reach a maximum size of 444 cm TL. The largest shark captured during our study was 464 cm TL but individuals >450 cm TL were extremely rare (0.005% of sharks captured). We conclude that tiger shark growth rates and maximum sizes in Hawaii are generally consistent with those in other regions, and hypothesize that a broad diet may help them to achieve this rapid growth by maximizing prey consumption rates.
doi:10.1371/journal.pone.0084799
PMCID: PMC3885620  PMID: 24416287
20.  Controlling fibrous capsule formation through long-term down-regulation of collagen type I (COL1A1) expression by nanofiber-mediated siRNA gene silencing 
Acta biomaterialia  2012;9(1):4513-4524.
The foreign body reaction often interferes with the long-term functionality and performance of implanted biomedical devices through fibrous capsule formation. While many implant modification techniques have been adopted in attempts to control fibrous encapsulation, the outcomes remained sub-optimal. Nanofiber scaffold-mediated RNA interference may serve as an alternative approach through the localized and sustained delivery of siRNA at implant sites. In this study, we investigated the efficacy of siRNA-PCLEEP (poly(caprolactone-co-ethylethylene phosphate) nanofibers in controlling fibrous capsule formation through the down-regulation of Collagen type I (COL1A1) in vitro and in vivo. By encapsulating complexes of COL1A1 siRNA with a transfection reagent (Transit TKO) or cell penetrating peptides (CPPs), CADY or MPG, within the nanofibers (550–650 nm in diameter), a sustained release of siRNA was obtained for at least 28 days (loading efficiency ~ 60–67%). Scaffold-mediated transfection significantly enhanced cellular uptake of oligonucleotides and prolonged in vitro gene silencing duration by at least 2–3 times as compared to conventional bolus delivery of siRNA (14 days vs 5–7 days by bolus delivery). In vivo subcutaneous implantation of siRNA scaffolds revealed a significant decrease in fibrous capsule thickness at weeks 2 and 4 as compared to plain nanofibers (p < 0.05). Taken together, the results demonstrated the efficacy of scaffold-mediated siRNA gene-silencing in providing effective long-term control of fibrous capsule formation.
doi:10.1016/j.actbio.2012.09.029
PMCID: PMC3523808  PMID: 23036951
RNA interference; Gene knockdown; Scaffold-mediated transfection; Cell penetrating peptides; Electrospinning
21.  Lymphocyte/Macrophage Interactions: Biomaterial Surface-Dependent Cytokine, Chemokine, and Matrix Protein Production 
Journal of biomedical materials research. Part A  2008;87(3):10.1002/jbm.a.31630.
The role of lymphocytes in the biological response to synthetic polymers is poorly understood despite the transient appearance of lymphocytes at the biomaterial implant site. To investigate cytokines, chemokines, and extracellular matrix (ECM) proteins produced by lymphocytes and macrophages in response to biomaterial surfaces, human peripheral blood monocytes and lymphocytes were co-cultured on polyethylene terephthalate (PET)-based material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic chemistries. Antibody array screening showed the majority of detected proteins are inflammatory mediators that guide the early inflammatory phases of wound healing. Proteomic ELISA quantification and adherent cell analysis were performed after 3, 7, and 10 days of culture. IL-2 and IFN-γ were not detected in any co-cultures suggesting lack of lymphocyte activation. The hydrophilic/neutral surfaces increased IL-8 relative to the hydrophobic PET surface (p<0.05). The hydrophilic/anionic surfaces promoted increased TNF-α over hydrophobic and cationic surfaces and increased MIP-1β compared to hydrophobic surfaces (p<0.05). Since enhanced macrophage fusion was observed on hydrophilic/anionic surfaces, the production of these cytokines likely plays an important role in the fusion process. The hydrophilic/cationic surface promoted IL-10 production and increased matrix metalloproteinase (MMP)-9/tissue inhibitor of MMP (TIMP) relative to hydrophilic/neutral and anionic surfaces (p<0.05). These results suggest hydrophilic/neutral and anionic surfaces promote pro-inflammatory responses and reduced degradation of the ECM, whereas the hydrophilic/cationic surfaces induce an anti-inflammatory response and greater MMP-9/TIMP with an enhanced potential for ECM breakdown. The study also underscores the usefulness of protein arrays in assessing the role of soluble mediators in the inflammatory response to biomaterials.
doi:10.1002/jbm.a.31630
PMCID: PMC3867010  PMID: 18200554
PET biomaterials; lymphocytes; macrophages; cytokines; matrix metalloproteinases
22.  The Foreign Body Reaction in T cell deficient mice 
Journal of biomedical materials research. Part A  2009;90(1):10.1002/jbm.a.32050.
The role/s of T lymphocytes in the foreign body response has not been thoroughly elucidated. Lymphocytes are known to augment macrophage adhesion and fusion in vitro. Furthermore T lymphocytes are a possible source of the cytokines, IL-4 and IL-13 which induce macrophage fusion. In this study we used BALB/c mice and BALB/c (nu/nu) nude mice to investgate foreign body giant cell (FBGC) formation in a T cell deficient setting. Mice were implanted with Elasthane 80A (PEU), silicone rubber (SR) or poly(ethylene terephthalate) (PET) for 7, 14, or 21 days using the cage implant system. Exudate cells and IL-4 and IL-13 levels in exudate supernatants were analyzed by flow cytometry and a multiplex immunoassay, respectively, at days 7, 14, and 21. Macrophage adhesion and fusion on material surfaces were analyzed using optical microscopy. T cell deficient mice had lower total leukocyte concentrations at the biomaterial implant site at all time points. Adherent cell density was comparable between normal and T cell deficient mice except in the PEU group at day 21. However, percent fusion, average nuclei per FBGC, and FBGC morphology was comparable between normal and T cell deficient mice. IL-4 was not detected in any samples, but IL-13 levels were also comparable between normal and T cell deficient mice indicating Th2 polarized T cells are not the sole source of this cytokine. We have shown that there are pathways that do not require thymus-matured T lymphocytes which lead to a normal foreign body response to biomaterials in a murine model.
doi:10.1002/jbm.a.32050
PMCID: PMC3864678  PMID: 18491378
foreign body response; IL-4; IL-13; T lymphocyte; biomaterials
23.  Paracrine and Juxtacrine Lymphocyte Enhancement of Adherent Macrophage and Foreign Body Giant Cell Activation 
Journal of biomedical materials research. Part A  2009;89(2):10.1002/jbm.a.31981.
Lymphocytes have been shown to be involved in modulating monocyte and macrophage behavior in the foreign body reaction. Lymphocyte effects on biomaterial-adherent macrophage and foreign body giant cell (FBGC) behavior were further investigated by culturing monocytes alone or together with lymphocytes, either in direct co-cultures or indirectly in transwells, on a series of polyethylene terephthalate (PET)-based photograft co-polymerized material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic chemistries. After periods of 3, 7, and 10 days, cytokine production was quantified by ELISA and normalized to adherent macrophage/FBGC density to yield a measure of adherent macrophage/FBGC activation. Interactions with lymphocytes enhanced adherent macrophage and FBGC production of pro-inflammatory IL-1β, TNF-α, IL-6, IL-8, and MIP-1β on the hydrophobic and hydrophilic/cationic surfaces but had no effect on anti-inflammatory IL-10 production indicating lymphocytes promote a pro-inflammatory response to biomaterials. Lymphocytes also did not significantly influence MMP-9, TIMP-1, and TIMP-2 production. Interactions through indirect (paracrine) signaling showed a significant effect in enhancing adherent macrophage/FBGC activation at early time points while interactions via direct (juxtacrine) mechanisms dominated at later time points. Biomaterial surface chemistries differentially affected the observed responses as hydrophilic/neutral and hydrophilic/anionic surfaces evoked the highest levels of activation relative to the other surfaces but did not facilitate lymphocyte enhancement of adherent macrophage/FBGC activation.
doi:10.1002/jbm.a.31981
PMCID: PMC3864690  PMID: 18437695
biomaterials; foreign body reaction; lymphocytes; macrophages; activation
24.  Quantitative in vivo Cytokine Analysis at Synthetic Biomaterial Implant Sites 
Journal of biomedical materials research. Part A  2009;89(1):10.1002/jbm.a.31939.
In order to further elucidate the foreign body reaction, investigation of cytokines at biomaterial implant sites was carried out using a multiplex immunoassay and ELISA. Macrophage activation cytokines (IL-1β, IL-6, TNFα), cytokines important for macrophage fusion (IL-4, IL-13), anti-inflammatory cytokines (IL-10, TGFβ), chemokines (GRO/KC, MCP-1), and the T cell activation cytokine IL-2 were quantified at biomaterial implant sites. Empty cages (controls) or cages containing synthetic biomedical polymer (Elasthane 80A (PEU), Silicone rubber (SR), or polyethylene terephthalate (PET)) were implanted subcutaneously in Sprague Dawley rats for 4, 7, or 14 days, and cytokines in exudate supernatants and macrophage surface adhesion and fusion were quantified. The presence of a polymer implant did not affect the levels of IL-1β, TGFβ, and MCP-1 in comparison to the control group. IL-2 was not detected in virtually any of the samples. Although the levels of IL-4, IL-13, IL-10, and GRO/KC were affected by polymer implantation, but not dependent on a specific polymer, IL-6 and TNFα were significantly greater in those animals implanted with PEU and SR, materials that do not promote fusion. The results indicate differential material dependent cytokine profiles are produced by surface adherent macrophages and foreign body giant cells in vivo.
doi:10.1002/jbm.a.31939
PMCID: PMC3864694  PMID: 18431759
cage implant system; multiplex immunoassay; cytokines; synthetic biomaterials
25.  Analysis of Deoxynivalenol and Deoxynivalenol-3-glucoside in Hard Red Spring Wheat Inoculated with Fusarium Graminearum 
Toxins  2013;5(12):2522-2532.
Deoxynivalenol (DON) is a mycotoxin affecting wheat quality. The formation of the “masked” mycotoxin deoxinyvalenol-3-glucoside (D3G) results from a defense mechanism the plant uses for detoxification. Both mycotoxins are important from a food safety point of view. The aim of this work was to analyze DON and D3G content in inoculated near-isogenic wheat lines grown at two locations in Minnesota, USA during three different years. Regression analysis showed positive correlation between DON content measured with LC and GC among wheat lines, locality and year. The relationship between DON and D3G showed a linear increase until a certain point, after which the DON content and the D3G increased. Wheat lines having higher susceptibility to Fusarium showed the opposite trend. ANOVA demonstrated that the line and location have a greater effect on variation of DON and D3G than do their interaction among years. The most important factor affecting DON and D3G was the growing location. In conclusion, the year, environmental conditions and location have an effect on the D3G/DON ratio in response to Fusarium infection.
doi:10.3390/toxins5122522
PMCID: PMC3873699  PMID: 24351715
fusarium; wheat; deoxinyvalenol; deoxynivalenol-3-glucoside

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