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1.  Novel Biomarkers of Arterial and Venous Ischemia in Microvascular Flaps 
PLoS ONE  2013;8(8):e71628.
The field of reconstructive microsurgery is experiencing tremendous growth, as evidenced by recent advances in face and hand transplantation, lower limb salvage after trauma, and breast reconstruction. Common to all of these procedures is the creation of a nutrient vascular supply by microsurgical anastomosis between a single artery and vein. Complications related to occluded arterial inflow and obstructed venous outflow are not uncommon, and can result in irreversible tissue injury, necrosis, and flap loss. At times, these complications are challenging to clinically determine. Since early intervention with return to the operating room to re-establish arterial inflow or venous outflow is key to flap salvage, the accurate diagnosis of early stage complications is essential. To date, there are no biochemical markers or serum assays that can predict these complications. In this study, we utilized a rat model of flap ischemia in order to identify the transcriptional signatures of venous congestion and arterial ischemia. We found that the critical ischemia time for the superficial inferior epigastric fasciocutaneus flap was four hours and therefore performed detailed analyses at this time point. Histolgical analysis confirmed significant differences between arterial and venous ischemia. The transcriptome of ischemic, congested, and control flap tissues was deciphered by performing Affymetrix microarray analysis and verified by qRT-PCR. Principal component analysis revealed that arterial ischemia and venous congestion were characterized by distinct transcriptomes. Arterial ischemia and venous congestion was characterized by 408 and 1536>2-fold differentially expressed genes, respectively. qRT-PCR was used to identify five candidate genes Prol1, Muc1, Fcnb, Il1b, and Vcsa1 to serve as biomarkers for flap failure in both arterial ischemia and venous congestion. Our data suggests that Prol1 and Vcsa1 may be specific indicators of venous congestion and allow clinicians to both diagnose and successfully treat microvascular complications before irreversible tissue damage and flap loss occurs.
PMCID: PMC3743756  PMID: 23977093
2.  9-Cis Retinoic Acid Promotes Lymphangiogenesis and Enhances Lymphatic Vessel Regeneration: Therapeutic Implications of 9-Cis Retinoic Acid for Secondary Lymphedema 
Circulation  2012;125(7):872-882.
The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction due to genetic defects or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swellings often associated with fibrosis and recurrent infections without available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema.
Methods and Results
We report that RAs promote proliferation, migration and tube formation of cultured lymphatic endothelial cells (LECs) by activating FGF-receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2 and the aurora kinases through both an Akt-mediated non-genomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals based on mouse trachea, matrigel plug and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating the experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration.
These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients.
PMCID: PMC3327127  PMID: 22275501
lymphangiogenesis; lymphedema; retinoic acids; lymphatic regeneration; therapy
3.  The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity 
Nature  2012;483(7391):603-607.
The systematic translation of cancer genomic data into knowledge of tumor biology and therapeutic avenues remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacologic annotation is available1. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number, and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacologic profiles for 24 anticancer drugs across 479 of the lines, this collection allowed identification of genetic, lineage, and gene expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Altogether, our results suggest that large, annotated cell line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of “personalized” therapeutic regimens2.
PMCID: PMC3320027  PMID: 22460905
4.  Interfering with Resistance to Smoothened Antagonists by Inhibition of the PI3K Pathway in Medulloblastoma 
Science translational medicine  2010;2(51):51ra70.
Mutations in Hedgehog (Hh) pathway genes, leading to constitutive activation of Smoothened (Smo), occur in medulloblastoma. Antagonists of Smo induce tumor regression in mouse models of medulloblastoma and hold great promise for treating this disease. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Here, we describe novel mechanisms of acquired resistance to Smo antagonists in medulloblastoma. NVP-LDE225, a potent and selective Smo antagonist, inhibits Hh signaling and induces tumor regressions in allograft models of medulloblastoma that are driven by mutations of Patched (Ptch), a tumor suppressor in the Hh pathway. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed distinct resistance mechanisms. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, or more rarely point mutations in Smo led to reactivated Hh signaling and restored tumor growth. Unexpectedly, analysis of pathway gene-expression signatures selectively deregulated in resistant tumors identified increased phosphoinositide 3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we demonstrated that the combination of NVP-LDE225 with the PI3K class I inhibitor NVP-BKM120 or the dual PI3K/mTOR inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma.
PMCID: PMC3422576  PMID: 20881279
5.  Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and -independent mechanisms 
Nature medicine  2011;17(9):1116-1120.
PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy1–4, making PI3K an attractive target for cancer therapy. Despite the great promise of targeted therapy, resistance often develops, resulting in treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CAH1047R. Surprisingly, most PIK3CAH1047R-driven mammary tumors recurred following PIK3CAH1047R inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of c-Met or c-Myc. While c-Met amplification allowed tumor survival dependent on activation of endogenous PI3K, tumors with c-Myc amplification became independent of the PI3K pathway. Functional analyses demonstrated that c-Myc contributed to oncogene independence and resistance to PI3K inhibition. Importantly, PI3KCA mutations and increased c-MYC levels co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.
PMCID: PMC3169724  PMID: 21822287
6.  Laboratory Studies of Feeding and Oviposition Preference, Developmental Performance, and Survival of the Predatory Beetle, Sasajiscymnus tsugae on Diets of the Woolly Adelgids, Adelges tsugae and Adelges piceae  
The suitability of the balsam woolly adelgid, Adelges piceae Ratzeburg (Hemiptera: Adelgidae) as an alternate mass rearing host for the adelgid predator, Sasajiscymnus tsugae Sasaji and McClure (Coleoptera: Coccinellidae) was studied in the laboratory. This predator is native to Japan and has been introduced to eastern hemlock, Tsuga canadensis (L.) Carrière (Pinales: Pinaceae), forests throughout the eastern United States for biological control of the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), also of Japanese origin. Feeding, oviposition, immature development, and adult long-term survival of S. tsugae were tested in a series of no choice (single-prey) and paired-choice experiments between the primary host prey, A. tsugae, and the alternate host prey, A. piceae. In paired-choice feeding tests, the predator did not discriminate between eggs of the two adelgid species, but in the no choice tests the predator did eat significantly more eggs of A. piceae than those of A. tsugae. S. tsugae accepted both test prey for oviposition and preferred to lay eggs on adelgid infested versus noninfested host plants. Overall oviposition rates were very low (< 1 egg per predator female) in the oviposition preference tests. Predator immature development rates did not differ between the two test prey, but only 60% of S. tsugae survived egg to adult development when fed A. piceae compared to 86% when fed A. tsugae. S. tsugae adult long-term survival was significantly influenced (positively and negatively) by prey type and the availability of a supplemental food source (diluted honey) when offered aestivating A. tsugae sistens nymphs or ovipositing aestivosistens A. piceae adults, but not when offered ovipositing A. tsugae sistens adults. These results suggest that the development of S. tsugae laboratory colonies reared on a diet consisting only of A. piceae may be possible, and that the biological control potential of the predator might be expanded to include management of A. piceae in Christmas tree plantations.
PMCID: PMC3385972  PMID: 21867435
Abies fraseri; alternate rearing host; balsam woolly adelgid; biological control; hemlock woolly adelgid; prey suitability; Tsuga canadensis
7.  Two Novel Techniques to Screen Abies Seedlings for Resistance to the Balsam Woolly Adelgid, Adelges piceae  
Since its introduction into the Southern Appalachians in the 1950s, the balsam woolly adelgid, Adelges piceae Ratzeburg (Hemiptera: Adelgidae), has devastated native populations of Fraser fir, Abies fraseri (Pursh) Poir. (Pinales: Pinaceae), and has become a major pest in Christmas tree plantations requiring expensive chemical treatments. Adelges piceae—resistant Fraser fir trees would lessen costs for the Christmas tree industry and assist in the restoration of native stands. Resistance screening is an important step in this process. Here, four studies directed toward the development of time— and cost—efficient techniques for screening are reported. In the first study, three methods to artificially infest seedlings of different ages were evaluated in a shade—covered greenhouse. Two—year—old seedlings had much lower infestation levels than 7 year—old seedlings. Placing infested bark at the base of the seedling was less effective than tying infested bark to the seedling or suspending infested bolts above the seedling. Although the two latter techniques resulted in similar densities on the seedlings, they each have positive and negative considerations. Attaching bark to uninfested trees is effective, but very time consuming. The suspended bolt method mimics natural infestation and is more economical than attaching bark, but care must be taken to ensure an even distribution of crawlers falling onto the seedlings. The second study focused on the density and distribution of crawlers falling from suspended bolts onto paper gridded into 7.6 × 7.6 cm cells. Crawler density in a 30 cm band under and to each side of the suspended bolt ranged from 400 to over 3000 crawlers per cell (1 to 55 crawlers per cm2). In the third study, excised branches from 4 year—old A. fraseri and A. vetchii seedlings were artificially infested with A. piceae to determine whether this technique may be useful for early resistance screening. The excised A. fraseri branches supported complete adelgid development (crawler to egg—laying adult), and very little adelgid development occurred on A. vetchii branches. The fourth study compared infestation levels and gouting response on excised versus intact branches of 4 year—old A. fraseri seedlings from three different seed sources, and excised branches from 4 year—old and 25 year—old trees. There were no differences in infestation levels between excised versus intact branches nor in very young versus mature trees; gouting response was observed only on intact branches.
PMCID: PMC3391932  PMID: 22239164
Abies fraseri; artificial infestation; excised branches; Fraser fir; host resistance
8.  Substance Use, Symptom, and Employment Outcomes of Persons With a Workplace Mandate for Chemical Dependency Treatment 
This study examined the role of workplace mandates to chemical dependency treatment in treatment adherence, alcohol and drug abstinence, severity of employment problems, and severity of psychiatric problems.
The sample included 448 employed members of a private, nonprofit U.S. managed care health plan who entered chemical dependency treatment with a workplace mandate (N=75) or without one (N=373); 405 of these individuals were followed up at one year (N=70 and N=335, respectively), and 362 participated in a five-year follow up (N=60 and N=302, respectively). Propensity scores predicting receipt of a workplace mandate were calculated. Logistic regression and ordinary least-squares regression were used to predict length of stay in chemical dependency treatment, alcohol and drug abstinence, and psychiatric and employment problem severity at one and five years.
Overall, participants with a workplace mandate had one- and five-year outcomes similar to those without such a mandate. Having a workplace mandate also predicted longer treatment stays and improvement in employment problems. When other factors related to outcomes were controlled for, having a workplace mandate predicted abstinence at one year, with length of stay as a mediating variable.
Workplace mandates can be an effective mechanism for improving work performance and other outcomes. Study participants who had a workplace mandate were more likely than those who did not have a workplace mandate to be abstinent at follow-up, and they did as well in treatment, both short and long term. Pressure from the workplace likely gets people to treatment earlier and provides incentives for treatment adherence.
PMCID: PMC2878200  PMID: 19411353
9.  The Specificity of the FOXL2 c.402C>G Somatic Mutation: A Survey of Solid Tumors 
PLoS ONE  2009;4(11):e7988.
A somatic mutation in the FOXL2 gene is reported to be present in almost all (97%; 86/89) morphologically defined, adult-type, granulosa-cell tumors (A-GCTs). This FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W). It was also found in a minority of other ovarian malignant stromal tumors, but not in benign ovarian stromal tumors or unrelated ovarian tumors or breast cancers.
Methodology/Principal Findings
Herein we studied other cancers and cell lines for the presence of this mutation. We screened DNA from 752 tumors of epithelial and mesenchymal origin and 28 ovarian cancer cell lines and 52 other cancer cell lines of varied origin. We found the FOXL2 c.402C>G mutation in an unreported A-GCT case and the A-GCT-derived cell line KGN. All other tumors and cell lines analyzed were mutation negative.
In addition to proving that the KGN cell line is a useful model to study A-GCTs, these data show that the c.402C>G mutation in FOXL2 is not commonly found in a wide variety of other cancers and therefore it is likely pathognomonic for A-GCTs and closely related tumors.
PMCID: PMC2777318  PMID: 19956657
10.  Comparison of antipsychotic medication effects on reducing violence in people with schizophrenia 
Violence is an uncommon but significant problem associated with schizophrenia.
To compare antipsychotic medications in reducing violence among patients with schizophrenia over 6 months, identify prospective predictors of violence and examine the impact of medication adherence on reduced violence.
Participants (n=1445) were randomly assigned to double-blinded treatment with one of five antipsychotic medications. Analyses are presented for the intention-to-treat sample and for patients completing 6 months on assigned medication.
Violence declined from 16% to 9% in the retained sample and from 19% to 14% in the intention-to-treat sample. No difference by medication group was found, except that perphenazine showed greater violence reduction than quetiapine in the retained sample. Medication adherence reduced violence, but not in patients with a history of childhood antisocial conduct. Prospective predictors of violence included childhood conduct problems, substance use, victimisation, economic deprivation and living situation. Negative psychotic symptoms predicted lower violence.
Newer antipsychotics did not reduce violence more than perphenazine. Effective antipsychotics are needed, but may not reduce violence unrelated to acute psychopathology.
PMCID: PMC2801826  PMID: 18700216
11.  Housing and use of leverage in mental health treatment 
BMC Psychiatry  2007;7(Suppl 1):S27.
PMCID: PMC3332843
12.  Mandated community treatment: a promising concept for world psychiatry? 
BMC Psychiatry  2007;7(Suppl 1):S7.
PMCID: PMC3332890
13.  Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene reduces the vasoconstrictive response 
Adeno-associated virus (AAV) has a number of attractive features for gene therapy including the ability to transduce nondividing cells and long term transgene expression.
To investigate whether the endothelial constitutive nitric oxide synthase (ecNOS) gene can be efficiently introduced into rat aortic segments using AAV vectors and thereby modulate the vasoconstrictive response.
Excised rat aortas were incubated with medium containing ecNOS-expressing AAV vectors (AAV-ecNOS). Expression of ecNOS in the aortic segments was evaluated by immunohistochemical staining. The isometric tension of the aortic segments transduced with AAV-ecNOS was measured.
Adventitial cells in rat aortic segments were efficiently transduced with AAV-ecNOS. The vasoconstrictive response induced by 30 mmol/L K+ was enhanced in endothelium-denuded aortic segments compared with intact aortic segments. However, in endothelium-denuded aortic segments transduced with AAV-ecNOS, the enhancement of the vasoconstrictive response disappeared. This effect induced by ecNOS gene transfer was abolished in the presence of the nitric oxide synthase inhibitor NG-monomethyl-l-arginine acetate.
These results show that ecNOS gene transfer using AAV vectors abolishes the pathological enhancement of the vasoconstrictive response in endothelium-denuded aortic segments.
PMCID: PMC2858966  PMID: 20428445
Endothelium; Gene transfer; Nitric oxide; Vasoconstriction
14.  An RNA fraction in chromatin of L-929 cells associated with DNA 
Nucleic Acids Research  1974;1(11):1421-1437.
The DNA of L-929 cell nuclei was examined for the presence of an RNA fraction hybridized to the DNA that could possibly serve a role in gene regulation. A low molecular weight ([unk]4.5S) RNA fraction was observed as an RNA-DNA complex in chromatin preparations subfractionated on a Cs2SO4 density gradient. This RNA does not appear to be covalently attached to the DNA. Neither does it appear to be attached to the DNA via a protein component. The RNA is resistant to ribonuclease H but is slowly attached by ribonuclease A + Tl. A role for, as well as the nature of the attachment of this RNA to DNA is suggested.
PMCID: PMC343422  PMID: 10793701
15.  Isopycnic centrifugation of sheared L-cell chromatin in metrizamide gradients 
Nucleic Acids Research  1974;1(11):1359-1370.
Isopycnic gradient centrifugation of L-929 cell chromatin in a 38% (W/V) metrizamide solution yields two distinct fractions. The fraction banding at a density of 1.24 gm/cm2 (H chromatin) contains about 10% of the DNA present in the fraction banding at a density of 1.18 gm/cm2 (L chromatin). Both fractions contain the same proportions of satellite to main band DNA's. Some differences can be seen in the DNA: protein ratios and types of proteins present in the H and L chromatin fractions.
PMCID: PMC343417  PMID: 10793696

Results 1-15 (15)