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1.  Comparisons of Thyroid Hormone, Intelligence, Attention, and Quality of Life in Children with Obstructive Sleep Apnea Hypopnea Syndrome before and after Endoscopic Adenoidectomy 
BioMed Research International  2015;2015:523716.
Objective. The aim of this study was to compare the differences in thyroid hormone, intelligence, attention, and quality of life (QoL) of children with obstructive sleep apnea hypopnea syndrome (OSAHS) before and after endoscopic adenoidectomy. Method. A total of 35 OSAHS children (21 males and 14 females with a mean age of 6.81 ± 1.08 years) were included in this study for analyzing the levels of thyroid hormone, intelligence, attention, and QoL. There were 22 children underwent endoscopic adenoidectomy with bilateral tonsillectomy (BT), while the other 13 children who underwent endoscopic adenoidectomy without bilateral tonsillectomy without BT. Results. Our results revealed no significant difference in serum free triiodothyronine (FT3), free thyroxine (FT4), and thyroid stimulating hormone (TSH) levels in OSAHS children before and after endoscopic adenoidectomy (all P > 0.05). However, there were significant differences in full-scale intelligence quotient (FIQ) (92.45 ± 5.88 versus 106.23 ± 7.39, P < 0.001), verbal intelligence quotient (VIQ) (94.17 ± 15.01 versus 103.91 ± 9.74, P = 0.006), and performance intelligence quotient (PIQ) (94.12 ± 11.04 versus 104.31 ± 10.05, P = 0.001), attention (98.48 ± 8.74 versus 106.87 ± 8.58, P < 0.001), and total OSA-18 scores (87.62 ± 17.15 versus 46.61 ± 10.15, P < 0.001) between before and after endoscopic adenoidectomy in OSAHS children. Conclusion. Our findings provided evidence that the intelligence, attention, and QoL of OSAHS children may be significantly improved after endoscopic adenoidectomy.
PMCID: PMC4310307  PMID: 25654109
2.  Towards accurate characterization of clonal heterogeneity based on structural variation 
BMC Bioinformatics  2014;15(1):299.
Recent advances in deep digital sequencing have unveiled an unprecedented degree of clonal heterogeneity within a single tumor DNA sample. Resolving such heterogeneity depends on accurate estimation of fractions of alleles that harbor somatic mutations. Unlike substitutions or small indels, structural variants such as deletions, duplications, inversions and translocations involve segments of DNAs and are potentially more accurate for allele fraction estimations. However, no systematic method exists that can support such analysis.
In this paper, we present a novel maximum-likelihood method that estimates allele fractions of structural variants integratively from various forms of alignment signals. We develop a tool, BreakDown, to estimate the allele fractions of most structural variants including medium size (from 1 kilobase to 1 megabase) deletions and duplications, and balanced inversions and translocations.
Evaluation based on both simulated and real data indicates that our method systematically enables structural variants for clonal heterogeneity analysis and can greatly enhance the characterization of genomically instable tumors.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2105-15-299) contains supplementary material, which is available to authorized users.
PMCID: PMC4165998  PMID: 25201439
Structural variation; Clonal heterogeneity; Variant allele fraction
3.  Alcohol Causes Alveolar Epithelial Oxidative Stress by Inhibiting the Nuclear Factor (Erythroid-Derived 2)–Like 2–Antioxidant Response Element Signaling Pathway 
Excessive alcohol use increases the risk of acute lung injury and pneumonia. Chronic alcohol ingestion causes oxidative stress within the alveolar space, including near depletion of glutathione (GSH), which impairs alveolar epithelial and macrophage function, in experimental animals and human subjects. However, the fundamental mechanism(s) by which alcohol induces such profound lung oxidative stress is unknown. Nuclear factor (erythroid-derived 2)–like 2 (Nrf2) is a redox-sensitive master transcription factor that regulates activation of the antioxidant response element (ARE). As the alveolar epithelium controls GSH levels within the alveolar space, we hypothesized that alcohol also decreases Nrf2 expression and/or activation within the alveolar epithelium. In this study, we determined that alcohol ingestion in vivo or direct alcohol exposure in vitro down-regulated the Nrf2–ARE pathway in lung epithelial cells, decreased the expression of antioxidant genes, and lowered intracellular GSH levels. RNA silencing of Nrf2 gene expression in alveolar epithelial cells in vitro decreased expression of these same antioxidant genes, and likewise lowered intracellular GSH levels, findings that mirrored the effects of alcohol. In contrast, treating alcohol-exposed alveolar epithelial cells in vitro with the Nrf2 activator, sulforaphane, preserved Nrf2 expression, ARE activation, intracellular GSH levels, and epithelial barrier function. These new experimental findings implicate down-regulation of the Nrf2–ARE signaling pathway as a fundamental mechanism by which alcohol causes profound oxidative stress and alveolar epithelial dysfunction, and suggest that treatments, such as sulforaphane, that activate this pathway could mitigate the pathophysiological consequences of alcohol on the lung and other organs.
PMCID: PMC4080903  PMID: 23306837
acute respiratory distress syndrome; glutathione; lung; redox signaling; oxidative stress
4.  Mechanism of blood-retinal barrier breakdown induced by HIV-1 (Review) 
Human immunodeficiency virus (HIV)-1 has been detected in ocular tissues; however, the mechanism of entry has not been established. It has been hypothesized that the blood-retinal barrier (BRB), a critical guardian against microbial invasion of the eye, may be compromised in the presence of HIV-1 in the eye. In vivo and in vitro model systems have shown that the breach of tight junctions induced by HIV-1-associated factors contributes to the breakdown of the BRB. The present study reviews the mechanism of tight junction disruption, focusing on signaling pathways, the expression of enzymes, including metalloproteinases, and cytokines that affect inflammation. The studied pathways may be potential targets for the prevention of ocular HIV complications.
PMCID: PMC3961112  PMID: 24660027
actin cytoskeleton; blood-retinal barrer; caveolin-1; matrix metalloproteinases; ocular HIV-1; tight junction
5.  BreakTrans: uncovering the genomic architecture of gene fusions 
Genome Biology  2013;14(8):R87.
Producing gene fusions through genomic structural rearrangements is a major mechanism for tumor evolution. Therefore, accurately detecting gene fusions and the originating rearrangements is of great importance for personalized cancer diagnosis and targeted therapy. We present a tool, BreakTrans, that systematically maps predicted gene fusions to structural rearrangements. Thus, BreakTrans not only validates both types of predictions, but also provides mechanistic interpretations. BreakTrans effectively validates known fusions and discovers novel events in a breast cancer cell line. Applying BreakTrans to 43 breast cancer samples in The Cancer Genome Atlas identifies 90 genomically validated gene fusions. BreakTrans is available at
PMCID: PMC4054677  PMID: 23972288
6.  Background mutations in parental cells account for most of the genetic heterogeneity of Induced Pluripotent Stem Cells 
Cell Stem Cell  2012;10(5):570-582.
To assess the genetic consequences of induced Pluripotent Stem Cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. This data suggests that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which “captures” their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.
PMCID: PMC3348423  PMID: 22542160
7.  Improvement in visual function and quality of life following a blindness prevention surgery program in a rural area of Eastern China 
The aim of this study was to assess the changes in visual function (VF) and quality of life (QOL) among patients following blindness prevention surgery in a rural area of Eastern China. The prospective study selected cataract patients via mobile eye screening camps. VF and QOL questionnaires originally developed by Fletcher et al were completed prior to and 6 months after surgery. Small-incision cataract surgery (SICS) with posterior chamber intraocular lens (IOL) implantation was performed on patients by a blindness prevention surgery group. The VF and QOL scores of 178 cataract patients preoperatively were 48.58±31.18 and 65.97±26.77, respectively. The scores decreased in proportion to decreasing vision status. The VF and QOL scale scores were significantly correlated with the vision grade of the patient (rVF=−17.2093, t=−10.87, P<0.001, rQOL=−13.1399, t=−8.87, P<0.001) and age (rVF=−0.6505, t=−3.87, P<0.001, rQOL=− 0.3309, t=−2.10, P=0.037). A total of 131 patients responded to the second survey, VF and QOL scores increased significantly over a six-month postoperative period (VF=83.21±16.40, P<0.001; QOL=86.53±16.33, P<0.001). The VF scale scores were correlated with the grade of vision and residence area, the QOL scale scores were correlated with the grade of vision and gender. The VF and QOL of patients were significantly improved by performing SICS with posterior chamber IOL implantation collectively in a short period in rural areas of Eastern China. It is important to follow-up cataract patients postoperatively as untreated complications of the surgery may affect the stability of VF and QOL postoperatively.
PMCID: PMC3702720  PMID: 23837062
prevent blindness; quality of life; visual function; cataract; surgery
8.  CREST maps somatic structural variation in cancer genomes with base-pair resolution 
Nature methods  2011;8(8):652-654.
We developed CREST (Clipping REveals STructure), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80% demonstrating that CREST had a high predictive accuracy.
PMCID: PMC3527068  PMID: 21666668
9.  A Multi-Compartment Segmentation Framework With Homeomorphic Level Sets 
The simultaneous segmentation of multiple objects is an important problem in many imaging and computer vision applications. Various extensions of level set segmentation techniques to multiple objects have been proposed; however, no one method maintains object relationships, preserves topology, is computationally efficient, and provides an object-dependent internal and external force capability. In this paper, a framework for segmenting multiple objects that permits different forces to be applied to different boundaries while maintaining object topology and relationships is presented. Because of this framework, the segmentation of multiple objects each with multiple compartments is supported, and no overlaps or vacuums are generated. The computational complexity of this approach is independent of the number of objects to segment, thereby permitting the simultaneous segmentation of a large number of components. The properties of this approach and comparisons to existing methods are shown using a variety of images, both synthetic and real.
PMCID: PMC3516193  PMID: 23223164
10.  Chronic alcohol ingestion exacerbates lung epithelial barrier dysfunction in HIV-1 transgenic rats 
Alcohol abuse and HIV-1 infection frequently co-exist and these individuals are at high risk for serious lung infections and respiratory failure. Although alcohol ingestion and HIV-1 transgene expression have been shown to independently cause oxidative stress and disrupt alveolar epithelial barrier function in experimental models, their interactive effects have not been examined.
Methods and Results
In this study we determined that chronic alcohol ingestion (12 wks) exacerbated the already significant defects in alveolar epithelial paracellular permeability and lung liquid clearance in HIV-1 transgenic rats. Further, immunocytochemical analyses of tight junction protein expression in primary alveolar epithelial cells showed that occludin and zonula occludens-1 (ZO-1) localization within the plasma membrane was more disrupted than in either condition alone, consistent with the observed defects in epithelial barrier function. Interestingly, expression of Nrf2, the transcription factor required to activate the antioxidant response element, was decreased in primary alveolar epithelial cells isolated from HIV-1 transgenic rats. In parallel, exposing lung epithelial cells in vitro to either alcohol or the HIV-related protein gp120 also decreased Nrf2 expression. Importantly, treatment with procysteine, which increases thiol antioxidants including glutathione, improved tight junction protein localization in the plasma membrane and restored alveolar epithelial barrier function in alcohol-fed HIV-1 transgenic rats.
These results provide novel evidence that HIV-related proteins and alcohol together causes more barrier dysfunction in the lung epithelium than either stress alone. However, these significant effects on the alveolar barrier can be mitigated by augmenting the thiol antioxidant pool, a strategy with potential clinical applications in subjects who are highly vulnerable to lung disease because of co-existent alcohol abuse and HIV infection.
PMCID: PMC3157600  PMID: 21569054
alveolar barrier function; tight junction proteins; Nrf2; procysteine; glutathione
11.  Clonal Architecture of Secondary Acute Myeloid Leukemia 
The New England Journal of Medicine  2012;366(12):1090-1098.
The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood.
We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations.
Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene.
Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.)
PMCID: PMC3320218  PMID: 22417201
12.  Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models 
To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models.
TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy.
After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575µm in thickness during the monitoring period. A 4-5 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared.
The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.
PMCID: PMC3428535  PMID: 22937499
tissue-engineered human corneal epithelium; limbal stem cell deficiency rabbit; lamellar keratoplasty; human corneal epithelial cells; denuded amniotic membrane; reconstruction
13.  Zinc supplementation restores PU.1 and Nrf2 nuclear binding in alveolar macrophages and improves redox balance and bacterial clearance in the lungs of alcohol-fed rats 
Chronic alcohol abuse causes oxidative stress, impairs alveolar macrophage immune function, and increases the risk of pneumonia and acute lung injury. Recently we determined that chronic alcohol ingestion in rats decreases zinc levels and macrophage function in the alveolar space; provocative findings in that zinc is essential for normal immune and antioxidant defenses. Alveolar macrophage immune function depends on stimulation by GM-CSF, which signals via the transcription factor PU.1. In parallel, the antioxidant response element signals via the transcription factor Nrf2. However, the role of zinc bioavailability on these signaling pathways within the alveolar space is unknown.
To determine the efficacy of dietary zinc supplementation on lung bacterial clearance and oxidative stress, we tested three different groups of rats: control-fed, alcohol-fed, and alcohol-fed with zinc supplementation. Rats were then inoculated with intratracheal Klebsiella pneumoniae and lung bacterial clearance was determined 24 hrs later. Isolated alveolar macrophages were isolated from uninfected animals and evaluated for oxidative stress and signaling through PU.1 and Nrf2.
Alcohol-fed rats had a 5-fold decrease in lung bacterial clearance compared to control-fed rats. Dietary zinc supplementation of alcohol-fed rats normalized bacterial clearance and mitigated oxidative stress in the alveolar space, as reflected by the relative balance of the thiol redox pair cysteine and cystine, and increased nuclear binding of both PU.1 and Nrf2 in alveolar macrophages from alcohol-fed rats.
Dietary zinc supplementation prevents alcohol-induced alveolar macrophage immune dysfunction and oxidative stress in a relevant experimental model, suggesting that such a strategy could decrease the risk of pneumonia and lung injury in individuals with alcohol use disorders.
PMCID: PMC3128659  PMID: 21447000
rat; macrophages; GM-CSF; oxidative stress; lung; bacterial infections; zinc
14.  A Multiple Geometric Deformable Model Framework for Homeomorphic 3D Medical Image Segmentation 
This paper presents a 3D segmentation framework for multiple objects or compartments embedded as level sets. Thanks to a compact representation of the level set functions of multiple objects, the framework guarantees no overlap and vacuum, and leads to a computationally efficient evolution scheme largely independent of the number of objects. Appropriate topology constraints ensure not only that the topology of each object remains the same, but that the relationship between objects is also maintained. The decomposition of objects makes the framework specifically attractive to the segmentation of related anatomical regions or the parcellation of an organ, where relationships must be maintained and different evolution forces are needed on different parts of the objects interface. Examples of 3D whole brain segmentation and thalamic parcellation demonstrate the potential of our method for such segmentation tasks.
PMCID: PMC3227018  PMID: 22140657
15.  Sequencing a mouse acute promyelocytic leukemia genome reveals genetic events relevant for disease progression 
The Journal of Clinical Investigation  2011;121(4):1445-1455.
Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). It is characterized by the t(15;17)(q22;q11.2) chromosomal translocation that creates the promyelocytic leukemia–retinoic acid receptor α (PML-RARA) fusion oncogene. Although this fusion oncogene is known to initiate APL in mice, other cooperating mutations, as yet ill defined, are important for disease pathogenesis. To identify these, we used a mouse model of APL, whereby PML-RARA expressed in myeloid cells leads to a myeloproliferative disease that ultimately evolves into APL. Sequencing of a mouse APL genome revealed 3 somatic, nonsynonymous mutations relevant to APL pathogenesis, of which 1 (Jak1 V657F) was found to be recurrent in other affected mice. This mutation was identical to the JAK1 V658F mutation previously found in human APL and acute lymphoblastic leukemia samples. Further analysis showed that JAK1 V658F cooperated in vivo with PML-RARA, causing a rapidly fatal leukemia in mice. We also discovered a somatic 150-kb deletion involving the lysine (K)-specific demethylase 6A (Kdm6a, also known as Utx) gene, in the mouse APL genome. Similar deletions were observed in 3 out of 14 additional mouse APL samples and 1 out of 150 human AML samples. In conclusion, whole genome sequencing of mouse cancer genomes can provide an unbiased and comprehensive approach for discovering functionally relevant mutations that are also present in human leukemias.
PMCID: PMC3069786  PMID: 21436584
16.  Zinc Deficiency Mediates Alcohol-Induced Alveolar Epithelial and Macrophage Dysfunction in Rats 
Chronic alcohol abuse impairs both alveolar epithelial and macrophage function, and renders individuals susceptible to acute lung injury, pneumonia, and other serious lung diseases. Zinc deficiency, which is known to impact both epithelial and immune cell functions, is also associated with alcohol abuse. In this study, chronic alcohol ingestion (6 wk) in rats altered expression of key zinc transporters and storage proteins in the small intestine and the lung, and decreased zinc levels in the alveolar compartment. Zinc supplementation of alveolar epithelial monolayers derived from alcohol-fed rats in vitro, or of the diets of alcohol-fed rats in vivo, restored alveolar epithelial barrier function, and these improvements were associated with salutary changes in tight junction protein expression and membrane localization. In parallel, dietary zinc supplementation increased intracellular zinc levels, GM-CSF receptor expression, and bacterial phagocytic capacity in the alveolar macrophages of alcohol-fed rats. Together, these studies implicate zinc deficiency as a novel mechanism mediating alcohol-induced alveolar epithelial and macrophage dysfunction. Importantly, these findings argue that dietary supplementation can overcome alcohol-induced zinc deficiency and restore alveolar epithelial and macrophage function, and therefore could be an effective treatment for the susceptible alcoholic lung phenotype.
PMCID: PMC2715909  PMID: 19109243
GM-CSF; phagocytosis; tight junctions; zinc transporters; metallothionein
17.  HIV-1–Transgene Expression in Rats Decreases Alveolar Macrophage Zinc Levels and Phagocytosis 
HIV-1 infection impairs alveolar macrophage immune function and renders patients susceptible to pneumonia by poorly understood mechanisms. Alveolar macrophage maturation and function depends on granulocyte-macrophage colony–stimulating factor (GM-CSF), which is produced and secreted by the alveolar epithelium. Macrophages respond to GM-CSF through the GM-CSF receptor (GM-CSFR), which has a binding subunit (GM-CSFRα) and a signaling subunit (GM-CSFRβ). In this study, we measured GM-CSFR expression and alveolar macrophage function in a transgene HIV-1 rat model (NL4-3Δ gag/pol); this construct bears a pro-virus with gag and pol deleted, but other HIV-1–related proteins, such as gp120 and Tat, are expressed, and the rats develop an AIDS-like phenotype as they age. We first determined that HIV-1–transgenic expression selectively decreased alveolar macrophage expression of GM-CSFRβ and impaired bacterial phagocytosis in vitro. Next, we examined the role of zinc (Zn) deficiency as a potential mechanism underlying these effects, and determined that HIV-1–transgenic rats have significantly lower levels of Zn in the alveolar space and macrophages. To test the direct effect of Zn deficiency on macrophage dysfunction, we treated rat alveolar macrophage cell line with a Zn chelator, N,N,N′,N′-tetrakis-(2-pyridyl-methyl) ethylenediamine, and this decreased GM-CSFRβ expression and phagocytosis. In parallel, treatment with Zn acetate in vitro for 48 hours restored intracellular Zn levels and phagocytic function in alveolar macrophages from HIV-1–transgenic rats. Taken together, these data suggest that pulmonary Zn deficiency could be one of the mechanisms by which chronic HIV-1 infection impairs alveolar macrophage immune function and renders these individuals susceptible to serious lung infections.
PMCID: PMC2542456  PMID: 18314538
AIDS; lung; monocyte/macrophages; phagocytosis; rodent
18.  The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells 
Bone  2008;43(1):48-54.
Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(−/−) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(−/−) mice showed significant mechanical repression of RANKL expression (p<0.05). Mechanical strain still increased NO production in the absence of eNOS, and was abolished by SMTC, a specific nNOS inhibitor. nNOS mRNA and protein expression were increased by strain in eNOS(−/−) but not in WT cells, revealing that nNOS was mechanically sensitive. When NO synthesis was blocked with either SMTC or siRNA targeting nNOS in eNOS(−/−) cells however, strain still was able to suppress RANKL expression by 34%. This indicated that strain suppression of RANKL can also occur through non-NO dependent pathways. While our results confirm the importance of NO in the mechanical control of skeletal remodeling, they also suggest alternative signaling pathways by which mechanical force can produce anti-catabolic effects on the skeleton.
PMCID: PMC2532985  PMID: 18440890
nNOS; eNOS; mechanical strain; RANKL; bone remodeling
19.  HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction 
HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined.
HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization.
Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.
PMCID: PMC2644707  PMID: 19193217
20.  Chronic alcohol ingestion alters claudin expression in the alveolar epithelium of rats 
Alcohol (Fayetteville, N.Y.)  2007;41(5):371-379.
Previously we determined that chronic alcohol ingestion (6 wks) in rats increases lung epithelial permeability in vivo ~5-6-fold and promotes flooding of the alveolar airspaces with proteinaceous fluid in response to stresses such as sepsis. In parallel, alveolar epithelial cells isolated from alcohol-fed rats fail to form tight monolayers in vitro, even when cultured for up to 8 days in the absence of alcohol. However, the molecular mechanisms underlying alcohol-induced permeability are unknown. Claudins are key components of tight junctions that restrict the paracellular movement of water, proteins, and solutes across cellular barriers including the alveolar epithelium. In this study, we examined the expression of multiple members of the claudin protein family in the lungs of alcohol-fed vs. control-fed rats (Lieber-DeCarli liquid diet with 36% of calories as alcohol vs. maltin-dextrin for 6 wks). We determined that chronic alcohol ingestion affected the expression of multiple claudins; most striking were decreases in claudin-1 and claudin-7, and an increase in claudin-5, in the whole lung and in alveolar epithelial monolayers derived from alcohol-fed rats. In parallel, immunocytochemistry of alveolar epithelial monolayers from alcohol-fed rats revealed abnormal intracellular accumulation of claudin-7 protein and relatively decreased localization to cell membranes. Claudin-1 and claudin-7 are relatively specific to alveolar epithelial type I pneumocytes that form the vast majority of the alveolar epithelial barrier in vivo, and increases in claudin-5 have been associated with increased epithelial permeability in other systems. Therefore, these findings suggest that changes in claudin expression in the alveolar epithelium produce a “leakier” phenotype that renders the alcoholic lung susceptible to alveolar flooding during acute inflammatory stresses.
PMCID: PMC2048749  PMID: 17889313
claudin; ARDS; ethanol; type II pneumocyte; type I pneumocyte; tight junction

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