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1.  Expression patterns, molecular markers and genetic diversity of insect-susceptible and resistant Barbarea genotypes by comparative transcriptome analysis 
BMC Genomics  2015;16(1):486.
Background
Barbarea vulgaris contains two genotypes: the glabrous type (G-type), which confers resistance to the diamondback moth (DBM) and other insect pests, and the pubescent type (P-type), which is susceptible to the DBM. Herein, the transcriptomes of P-type B. vulgaris before and after DBM infestation were subjected to Illumina (Solexa) pyrosequencing and comparative analysis.
Results
5.0 gigabase pairs of clean nucleotides were generated. Non-redundant unigenes (33,721) were assembled and 94.1 % of them were annotated. Compared with our previous G-type transcriptome, the expression patterns of many insect responsive genes, including those related to secondary metabolism, phytohormones and transcription factors, which were significantly induced by DBM in G-type plants, were less sensitive to DBM infestation in P-type plants. The genes of the triterpenoid saponin pathway were identified in both G- and P-type plants. The upstream genes of the pathway showed similar expression patterns between the two genotypes. However, gene expression for two downstream enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclase (OSC), were significantly upregulated in the P-type compared with the G-type plant. The homologous genes from P- and G-type plants were detected by BLAST unigenes with a cutoff level E-value < e−10. 12,980 gene families containing 26,793 P-type and 36,944 G-type unigenes were shared by the two types of B. vulgaris. 38,397 single nucleotide polymorphisms (SNPs) were found in 9,452 orthologous genes between the P- and G-type plants. We also detected 5,105 simple sequence repeats (SSRs) in the B. vulgaris transcriptome, comprising mono-nucleotide-repeats (2,477; 48.5 %) and triple-nucleotide-repeats (1,590; 31.1 %). Of these, 1,657 SSRs displayed polymorphisms between the P- and G-type. Consequently, 913 SSR primer pairs were designed with a resolution of more than two nucleotides. We randomly chose 30 SSRs to detect the genetic diversity of 32 Barbarea germplasms. The distance tree showed that these accessions were clearly divided into groups, with the G-type grouping with available Western and Central European B. vulgaris accessions in contrast to the P-type accession, B. stricta and B. verna.
Conclusions
These data represent useful information for pest-resistance gene mining and for the investigation of the molecular basis of plant-pest interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1609-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1609-y
PMCID: PMC4487577  PMID: 26126637
Barbarea vulgaris; Transcriptome; Diamondback moth; Expression pattern; Molecular marker; Genetic diversity; Saponin biosynthesis
4.  The Role of an Integrated Care Model for Kidney Disease in the Development of Peritoneal Dialysis: A Single-Center Experience in China 
Peritoneal dialysis plays a crucial role in the integrated care of patients with end-stage renal disease (ESRD). In this paper, we retrospectively analyzed the quality indicators of peritoneal dialysis (PD) in 712 patients from our center who underwent PD between 2004 and 2011. In 43% of patients, follow-up was undertaken every 3 months at our outpatient department, and 54% patients were followed up by both our hospital and other local hospitals. The patient survival rate at 1, 3 and 5 years was 96.3%, 85.4% and 76.2%, respectively. The technique survival (excludes death/transplantation) at 1, 3 and 5 years was 95.1%, 87.7% and 79.6%, respectively. Fluid overload occurred in 29.2% of patients and was one of the major reasons for discontinuing PD. The peritonitis rate in our center decreased to 0.16 episodes/year in 2011. In addition, since our center is one of the largest integrated-treatment centers for ESRD in China, we have developed a multilevel care program in Zhejiang Province, which resulted in rapid growth of PD in our province in recent years.
doi:10.3747/pdi.2013.00124
PMCID: PMC4076965  PMID: 24962964
Integrated care model; peritoneal dialysis; technique survival
5.  Transforming growth factor-beta-1 is a serum biomarker of radiation-induced pneumonitis in esophageal cancer patients treated with thoracic radiotherapy: preliminary results of a prospective study 
OncoTargets and therapy  2015;8:1129-1136.
Objective
To examine the relationship between cytokine levels of transforming growth factor-beta-1 (TGF-β1), interleukin-1 beta (IL-1β), and angiotensin-converting enzyme (ACE) in the plasma of esophageal carcinoma patients and radiation-induced pneumonitis (RP).
Materials and methods
Sixty-three patients with esophageal carcinoma were treated with three-dimensional conformal radiotherapy (RT) using the Elekta Precise treatment planning system with a prescribed dose of 50–70 Gy. Dose–volume histograms were collected from three-dimensional conformal RT to determine the volume percentage of the lung received V5, V10, V20, and the normal tissue complication probability. RP was diagnosed based on computed tomography imaging, respiratory symptoms, and signs. The severity of radiation-induced lung toxicity was determined using the Lent–Soma scale defined by the Radiation Therapy Oncology Group. Plasma samples obtained before RT, during RT (at 40 Gy), and at 1 day, 1 month, and 3 months after RT were assayed for TGF-β1, IL-1β, and ACE levels by enzyme-linked immunosorbent assay.
Results
From the 63 patients, 17 (27%) developed RP, and 13 (21%) had RP of grade I and four (6%) had grade II or higher. We found plasma TGF-β1 levels were elevated in the patients that had RP when compared with the other 46 patients who did not have RP. The plasma IL-1β levels were not changed. The ACE levels were significantly lower in the 17 patients with RP compared to the 46 patients without RP throughout the RT. As expected, RP is associated with a higher dose of irradiation (>60 Gy); no other factors, including dose–volume histogram, age, sex, smoking status, location of tumor, and methods of treatment, are associated with RP.
Conclusion
Elevated plasma TGF-β1 levels can be used as a marker for RP.
doi:10.2147/OTT.S79433
PMCID: PMC4446015  PMID: 26056477
radiation-induced pneumonitis; esophageal carcinoma; TGF-β1; IL-1β; ACE
6.  Extreme expansion of NBS-encoding genes in Rosaceae 
BMC Genetics  2015;16:48.
Background
Nucleotide binding site leucine-rich repeats (NBS-LRR) genes encode a large class of disease resistance (R) proteins in plants. Extensive studies have been carried out to identify and investigate NBS-encoding gene families in many important plant species. However, no comprehensive research into NBS-encoding genes in the Rosaceae has been performed.
Results
In this study, five whole-genome sequenced Rosaceae species, including apple, pear, peach, mei, and strawberry, were analyzed to investigate the evolutionary pattern of NBS-encoding genes and to compare them to those of three Cucurbitaceae species, cucumber, melon, and watermelon. Considerable differences in the copy number of NBS-encoding genes were observed between Cucurbitaceae and Rosaceae species. In Rosaceae species, a large number and a high proportion of NBS-encoding genes were observed in peach (437, 1.52%), mei (475, 1.51%), strawberry (346, 1.05%) and pear (617, 1.44%), and apple contained a whopping 1303 (2.05%) NBS-encoding genes, which might be the highest number of R-genes in all of these reported diploid plant. However, no more than 100 NBS-encoding genes were identified in Cucurbitaceae. Many more species-specific gene families were classified and detected with the signature of positive selection in Rosaceae species, especially in the apple genome.
Conclusions
Taken together, our findings indicate that NBS-encoding genes in Rosaceae, especially in apple, have undergone extreme expansion and rapid adaptive evolution. Useful information was provided for further research on the evolutionary mode of disease resistance genes in Rosaceae crops.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-015-0208-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12863-015-0208-x
PMCID: PMC4417205  PMID: 25935646
NBS-encoding gene; Rosaceae; Cucurbitaceae; Expansion; Rapid evolution
7.  Effects of JWA, XRCC1 and BRCA1 mRNA expression on molecular staging for personalized therapy in patients with advanced esophageal squamous cell carcinoma 
BMC Cancer  2015;15:331.
Background
DNA damage repair genes JWA, XRCC1 and BRCA1 were associated with clinical outcomes and could convert the response to the cisplatin-based therapy in some carcinomas. The synergistic effects of JWA, XRCC1 and BRCA1 mRNA expression on personalized therapy remain unknown in advanced esophageal squamous cell carcinoma (ESCC).
Methods
We employed quantitative real-time polymerase chain reaction (qPCR) to determine the expression of JWA, XRCC1 and BRCA1 mRNA in paraffin-embedded specimen from 172 patients with advanced ESCC who underwent the first-line cisplatin-or docetaxel-based treatments.
Results
High JWA or XRCC1mRNA expression was correlated with longer median overall survival (mOS) in all the patients (both P < 0.001) or in subgroups with different regimens (all P < 0.05), but not correlated with response rate (RR, all P > 0.05). Multivariate analysis revealed that high JWA (HR 0.22; 95% CI 0.13-0.37; P < 0.001) or XRCC1 (HR 0.36; 95% CI 0.21-0.63; P < 0.001) mRNA expression emerged as the independent prognostic factors for ESCC patients in this cohort. But no significant difference in prognostic efficacy was found between JWA plus XRCC1 and JWA alone through ROC analysis. Further subgroup analysis showed cisplatin-based treatments could improve mOS of patients with low JWA expression (P < 0.05), especially in those with low BRCA1 expression simultaneously (P < 0.001); while in patients with high JWA expression, high BRCA1 mRNA expression was correlated with increased mOS in docetaxel-based treatments (P = 0.044).
Conclusion
JWA, XRCC1and BRCA1 mRNA expression could be used as predictive markers in molecular staging for personalized therapy in patients with advanced ESCC who received first-line cisplatin- or docetaxel-based treatments.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1364-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1364-0
PMCID: PMC4469327  PMID: 25925371
8.  miR-15a/16 are upreuglated in the serum of neonatal sepsis patients and inhibit the LPS-induced inflammatory pathway 
Infection in neonates, particular the neonatal sepsis continues to be a global problem with significant morbidity and mortality. The diagnosis of neonatal sepsis is complicated by nonspecific clinical symptomatology, a high-false negative rate, and a delay in obtaining blood culture results. MicroRNAs (miRNAs) have recently been used as finger prints for sepsis, and have been validated to be potential sepsis biomarker recently. In the present study, we investigated the level of several miRNAs, such as miR-15a, miR-16, miR-15b, and miR-223, which have been identified as a biomarker in adult sepsis, in neonatal sepsis patients, and then we analyzed the association of miR-15a/16 with the patient prognosis. Results demonstrated that the level of miR-15a/16 was up-regulated in neonatal sepsis patients than in normal neonatal subjects; however, no statistical difference was disclosed in the miR-15b and miR-223 level between two groups. And the ROC analysis indicated the miR-15a and miR-16 were potent fingerprints for diagnosing neonate sepsis. In order to explore the miR-15a/16 function on the lipopolysaccharide (LPS)-induced inflammatory pathway, the mice macrophage RAW264.7 cells were transiently transfected with miR-15a/16 mimics. And it was demonstrated that the miR-15a/16 transfection down-regulated the Toll-like receptor 4 (TLR4) and Interleukin-1 receptor-associated kinase 1 (IRAK-1) transcription level with a statistical difference in the LPS treated cells. And the suppression capability of miR-15a/16 on the expression of TLR-4 and IRAK-1 were evaluated by western blot. Thus, in present study, we identified miR-15a/16 as potential biomarker for the diagnosis and prognosis of neonatal sepsis, and the upregulated miR-15a/16 downregulated the LPS-induced inflammatory pathway.
PMCID: PMC4483976  PMID: 26131152
miR-15a/16; neonatal sepsis; TLR4; IRAK-1
9.  UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions 
Highlights
•Growing cells incorporate thio-iodo-deoxyuridine and thio-bromo-deoxyuridine into DNA.•They are non-toxic but act as powerful UVA photosensitisers.•UVA lesions include DNA-protein and DNA–DNA crosslinks.•Singlet oxygen is involved in the formation of this potentially lethal damage.•Thio-halo-deoxynucleosides offer a potential selective therapeutic option.
Photochemotherapy, the combination of a photosensitiser and ultraviolet (UV) or visible light, is an effective treatment for skin conditions including cancer. The high mutagenicity and non-selectivity of photochemotherapy regimes warrants the development of alternative approaches. We demonstrate that the thiopyrimidine nucleosides 5-bromo-4-thiodeoxyuridine (SBrdU) and 5-iodo-4-thiodeoxyuridine (SIdU) are incorporated into the DNA of cultured human and mouse cells where they synergistically sensitise killing by low doses of UVA radiation. The DNA halothiopyrimidine/UVA combinations induce DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, nucleobase damage and lesions that resemble UV-induced pyrimidine(6-4)pyrimidone photoproducts. These are potentially lethal DNA lesions and cells defective in their repair are hypersensitive to killing by SBrdU/UVA and SIdU/UVA. DNA SIdU and SBrdU generate lethal DNA photodamage by partially distinct mechanisms that reflect the different photolabilities of their C–I and C–Br bonds. Although singlet oxygen is involved in photolesion formation, DNA SBrdU and SIdU photoactivation does not detectably increase DNA 8-oxoguanine levels. The absence of significant collateral damage to normal guanine suggests that UVA activation of DNA SIdU or SBrdU might offer a strategy to target hyperproliferative skin conditions that avoids the extensive formation of a known mutagenic DNA lesion.
doi:10.1016/j.jphotobiol.2015.02.012
PMCID: PMC4376468  PMID: 25747491
10.  Air pollution and respiratory symptoms among children with asthma: Vulnerability by corticosteroid use and residence area☆ 
Rationale
Information on how ambient air pollution affects susceptible populations is needed to ensure protective air quality standards.
Objectives
To estimate the effect of community-level ambient particulate matter (PM) and ozone (O3) on respiratory symptoms among primarily African-American and Latino, lower-income asthmatic children living in Detroit, Michigan and to evaluate factors associated with heterogeneity in observed health effects.
Methods
A cohort of 298 children with asthma was studied prospectively from 1999 to 2002. For 14 days each season over 11 seasons, children completed a respiratory symptom diary. Simultaneously, ambient pollutant concentrations were measured at two community-level monitoring sites. Logistic regression models using generalized estimating equations were fit for each respiratory symptom in single pollutant models, looking for interactions by area or by corticosteroid use, a marker of more severe asthma. Exposures of interest were: daily concentrations of PM<10 μm, <2.5 μm, and between 10 and 2.5 μm in aerodynamic diameter (PM10, PM2.5, and PM10–2.5, respectively), the daily 8-hour maximum concentration of O3 (8HrPeak), and the daily 1-hour maximum concentration of O3 (1HrPeak).
Results
Outdoor PM2.5, PM10, 8HrPeak, and 1HrPeak O3 concentrations were associated with increased odds of respiratory symptoms, particularly among children using corticosteroid medication and among children living in the southwest community of Detroit. Similar patterns of associations were not seen with PM10–2.5.
Conclusions
PM2.5 and O3 at levels near or below annual standard levels are associated with negative health impact in this population of asthmatic children. Variation in effects within the city of Detroit and among the subgroup using steroids emphasizes the importance of spatially refined exposure assessment and the need for further studies to elucidate mechanisms and effective risk reduction interventions.
doi:10.1016/j.scitotenv.2012.11.070
PMCID: PMC4327853  PMID: 23273373
Asthma; Child; Community-based participatory research; Particulate matter; Ozone; Vulnerable populations
11.  Electronic Structure Engineering of Cu2O Film/ZnO Nanorods Array All-Oxide p-n Heterostructure for Enhanced Photoelectrochemical Property and Self-powered Biosensing Application 
Scientific Reports  2015;5:7882.
We have engineered the electronic structure at the interface between Cu2O and ZnO nanorods (NRs) array, through adjusting the carrier concentration of Cu2O. The electrodeposition of Cu2O at pH 11 acquired the highest carrier concentration, resulting in the largest interfacial electric field between Cu2O and ZnO, which finally led to the highest separation efficiency of photogenerated charge carriers. The optimized Cu2O/ZnO NRs array p-n heterostructures exhibited enhanced PEC performance, such as elevated photocurrent and photoconversion efficiency, as well as excellent sensing performance for the sensitive detection of glutathione (GSH) in PBS buffer even at applied bias of 0 V which made the device self-powered. Besides, the favorable selectivity, high reproducibility and extremely wide detection range, make such heterostructure a promising candidate for PEC biosensing applications, probably for the extended field of PEC water splitting or other solar photovoltaic beacons.
doi:10.1038/srep07882
PMCID: PMC4298735  PMID: 25600940
12.  Causes and consequences of crossing-over evidenced via a high-resolution recombinational landscape of the honey bee 
Genome Biology  2015;16(1):15.
Background
Social hymenoptera, the honey bee (Apis mellifera) in particular, have ultra-high crossover rates and a large degree of intra-genomic variation in crossover rates. Aligned with haploid genomics of males, this makes them a potential model for examining the causes and consequences of crossing over. To address why social insects have such high crossing-over rates and the consequences of this, we constructed a high-resolution recombination atlas by sequencing 55 individuals from three colonies with an average marker density of 314 bp/marker.
Results
We find crossing over to be especially high in proximity to genes upregulated in worker brains, but see no evidence for a coupling with immune-related functioning. We detect only a low rate of non-crossover gene conversion, contrary to current evidence. This is in striking contrast to the ultrahigh crossing-over rate, almost double that previously estimated from lower resolution data. We robustly recover the predicted intragenomic correlations between crossing over and both population level diversity and GC content, which could be best explained as indirect and direct consequences of crossing over, respectively.
Conclusions
Our data are consistent with the view that diversification of worker behavior, but not immune function, is a driver of the high crossing-over rate in bees. While we see both high diversity and high GC content associated with high crossing-over rates, our estimate of the low non-crossover rate demonstrates that high non-crossover rates are not a necessary consequence of high recombination rates.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0566-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0566-0
PMCID: PMC4305242  PMID: 25651211
13.  Vimentin Knockdown Decreases Corneal Opacity 
Purpose.
Wound induced corneal fibrosis can lead to permanent visual impairment. Keratocyte activation and differentiation play a key role in fibrosis, and vimentin, a major structural type III intermediate filament, is a required component of this process. The purpose of our study was to develop a nonviral therapeutic strategy for treating corneal fibrosis in which we targeted the knockdown of vimentin.
Methods.
To determine the duration of plasmid expression in corneal keratocytes, we injected a naked plasmid expressing green fluorescent protein (GFP; pCMV-GFP) into an unwounded mouse corneal stroma. We then injected pCMV-GFP or plasmids expressing small hairpin RNA in the corneal wound injury model (full-thickness corneal incision) to evaluate opacification.
Results.
GFP expression peaked between days 1 and 3 and had prominent expression for 15 days. In the corneal wound injury model, we found that the GFP-positive cells demonstrated extensive dendritic-like processes that extended to adjacent cells, whereas the vimentin knockdown model showed significantly reduced corneal opacity.
Conclusions.
These findings suggest that a nonviral gene therapeutic approach has potential for treating corneal fibrosis and ultimately reducing scarring.
Corneal fibrosis often results from infection, surgery complications, burns and traumatic eye injury; and corneal opacification is the 2nd leading cause of worldwide blindness. Knockdown of vimentin using plasmid-mediated shRNA expression showed reduction in scarring after a full-thickness incision.
doi:10.1167/iovs.13-13494
PMCID: PMC4078947  PMID: 24854859
corneal fibroblasts; corneal wound healing; keratocytes
14.  Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light–dark cycle 
BMC Genomics  2014;15(1):1185.
Background
Cyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light–dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.
Results
By combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light–dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid.
Conclusions
This investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1185) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1185
PMCID: PMC4320622  PMID: 25547186
Cyanothece; Cyanobacteria; RNA-Seq; N2 fixation; Proteomics; Butanol; CRISPR
15.  Hepatocyte Growth Factor Gene-Modified Adipose-Derived Mesenchymal Stem Cells Ameliorate Radiation Induced Liver Damage in a Rat Model 
PLoS ONE  2014;9(12):e114670.
Liver damage caused by radiotherapy is associated with a high mortality rate, but no established treatment exists. Adipose-derived mesenchymal stem cells (ADSCs) are capable of migration to injured tissue sites, where they aid in the repair of the damage. Hepatocyte growth factor (HGF) is critical for damage repair due to its anti-apoptotic, anti-fibrotic and cell regeneration-promoting effects. This study was performed to investigate the therapeutic effects of HGF-overexpressing ADSCs on radiation-induced liver damage (RILD). ADSCs were infected with a lentivirus encoding HGF and HGF-shRNA. Sprague-Dawley (SD) rats received 60Gy of irradiation to induce liver injury and were immediately given either saline, ADSCs, ADSCs + HGF or ADSCs + shHGF. Two days after irradiation, a significant reduction in apoptosis was observed in the HGF-overexpressing ADSC group compared with the RILD group, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Scanning electron microscopy showed chromatin condensation after irradiation, which was ameliorated in the group that received ADSCs and was reversed in the group that received HGF-overexpressing ADSCs. HGF-overexpressing ADSCs ameliorated radiation- induced liver fibrosis through down regulation of α-SMA and fibronectin. Hepatocyte regeneration was significantly improved in rats treated with ADSCs compared with rats from the RILD group), as assessed by Ki-67 immunohistochemistry. Rats that received HGF-overexpressing ADSCs showed an even greater level of hepatocyte regeneration. HGF-overexpressing ADSCs completely blocked the radiation-induced increase in the enzymes ALT and AST. The effect of mitigating RILD was compromised in the ADSC + shHGF group compared with the ADSC group. Altogether, these results suggest that HGF-overexpressing ADSCs can significantly improve RILD in a rat model, which may serve as a valuable therapeutic alternative.
doi:10.1371/journal.pone.0114670
PMCID: PMC4264768  PMID: 25501583
16.  The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity 
Journal of Bacteriology  2014;196(4):840-849.
Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H2 produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H2 production when grown under N2-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N2-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H2 produced by the nitrogenase.
doi:10.1128/JB.01248-13
PMCID: PMC3911175  PMID: 24317398
17.  A mechanically stabilized receptor–ligand flex-bond important in the vasculature 
Nature  2010;466(7309):992-995.
Haemostasis in the arteriolar circulation mediated by von Willebrand factor (VWF) binding to platelets is an example of an adhesive interaction that must withstand strong hydrodynamic forces acting on cells. VWF is a concatenated, multifunctional protein that has binding sites for platelets as well as subendothelial collagen1,2. Binding of the A1 domain in VWF to the glycoprotein Ib a subunit (GPIba) on the surface of platelets mediates crosslinking of platelets to one another and the formation of a platelet plug for arterioles3,4. The importance of VWF is illustrated by its mutation in von Willebrand disease, a bleeding diathesis1. Here, we describe a novel mechanochemical specialization of the A1–GPIbα bond for force-resistance. We have developed a method that enables, for the first time, repeated measurements of the binding and unbinding of a receptor and ligand in a single molecule (ReaLiSM). We demonstrate two states of the receptor-ligand bond, that is, a flex-bond. One state is seen at low force; a second state begins to engage at 10 pN with a ∼20-fold longer lifetime and greater force resistance. The lifetimes of the two states, how force exponentiates lifetime, and the kinetics of switching between the two states are all measured. For the first time, single-molecule measurements on this system are in agreement with bulk phase measurements. The results have important implications not only for how platelets bound to VWF are able to resist force to plug arterioles, but also how increased flow activates platelet plug formation.
doi:10.1038/nature09295
PMCID: PMC4117310  PMID: 20725043
18.  Neuropilin-2 promotes extravasation and metastasis by interacting with endothelial α5 integrin 
Cancer research  2013;73(14):4579-4590.
Metastasis, the leading cause of cancer death, requires tumor cell intravasation, migration through the bloodstream, arrest within capillaries, and extravasation to invade distant tissues. Few mechanistic details have been reported thus far regarding the extravasation process or re-entry of circulating tumor cells at metastatic sites. Here, we demonstrate that neuropilin-2 (NRP-2), a multi-functional non-kinase receptor for semaphorins, vascular endothelial growth factor (VEGF), and other growth factors, expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular extravasation and metastasis in zebrafish and murine xenograft models of clear cell renal cell carcinoma (RCC) and pancreatic adenocarcinoma. In tissue from RCC patients, NRP-2 expression is positively correlated with tumor grade and highest in metastatic tumors. In a prospectively acquired cohort of patients with pancreatic cancer, high NRP-2 expression co-segregated with poor prognosis. Through biochemical approaches as well as Atomic Force Microscopy (AFM), we describe a unique mechanism through which NRP-2 expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular adhesion and extravasation. Taken together, our studies reveal a clinically significant role of NRP-2 in cancer cell extravasation and promotion of metastasis.
doi:10.1158/0008-5472.CAN-13-0529
PMCID: PMC3774599  PMID: 23689123
Neuropilin; metastasis; integrin; renal cell carcinoma; pancreatic cancer; endothelial cells; adhesion; extravasation
19.  Radial Scars and Subsequent Breast Cancer Risk: A Meta-Analysis 
PLoS ONE  2014;9(7):e102503.
Background
The relationship between radial scars and breast cancer is unclear, as the results of different studies are inconsistent. We aim to solve the controversy and assess the breast cancer risk of radial scars.
Methods
Case-control or cohort studies about radial scars and breast cancer risk published in PubMed, Web of Science and the Cochrane Library from 2000 to 2013 were searched. Heterogeneity for the eligible data was assessed and a pooled odds ratio (OR) with 95% confidence interval (CI) was calculated.
Results
Five observational studies involving 2521 cases and 20290 controls were included in our study. From pooled analysis, radial scars were found to have a 1.33 fold increased risk of breast cancer, but which was not significant (P = 0.138). Sample size contributed to heterogeneity. In subgroup analysis, the results pooled from studies with sample size >2000 show that presence of radial scars was associated with 1.6 times breast cancer risk compared to absence of radial scars. Radial scars increased the risk of breast cancer among women with proliferative disease without atypia, but no significant association between radial scars and carcinoma was noted among women with atypical hyperplasia.
Conclusions
Radial scars tend to be associated with an increased breast cancer risk. Radial scars should be considered among women with proliferative disease without atypia, while atypical hyperplasia is still the primary concern among women with both radial scars and atypical hyperplasia.
doi:10.1371/journal.pone.0102503
PMCID: PMC4097058  PMID: 25019286
20.  Presurgical Corticosteroid Treatment Improves Corneal Transplant Survival in Mice 
Cornea  2013;32(12):1591-1598.
Purpose
To examine the effects of presurgical corticosteroid treatment for normal-risk penetrating keratoplasty (NRPK), high-risk penetrating keratoplasty (HRPK), and high-risk penetrating keratoplasty plus lensectomy.
Methods
We used 3 corneal transplantation models (NRPK, HRPK, and high-risk penetrating keratoplasty plus lensectomy). For each model, we tried to compare the effect of corticosteroid treatment according to different timetables as follows: The first trial began with a corticosteroid injection given 2 weeks before the PK and continued until 4 weeks after the PK (group 1). The second trial started with a corticosteroid injection given on the day of the PK and continued for 4 weeks after the PK (group 2). The third trial started with a corticosteroid injection administered on the day of the PK and continued for 8 weeks after the PK (group 3). After harvesting and immunostaining of corneas, graft survival, neovascularization (NV), and lymphangiogenesis (LY) were compared among the groups. A P value <0.05 was considered as being statistically significant.
Results
With respect to graft survival, group 1 had improved graft survival compared with that of group 3 in the HRPK model (P = 0.025). In all the 3 PK models, groups 2 and 3 demonstrated a similar graft survival (P > 0.05). With respect to NV and LY, in NRPK, group 1 showed less NV than did group 2 (P < 0.001) and group 3 (P = 0.016). In HRPK, group 1 also demonstrated less NV and LY than did group 3 (P = 0.045 and 0.044, respectively).
Conclusions
The initiation time point of the corticosteroid treatment is important for graft survival. Corticosteroid pretreatment is an effective means to increase graft survival for HRPK and to decrease NV and LY for both NRPK and HRPK.
doi:10.1097/ICO.0b013e31829ebb0d
PMCID: PMC4086307  PMID: 24005616
corticosteroid pretreatment; angiogenesis; penetrating keratoplasty
21.  TCF7L2 Variation and Proliferative Diabetic Retinopathy 
Diabetes  2013;62(7):2613-2617.
Proliferative diabetic retinopathy (PDR) is the most severe vision-threatening complication of diabetes. For investigation of genetic association between TCF7L2 and PDR in Caucasian type 2 diabetes mellitus (T2DM) and its functional consequences, 383 T2DM patients with PDR (T2DM-PDR) and 756 T2DM patients without diabetic retinopathy (T2DM–no DR) were genotyped with rs7903146 in TCF7L2. We found that risk allele (T) frequency of rs7903146 was significantly higher in T2DM-PDR patients (allelic P = 2.52E-04). In lymphoblastoid cells induced to undergo endoplasmic reticulum (ER) stress by treatment of tunicamycin, higher fold change of TCF7L2 and VEGFA mRNA levels were observed in rs7903146-TT cells than in rs7903146-CC cells (P = 0.02 for TCF7L2; P = 0.004 for VEGFA), suggesting that ER stress plays a role in PDR pathogenesis. Silencing TCF7L2 resulted in decreased mRNA levels of both TCF7L2 and VEGFA (P < 0.001). Retinas of oxygen-induced retinopathy mice (a model for PDR) had higher TCF7L2 and VEGFA mRNA levels than those of controls (P = 2.9E-04 for TCF7L2; P = 1.9E-07 for VEGFA). Together, data from our study show that TCF7L2-rs7903146 is associated with PDR in Caucasian T2DM and suggest that TCF7L2 promotes pathological retinal neovascularization via ER stress–dependent upregulation of VEGFA.
doi:10.2337/db12-1093
PMCID: PMC3712060  PMID: 23434931
22.  SIRT1 pathway dysregulation in the smoke-exposed airway epithelium and lung tumor tissue 
Cancer research  2012;72(22):5702-5711.
Cigarette smoke produces a molecular “field of injury” in epithelial cells lining the respiratory tract. However, the specific signaling pathways that are altered in the airway of smokers and the signaling processes responsible for the transition from smoking-induced airway damage to lung cancer remain unknown. In this study, we use a genomic approach to study the signaling processes associated with tobacco smoke exposure and lung cancer. First, we developed and validated pathway-specific gene expression signatures in bronchial airway epithelium that reflect activation of signaling pathways relevant to tobacco-exposure including ATM, BCL2, GPX1, NOS2, IKBKB, and SIRT1. Using these profiles and four independent gene expression datasets, we found that SIRT1 activity is significantly up-regulated in cytologically normal bronchial airway epithelial cells from active smokers compared to non-smokers. In contrast, this activity is strikingly down-regulated in non-small cell lung cancer. This pattern of signaling modulation was unique to SIRT1, and down-regulation of SIRT1 activity is confined to tumors from smokers. Decreased activity of SIRT1 was validated using genomic analyses of mouse models of lung cancer and biochemical testing of SIRT1 activity in patient lung tumors. Together, our findings indicate a role of SIRT1 in response to smoke and a potential role in repressing lung cancer. Further, our findings suggest that the airway gene-expression signatures derived in this study can provide novel insights into signaling pathways altered in the “field of inury” induced by tobacco smoke and thus may impact strategies for prevention of tobacco-related lung cancer.
doi:10.1158/0008-5472.CAN-12-1043
PMCID: PMC4053174  PMID: 22986747
23.  A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment 
Rationale: Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function.
Objectives: We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy.
Methods: Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays.
Measurements and Main Results: We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts.
Conclusions: Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD.
doi:10.1164/rccm.201208-1449OC
PMCID: PMC3707363  PMID: 23471465
chronic obstructive pulmonary disease; gene expression profiling; biologic markers
24.  Targeted Intraceptor Nanoparticle Therapy Reduces Angiogenesis and Fibrosis in Primate & Murine Macular Degeneration 
ACS nano  2013;7(4):3264-3275.
Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry©, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.
doi:10.1021/nn305958y
PMCID: PMC3634882  PMID: 23464925
nanoparticles; gene delivery; choroidal neovascularization models; anti-VEGF therapy; angiogenesis
25.  Novel mutations of the RS1 gene in a cohort of Chinese families with X-linked retinoschisis 
Molecular Vision  2014;20:132-139.
Purpose
X-linked retinoschisis is a retinal dystrophy caused by mutations in the RS1 gene in Xp22.1. These mutations lead to schisis (splitting) of the neural retina and subsequent reduction in visual acuity in affected men (OMIM # 312700). The aim of this study was to identify the RS1 gene mutations in a cohort of Chinese patients with X-linked retinoschisis, and to describe the associated phenotypes.
Methods
Patients and unaffected individuals from 16 unrelated families underwent detailed ophthalmic examinations. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. All exons including the exon-intron boundaries of the RS1 gene, were amplified by PCR and the products were analyzed by direct sequencing. Long-range PCR followed by DNA sequencing was used to define the breakpoints of the large deletion.
Results
Sixteen male individuals from 16 families were diagnosed with retinoschisis by clinical examination. The median age at review was 13.2 years (range: 5–34 years); the median best-corrected visual acuity upon review was 0.26 (range 0.02–1.0). Foveal schisis was found in 82.8% of the eyes (24/29) while peripheral schisis was present in 27.5% of the eyes (8/29). Sequencing of the RS1 gene identified 16 mutations, nine of which were novel. The mutations included eight missense mutations, all located in exons 4–6 (50.0%), two nonsense mutations (12.5%), four small deletions or insertions (25.0%), one splice site mutation (6.25%), and one large genomic deletion that included exon1 (6.25%).
Conclusions
The mutations found in our study broaden the spectrum of RS1 mutations. The identification of the specific mutation in each pedigree will allow future determination of female carrier status for genetic counseling purposes.
PMCID: PMC3913487  PMID: 24505212

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