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1.  The Draft Genome Sequence of European Pear (Pyrus communis L. ‘Bartlett’) 
PLoS ONE  2014;9(4):e92644.
We present a draft assembly of the genome of European pear (Pyrus communis) ‘Bartlett’. Our assembly was developed employing second generation sequencing technology (Roche 454), from single-end, 2 kb, and 7 kb insert paired-end reads using Newbler (version 2.7). It contains 142,083 scaffolds greater than 499 bases (maximum scaffold length of 1.2 Mb) and covers a total of 577.3 Mb, representing most of the expected 600 Mb Pyrus genome. A total of 829,823 putative single nucleotide polymorphisms (SNPs) were detected using re-sequencing of ‘Louise Bonne de Jersey’ and ‘Old Home’. A total of 2,279 genetically mapped SNP markers anchor 171 Mb of the assembled genome. Ab initio gene prediction combined with prediction based on homology searching detected 43,419 putative gene models. Of these, 1219 proteins (556 clusters) are unique to European pear compared to 12 other sequenced plant genomes. Analysis of the expansin gene family provided an example of the quality of the gene prediction and an insight into the relationships among one class of cell wall related genes that control fruit softening in both European pear and apple (Malus×domestica). The ‘Bartlett’ genome assembly v1.0 (http://www.rosaceae.org/species/pyrus/pyrus_communis/genome_v1.0) is an invaluable tool for identifying the genetic control of key horticultural traits in pear and will enable the wide application of marker-assisted and genomic selection that will enhance the speed and efficiency of pear cultivar development.
doi:10.1371/journal.pone.0092644
PMCID: PMC3974708  PMID: 24699266
2.  Molecular and Metabolic Mechanisms of Cardiac Dysfunction in Diabetes 
Life sciences  2012;92(11):601-608.
Diabetes mellitus type 2 (T2DM) is a widespread chronic medical condition with prevalence bordering on the verge of an epidemic. It is of great concern that cardiovascular disease is more common in patients with diabetes than the non-diabetic population. While hypertensive and ischemic heart disease is more common in diabetic patients, there is another type of heart disease in diabetes that is not associated with hypertension or coronary artery disease. This muscle functional disorder is termed “diabetic cardiomyopathy”. Diastolic dysfunction characterized by impaired diastolic relaxation time and reduced contractility precedes systolic dysfunction and is the main pathogenic hallmark of this condition. Even though the pathogenesis of “diabetic cardiomyopathy” is still controversial, impaired cardiac insulin sensitivity and metabolic overload are emerging as major molecular and metabolic mechanisms for cardiac dysfunction. Systemic insulin resistance, hyperinsulinemia, dysregulation of adipokine secretion, increases in circulating levels of inflammatory mediators, aberrant activation of renin angiotensin aldosterone system (RAAS), and increased oxidative stress contribute dysregulated insulin and metabolic signaling in the heart and development of diastolic dysfunction. In addition, maladaptive calcium homeostasis and endothelial cell dysregulation endoplasmic reticular stress play a potential role in cardiomyocyte fibrosis/diastolic dysfunction. In this review, we will focus on emerging molecular and metabolic pathways underlying cardiac dysfunction in diabetes. Elucidation of these mechanisms should provide a better understanding of the various cardiac abnormalities associated with diastolic dysfunction and its progression to systolic dysfunction and heart failure.
doi:10.1016/j.lfs.2012.10.028
PMCID: PMC3594135  PMID: 23147391
Diabetes; RAAS; Cardiac Dysfunction; Cardiorenal Metabolic syndrome; Diabetic Cardiomyopathy; Insulin Resistance
3.  Heterologous Protein Secretion in Lactobacilli with Modified pSIP Vectors 
PLoS ONE  2014;9(3):e91125.
We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in Lactobacillus strains representing 14 species using pSIP411, which harbors the broad-host-range Lactococcus lactis SH71rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range L. plantarum 256rep replicon with SH71rep and transformed into strains of five different species of Lactobacillus. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of Lactobacillus species.
doi:10.1371/journal.pone.0091125
PMCID: PMC3948729  PMID: 24614815
4.  Risk Factors for Aborted Cardiac Arrest and Sudden Cardiac Death in Children with the Congenital Long-QT Syndrome 
Circulation  2008;117(17):2184-2191.
Background
The congenital long-QT syndrome (LQTS) is an important cause of sudden cardiac death (SCD) in children without structural heart disease. However, specific risk factors for life-threatening cardiac events in children with this genetic disorder have not been identified
Methods and Results
Cox proportional hazards regression modeling was used to identify risk factors for aborted cardiac arrest (ACA) or SCD in 3,015 LQTS children from the International LQTS Registry who were followed up from age 1 through 12 years. The cumulative probability of the combined end point was significantly higher in males (5%) than in females (1%; p<0.001). Risk factors for ACA or SCD during childhood included QTc duration >500 msec (HR=2.72 [95%CI 1.50 - 4.92]; p=0.001) and prior syncope (recent syncope [<2 years]: HR=6.16 [95%CI 3.41 - 11.15], p<0.001; remote syncope [≥2 years]: HR=2.67 [95% CI 1.22 - 5.85], p=0.01) in males, whereas prior syncope was the only significant risk factor among females (recent syncope: HR=27.82 [95%CI 9.72 - 79.60], p<0.001]; remote syncope: HR=12.04 [95%CI 3.79 - 38.26], p<0.001). β-blocker therapy was associated with a significant 53% reduction in the risk of ACA or SCD (p=0.01).
Conclusions
LQTS males experience a significantly higher rate of fatal or near-fatal cardiac events than females during childhood. A QTc duration >500 msec and a history of prior syncope identify risk in males, whereas prior syncope is the only significant risk factor among females. β-blocker therapy is associated with a significant reduction in the risk of life-threatening cardiac events during childhood.
doi:10.1161/CIRCULATIONAHA.107.701243
PMCID: PMC3944375  PMID: 18427136
long-QT syndrome; risk factors; sudden death
5.  Establishment of a Simple Lactobacillus plantarum Cell Consortium for Cellulase-Xylanase Synergistic Interactions 
Applied and Environmental Microbiology  2013;79(17):5242-5249.
Lactobacillus plantarum is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into L. plantarum. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by L. plantarum at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD600) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of L. plantarum for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.
doi:10.1128/AEM.01211-13
PMCID: PMC3753964  PMID: 23811500
6.  New Type of Antimicrobial Protein Produced by the Plant Pathogen Clavibacter michiganensis subsp. michiganensis 
Applied and Environmental Microbiology  2013;79(18):5721-5727.
It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC50) of approximately 10 pmol, but was not toxic to potato leaves or tubers. CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum, CmmAMP-1 may be of interest in combating potato ring rot disease.
doi:10.1128/AEM.01065-13
PMCID: PMC3754172  PMID: 23851100
7.  Cardiac work-up protocol for liver transplant candidates: Experience from a single liver transplant centre 
BACKGROUND:
Ischemic cardiac events can cause significant morbidity and mortality postliver transplantation; however, no validated protocols to screen patients before transplantation exist.
OBJECTIVES:
To report the introduction of a noninvasive cardiac screening protocol used at the Liver Unit, University of Calgary (Calgary, Alberta); to determine whether the protocol decreases use of coronary angiograms; and to compare cardiac outcomes using the new protocol with an appropriately matched historical control group.
METHODS:
A new cardiac screening protocol was introduced into the program in 2005, which uses perfusion scintigraphy to screen high-risk cardiac patients, reserving coronary angiograms for abnormal results. Transplanted patients screened using this protocol were compared with matched historical controls. Electronic charts were reviewed for cardiac outcomes intra- and postliver transplantation.
RESULTS:
A total of 396 patients were screened between April 2005 and February 2009. Eighty-two were transplanted by February 2009 and included in the study. Eighty-one patients were successfully matched according to age, sex, cardiac history and presence of diabetes. Twelve of 82 (14.6%) and 11 of 81 (13.6%) in the study and control groups, respectively, underwent coronary angiograms (P=0.85). Coronary artery disease was found in six of 12 (50.0%) study patients and three of 11 (27.3%) control patients who underwent coronary angiography (P=0.27). The mean (± SD) length of the follow-up period was 1.87±0.91 years and 4.45±1.89 years in the study and control groups, respectively. One of 81 in the control group and zero of 82 in the study group experienced an acute coronary syndrome event postoperatively.
CONCLUSIONS:
Coronary events are infrequent in liver transplant recipients. The described protocol is an effective method of coronary artery disease screening before liver transplant but does not reduce the number of cardiac investigations performed.
PMCID: PMC3495698  PMID: 23166904
Acute coronary syndrome; Coronary angiogram; Coronary artery disease; Liver transplantation; Myocardial perfusion imaging
8.  Clinical, Pathologic, and Functional Outcomes After Nephron-Sparing Surgery in Patients with a Solitary Kidney: A Multicenter Experience 
Journal of Endourology  2012;26(10):1361-1366.
Abstract
Background and Purpose
Surgical management of a renal neoplasm in a solitary kidney is a balance between oncologic control and preservation of renal function. We analyzed patients with a renal mass in a solitary kidney undergoing nephron-sparing procedures to determine perioperative, oncologic, and renal functional outcomes.
Patients and Methods
A multicenter study was performed from 12 institutions. All patients with a functional or anatomic solitary kidney who underwent nephron-sparing surgery for one or more renal masses were included. Tumor size, complications, and recurrence rates were recorded. Renal function was assessed with serum creatinine level and estimated glomerular filtration rate.
Results
Ninety-eight patients underwent 105 ablations, and 100 patients underwent partial nephrectomy (PN). Preoperative estimated glomerular filtration rate (eGFR) was similar between the groups. Tumors managed with PN were significantly larger than those managed with ablation (P<0.001). Ablations were associated with a lower overall complication rate (9.5% vs 24%, P=0.01) and higher local recurrence rate (6.7% vs 3%, P=0.04). Eighty-four patients had a preoperative eGFR ≥60 mL/min/1.73 m2. Among these patients, 19 (23%) fell below this threshold after 3 months and 15 (18%) at 12 months. Postoperatively, there was no significant difference in eGFR between the groups.
Conclusions
Extirpation and ablation are both reasonable options for treatment. Ablation is more minimally invasive, albeit with higher recurrence rates compared with PN. Postoperative renal function is similar in both groups and is not affected by surgical approach.
doi:10.1089/end.2012.0114
PMCID: PMC3727568  PMID: 22667344
9.  Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue 
PLoS ONE  2013;8(9):e76039.
Low-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto- and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes.
doi:10.1371/journal.pone.0076039
PMCID: PMC3779194  PMID: 24073286
10.  MAGE-A3-specific anticancer immunotherapy in the clinical practice 
Oncoimmunology  2013;2(10):e25995.
Antigen-specific immunotherapy may offer a unique approach to fight cancer. We have demonstrated that specific immunotherapeutic regimens involving recombinant melanoma antigen family A3 (MAGE-A3) and different immunostimulants exert clinical anticancer activity. In particular, the combination of recombinant MAGE-A3 and AS15, a multicomponent immunostimulant, was found to elicit robust antigen-specific immune responses.
doi:10.4161/onci.25995
PMCID: PMC3827067  PMID: 24244898
biomarkers; cancer; immunotherapy; immunization; MAGE-A3
11.  Increased Duration of Dual Pegylated Interferon and Ribavirin Therapy for Genotype 1 Hepatitis C Post-liver Transplantation Increases Sustained Virologic Response: A Retrospective Review 
Background/Aim:
In patients with advanced post-transplant hepatitis C virus (HCV) recurrence, antiviral treatment (AVT) with interferon and ribavirin is indicated to prevent graft failure. The aim of this study was to determine and report Canadian data with respect to the safety, efficacy, and spontaneous virologic response (SVR) predictors of AVT among transplanted patients with HCV recurrence.
Patients and Methods:
A retrospective chart review was performed on patients transplanted in London, Ontario and Edmonton, Alberta from 2002 to 2012 who were treated for HCV. Demographic, medical, and treatment information was collected and analyzed.
Results:
A total of 85 patients with HCV received pegylated interferon with ribavirin post-liver transplantation and 28 of the 65 patients (43%) with genotype 1 achieved SVR. Of the patients having genotype 1 HCV who achieved SVR, there was a significantly lower stage of fibrosis (1.37 ± 0.88 vs. 1.89 ± 0.96; P = 0.03), increased ribavirin dose (total daily dose 1057 ± 230 vs. 856 ± 399 mg; P = 0.02), increased rapid virologic response (RVR) (6/27 vs. 0/31; P = 0.05), increased early virologic response (EVR) (28/28 vs. 18/35; P = 0.006), and longer duration of therapy (54.7 ± 13.4 weeks vs. 40.2 ± 18.7; P = 0.001). A logistic regression model using gender, age, RVR, EVR, anemia, duration of therapy, viral load, years’ post-transplant, and type of organ (donation after cardiac death vs. donation after brain death) significantly predicted SVR (P < 0.001), with duration of therapy having a significant odds ratio of 1.078 (P = 0.007).
Conclusions:
This study identified factors that predict SVR in HCV-positive patients who received dual therapy post-transplantation. Extending therapy from 48 weeks to 72 weeks of dual therapy is associated with increased SVR rates. Future studies examining the role of extended therapy are needed to confirm these findings, since the current study is a retrospective one.
doi:10.4103/1319-3767.118133
PMCID: PMC3793474  PMID: 24045596
Hepatitis C virus; liver transplant; retrospective study
12.  The Novel Angiotensin II Receptor Blocker Azilsartan Medoxomil Ameliorates Insulin Resistance Induced by Chronic Angiotensin II Treatment in Rat Skeletal Muscle 
Cardiorenal Medicine  2013;3(2):154-164.
Angiotensin receptor (type 1) blockers (ARBs) can reduce both hypertension and insulin resistance induced by local and systemic activation of the renin-angiotensin-aldosterone system. The effectiveness of azilsartan medoxomil (AZIL-M), a novel imidazole-based ARB, to facilitate metabolic improvements in conditions of angiotensin II (Ang II)-associated insulin resistance is currently unknown. The aim of this study was to determine the impact of chronic AZIL-M treatment on glucose transport activity and key insulin signaling elements in red skeletal muscle of Ang II-treated rats. Male Sprague-Dawley rats were treated for 8 weeks with or without Ang II (200 ng/kg/min) combined with either vehicle or AZIL-M (1 mg/kg/day). Ang II induced significant (p < 0.05) increases in blood pressure, which were completely prevented by AZIL-M. Furthermore, Ang II reduced insulin-mediated glucose transport activity in incubated soleus muscle, and AZIL-M co-treatment increased this parameter. Moreover, AZIL-M treatment of Ang II-infused animals increased the absolute phosphorylation of insulin signaling molecules, including Akt [both Ser473 (81%) and Thr308 (23%)] and AS160 Thr642 (42%), in red gastrocnemius muscle frozen in situ. Absolute AMPKα (Thr172) phosphorylation increased (98%) by AZIL-M treatment, and relative Thr389 phosphorylation of p70 S6K1, a negative regulator of insulin signaling, decreased (51%) with AZIL-M treatment. These results indicate that ARB AZIL-M improves the in vitro insulin action on glucose transport in red soleus muscle and the functionality of the Akt/AS160 axis in red gastrocnemius muscle in situ in Ang II-induced insulin-resistant rats, with the latter modification possibly associated with enhanced AMPKα and suppressed p70 S6K1 activation.
doi:10.1159/000353155
PMCID: PMC3721134  PMID: 23922555
Angiotensin II; Glucose transport; Red skeletal muscle; Akt; AS160; AMPK; p70 S6K1

13.  Mutations in Cytoplasmic Loops of the KCNQ1 Channel and the Risk of Life-Threatening Events: Implications for Mutation-Specific Response to Beta-Blocker Therapy in Type-1 Long QT Syndrome 
Circulation  2012;125(16):1988-1996.
Background
β-adrenergic stimulation is the main trigger for cardiac events in type-1 long QT syndrome (LQT1). We evaluated a possible association between ion channel response to β-adrenergic stimulation and clinical response to β-blocker therapy according to mutation location.
Methods and Results
The study sample comprised 860 patients with genetically-confirmed mutations in the KCNQ1 channel. Patients were categorized into carriers of missense mutations located in the cytoplasmic loops (C-loops), membrane spanning domain, C/N-terminus, and non-missense mutations. There were 27 aborted cardiac arrest [ACA] and 78 sudden cardiac death [SCD] events from birth through age 40 years. After multivariable adjustment for clinical factors, the presence of C-loop mutations was associated with the highest risk for ACA or SCD (hazard ratio [95% confidence interval] vs. non-missense mutations = 2.75 [1.29-5.86, P=0.009]). β-blocker therapy was associated with a significantly greater reduction in the risk of ACA or SCD among patients with C-loop mutations than among all other patients (hazard ratios = 0.12 [0.02-0.73, P=0.02] and 0.82 [0.31-2.13, P=0.68], respectively; P-for interaction = 0.04). Cellular expression studies showed that membrane spanning and C-loop mutations produced a similar decrease in current, but only C-loop mutations showed a pronounced reduction in channel activation in response to β-adrenergic stimulation.
Conclusions
Patients with C-loop missense mutations in the KCNQ1 channel exhibit a high-risk for life-threatening events and derive a pronounced benefit from treatment with β-blockers. Reduced channel activation following sympathetic activation can explain the increased clinical risk and response to therapy in patients with C-loop mutations.
doi:10.1161/CIRCULATIONAHA.111.048041
PMCID: PMC3690492  PMID: 22456477
beta-blockers; ion channels; long QT syndrome; mutation
14.  Risk of Syncope in Family Members Who Are Genotype Negative for a Family-Associated Long QT Syndrome Mutation 
Background
Current clinical diagnosis of long-QT syndrome (LQTS) includes genetic testing of family members of mutation positive patients. The present study was designed to assess the clinical course of individuals who are found negative for the LQTS-causing mutation in their families.
Methods and Results
Multivariate Cox proportional hazards model was used to assess the risk for cardiac events (comprising syncope, aborted cardiac arrest [ACA], or sudden cardiac death [SCD]) from birth through age 40 years among 1828 subjects from the LQTS Registry who were found negative for their family LQTS-causing mutation. The median QTc of study subjects was 423 msec (interquartile-range: 402–442 msec). The cumulative probability of a first syncope through age 40 years was 15%. However, only 2 patients (0.1%) experienced ACA and none died suddenly during follow-up. Independent risk factors for syncope in genotype negative subjects included female gender (HR 1.60, p = 0.002), prolonged QTc (HR = 1.63 per 100 msec increment, p = 0.02), family history of ACA or SCD (HR = 1.89, p = 0.002), and LQT2 vs. LQT1 family mutation (HR = 1.41, p = 0.03). Subgroup analysis showed that the presence of the K897T polymorphism in the LQT2 gene in an affected family was associated with an 11-fold (p = 0.001) increase in the risk of recurrent syncope in genotype negative subjects.
Conclusions
Our findings suggest that cardiac events among genotype-negative family members of LQTS patients are dominated by nonfatal syncopal episodes without occurrence of sudden cardiac death. The risk for nonfatal events in this population may be mediated by the presence of common polymorphisms in LQTS genes.
doi:10.1161/CIRCGENETICS.111.960179
PMCID: PMC3690290  PMID: 21831960
gene mutation; genetic polymorphisms; long-QT syndrome; sudden cardiac death arrhythmia; syncope
15.  Transcriptional Dominance of Pax7 in Adult Myogenesis is Due to High-Affinity Recognition of Homeodomain Motifs 
Developmental Cell  2012;22(6):1208-1220.
SUMMARY
Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome wide binding-site analysis we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrates distinct attributes of Pax7 function and provides mechanistic insight into the non-redundancy of Pax3 and Pax7 in muscle development.
doi:10.1016/j.devcel.2012.03.014
PMCID: PMC3376216  PMID: 22609161
16.  MOLECULAR CHARACTERIZATION OF KIDNEY CANCER: ASSOCIATION OF HYALURONIC ACID FAMILY WITH HISTOLOGICAL SUBTYPES AND METASTASIS 
Cancer  2011;118(9):2394-2402.
Background
Molecular profiling of renal cell carcinomas (RCC) may improve the distinction between oncocytoma and malignant RCC subtypes and aid in early detection of metastasis. Hyaluronic acid (HA) family includes HA-synthases (HAS1, HAS2, HAS3), hyaluronidases (HYAL-1, HYAL-2, HYAL-3, HYAL-4, PH20, HYAL-P1), and HA receptors (CD44s, CD44v and RHAMM). HA family members promote tumor growth and metastasis. We evaluated the expression of HA family members in kidney specimens.
Methods
Using quantitative PCR, mRNA levels of twelve HA family members were measured in tumor specimens obtained from 86 consecutive patients undergoing nephrectomy; 80 of them also provided normal specimens. Mean and median follow-up: 15.2 ± 8.8 and 13.8 months. RCC specimens included: clear cell RCC (ccRCC): 65; papillary: 10; chromophobe: 5; oncocytoma: 6; metastasis (+): 17.
Results
Median HAS1, CD44s and RHAMM transcript levels were 3–25 elevated in ccRCC, papillary and chromophobe tumors when compared to normal tissues. HYAL-4, CD44s and RHAMM levels were 4–12-fold elevated in ccRCC and papillary tumors when compared to oncocytomas; only HYAL-4 levels distinguished between chromophobe and oncocytoma (P=0.009). CD44s and RHAMM levels were significantly higher in tumors < 4-cm (510±611; 19.6±20.8, respectively) when compared to oncocytoma (46.4±20; 3.8±2.5; P≤0.006). In univariate and multivariate analyses, CD44s (P<0.0001), RHAMM (P<0.0001), stage, tumor size, and/or renal vein involvement significantly associated with metastasis. The combined CD44s+RHAMM marker had 82% sensitivity and 86% specificity to predict metastasis.
Conclusion
CD44s and RHAMM levels distinguish between oncocytoma and RCC subtypes regardless of tumor size and are potential predictors of RCC metastasis.
doi:10.1002/cncr.26520
PMCID: PMC3232339  PMID: 21887686
Prognostic markers; HA-synthase; Hyaluronidase; HA-receptors; Renal cell carcinoma; metastasis; oncocytoma
17.  Semidiurnal Temperature Changes Caused by Tidal Front Movements in the Warm Season in Seabed Habitats on the Georges Bank Northern Margin and Their Ecological Implications 
PLoS ONE  2013;8(2):e55273.
Georges Bank is a large, shallow feature separating the Gulf of Maine from the Atlantic Ocean. Previous studies demonstrated a strong tidal-mixing front during the warm season on the northern bank margin between thermally stratified water in the Gulf of Maine and mixed water on the bank. Tides transport warm water off the bank during flood tide and cool gulf water onto the bank during ebb tide. During 10 days in August 2009, we mapped frontal temperatures in five study areas along ∼100 km of the bank margin. The seabed “frontal zone”, where temperature changed with frontal movment, experienced semidiurnal temperature maxima and minima. The tidal excursion of the frontal boundary between stratified and mixed water ranged 6 to 10 km. This “frontal boundary zone” was narrower than the frontal zone. Along transects perpendicular to the bank margin, seabed temperature change at individual sites ranged from 7.0°C in the frontal zone to 0.0°C in mixed bank water. At time series in frontal zone stations, changes during tidal cycles ranged from 1.2 to 6.1°C. The greatest rate of change (−2.48°C hr−1) occurred at mid-ebb. Geographic plots of seabed temperature change allowed the mapping of up to 8 subareas in each study area. The magnitude of temperature change in a subarea depended on its location in the frontal zone. Frontal movement had the greatest effect on seabed temperature in the 40 to 80 m depth interval. Subareas experiencing maximum temperature change in the frontal zone were not in the frontal boundary zone, but rather several km gulfward (off-bank) of the frontal boundary zone. These results provide a new ecological framework for examining the effect of tidally-driven temperature variability on the distribution, food resources, and reproductive success of benthic invertebrate and demersal fish species living in tidal front habitats.
doi:10.1371/journal.pone.0055273
PMCID: PMC3566201  PMID: 23405129
18.  Surface Display of N-Terminally Anchored Invasin by Lactobacillus plantarum Activates NF-κB in Monocytes 
Applied and Environmental Microbiology  2012;78(16):5864-5871.
The probiotic lactic acid bacterium Lactobacillus plantarum is a potential delivery vehicle for mucosal vaccines because of its generally regarded as safe (GRAS) status and ability to persist at the mucosal surfaces of the human intestine. However, the inherent immunogenicity of vaccine antigens is in many cases insufficient to elicit an efficient immune response, implying that additional adjuvants are needed to enhance the antigen immunogenicity. The goal of the present study was to increase the proinflammatory properties of L. plantarum by expressing a long (D1 to D5 [D1-D5]) and a short (D4-D5) version of the extracellular domain of invasin from the human pathogen Yersinia pseudotuberculosis. To display these proteins on the bacterial surface, four different N-terminal anchoring motifs from L. plantarum were used, comprising two different lipoprotein anchors, a transmembrane signal peptide anchor, and a LysM-type anchor. All these anchors mediated surface display of invasin, and several of the engineered strains were potent activators of NF-κB when interacting with monocytes in cell culture. The most distinct NF-κB responses were obtained with constructs in which the complete invasin extracellular domain was fused to a lipoanchor. The proinflammatory L. plantarum strains constructed here represent promising mucosal delivery vehicles for vaccine antigens.
doi:10.1128/AEM.01227-12
PMCID: PMC3406107  PMID: 22706054
19.  Mineralocorticoid Receptor-Dependent Proximal Tubule Injury Is Mediated by a Redox-Sensitive mTOR/S6K1 Pathway 
American Journal of Nephrology  2011;35(1):90-100.
Background/Aims
The mammalian target of rapamycin (mTOR) is a serine kinase that regulates phosphorylation (p) of its target ribosomal S6 kinase (S6K1), whose activation can lead to glomerular and proximal tubular cell (PTC) injury and associated proteinuria. Increased mTOR/S6K1 signaling regulates signaling pathways that target fibrosis through adherens junctions. Recent data indicate aldosterone signaling through the mineralocorticoid receptor (MR) can activate the mTOR pathway. Further, antagonism of the MR has beneficial effects on proteinuria that occur independent of hemodynamics.
Methods
Accordingly, hypertensive transgenic TG(mRen2)27 (Ren2) rats, with elevated serum aldosterone and proteinuria, and age-matched Sprague-Dawley rats were treated with either a low dose (1 mg/kg/day) or a conventional dose (30 mg/kg/day) of spironolactone (MR antagonist) or placebo for 3 weeks.
Results
Ren2 rats displayed increases in urine levels of the PTC brush border lysosomal enzyme N-acetyl-β-aminoglycosidase (β-NAG) in conjunction with reductions in PTC megalin, the apical membrane adherens protein T-cadherin and basolateral α-(E)-catenin, and fibrosis. In concert with these abnormalities, Ren2 renal cortical tissue also displayed increased Ser2448 (p)/activation of mTOR and Thr389 (p)-S6K1 and increased 3-nitrotyrosine (3-NT) content, a marker for peroxynitrite. Low-dose spironolactone had no effect on blood pressure but decreased proteinuria and β-NAG comparable to a conventional dose of this MR antagonist. Both doses of spironolactone attenuated ultrastructural maladaptive alterations and led to comparable reductions in (p)-mTOR/(p)-S6K1, 3-NT, fibrosis, and increased expression of α-(E)-catenin, T- and N-cadherin.
Conclusions
Thereby, MR antagonism improves proximal tubule integrity by targeting mTOR/S6K1 signaling and redox status independent of changes in blood pressure.
doi:10.1159/000335079
PMCID: PMC3316484  PMID: 22205374
Cadherin; Megalin; β-NAG; Proteinuria
20.  The Role of Tissue Renin-Angiotensin-Aldosterone System in the Development of Endothelial Dysfunction and Arterial Stiffness 
Epidemiological studies support the notion that arterial stiffness is an independent predictor of adverse cardiovascular events contributing significantly to systolic hypertension, impaired ventricular-arterial coupling and diastolic dysfunction, impairment in myocardial oxygen supply and demand, and progression of kidney disease. Although arterial stiffness is associated with aging, it is accelerated in the presence of obesity and diabetes. The prevalence of arterial stiffness parallels the increase of obesity that is occurring in epidemic proportions and is partly driven by a sedentary life style and consumption of a high fructose, high salt, and high fat western diet. Although the underlying mechanisms and mediators of arterial stiffness are not well understood, accumulating evidence supports the role of insulin resistance and endothelial dysfunction. The local tissue renin-angiotensin-aldosterone system (RAAS) in the vascular tissue and immune cells and perivascular adipose tissue is recognized as an important element involved in endothelial dysfunction which contributes significantly to arterial stiffness. Activation of vascular RAAS is seen in humans and animal models of obesity and diabetes, and associated with enhanced oxidative stress and inflammation in the vascular tissue. The cross talk between angiotensin and aldosterone underscores the importance of mineralocorticoid receptors in modulation of insulin resistance, decreased bioavailability of nitric oxide, endothelial dysfunction, and arterial stiffness. In addition, both innate and adaptive immunity are involved in this local tissue activation of RAAS. In this review we will attempt to present a unifying mechanism of how environmental and immunological factors are involved in this local tissue RAAS activation, and the role of this process in the development of endothelial dysfunction and arterial stiffness and targeting tissue RAAS activation.
doi:10.3389/fendo.2013.00161
PMCID: PMC3810594  PMID: 24194732
renin-angiotensin-aldosterone system; arterial stiffness; insulin resistance; endothelial dysfunction; obesity; diabetes
21.  Clinical Implications for Patients with Long QT Syndrome Who Experience a Cardiac Event During Infancy 
Objectives
This study was designed to evaluate the clinical and prognostic aspects of long QT syndrome-related cardiac events that occur in the first year of life (infancy).
Background
The clinical implications for patients with long QT syndrome who experience cardiac events in infancy have not been studied previously.
Methods
The study population of 3,323 patients with QTc ≥ 450 ms enrolled in the International LQTS Registry involved 20 patients with sudden cardiac death (SCD), 16 patients with aborted cardiac arrest (ACA), 34 patients with syncope, and 3,253 patients who were asymptomatic during the first year of life.
Results
The risk factors for a cardiac event among 212 patients who had an ECG recorded in the first year of life included QTc≥500ms, heart rate ≤100bpm, and female sex. ACA before age 1 year was associated with a hazard ratio of 23.4 (p<0.01) for ACA or SCD during age 1-10 years. During the 10-year follow-up after infancy, beta-blocker therapy was associated with a significant reduction in ACA/SCD only in those with a syncopal episode within 2 years before ACA/SCD, but not for those who survived ACA in infancy.
Conclusions
Patients with LQTS who experience ACA during the first year of life are at very high-risk for subsequent ACA or death during their next 10 years of life, and beta-blockers may not be effective in preventing fatal or near fatal cardiac events in this small but high-risk subset.
doi:10.1016/j.jacc.2009.05.029
PMCID: PMC3517782  PMID: 19695463
Long QT Syndrome; Genetics; Infants; Risk Stratification
22.  OVA-induced airway hyperresponsiveness alters murine heart rate variability and body temperature 
Altered autonomic (ANS) tone in chronic respiratory disease is implicated as a factor in cardiovascular co-morbidities, yet no studies address its impact on cardiovascular function in the presence of murine allergic airway (AW) hyperresponsiveness (AHR). Since antigen (Ag)-induced AHR is used to model allergic asthma (in which ANS alterations have been reported), we performed a pilot study to assess measurement feasibility of, as well as the impact of allergic sensitization to ovalbumin (OVA) on, heart rate variability (HRV) in a murine model. Heart rate (HR), body temperature (TB), and time- and frequency-domain HRV analyses, a reflection of ANS control, were obtained in chronically instrumented mice (telemetry) before, during and for 22 h after OVA or saline aerosolization in sensitized (OVA) or Alum adjuvant control exposed animals. OVA mice diverged significantly from Alum mice with respect to change in HR during aerosol challenge (P < 0.001, Two-Way ANOVA; HR max change Ctrl = +80 ± 10 bpm vs. OVA = +1 ± 23 bpm, mean ± SEM), and displayed elevated HR during the subsequent dark cycle (P = 0.006). Sensitization decreased the TB during aerosol challenge (P < 0.001). Sensitized mice had decreased HRV prior to challenge (SDNN: P = 0.038; Low frequency (LF) power: P = 0.021; Low/high Frequency (HF) power: P = 0.042), and increased HRV during Ag challenge (RMSSD: P = 0.047; pNN6: P = 0.039). Sensitized mice displayed decreased HRV subsequent to OVA challenge, primarily in the dark cycle (RMSSD: P = 0.018; pNN6: P ≤ 0.001; LF: P ≤ 0.001; HF: P = 0.040; LF/HF: P ≤ 0.001). We conclude that implanted telemetry technology is an effective method to assess the ANS impact of allergic sensitization. Preliminary results show mild sensitization is associated with reduced HRV and a suppression of the acute TB-response to OVA challenge. This approach to assess altered ANS control in the acute OVA model may also be beneficial in chronic AHR models.
doi:10.3389/fphys.2012.00456
PMCID: PMC3514704  PMID: 23227012
airway hyperresponsiveness; mouse; ovalbumin; heart rate variability; autonomic control; allergic sensitization; asthma; cardiovascular comorbidity
23.  Antibiofilm activity of coconut (Cocos nucifera Linn.) husk fibre extract 
In this study, antibiofilm activity of coconut husk extract (CHE) was tested by various assays in the laboratory. The effects of CHE on extracellular polymeric substance (EPS) production, hydrophobicity and adhesion ability of Pseudomonas sp., Alteromonas sp. and Gallionella sp. and the antimicrobial activity of the extract against these bacteria were assessed. CHE was found to possess antibacterial activity against all the bacterial strains and affected the EPS production. The CHE affected the growth of the biofilm-forming bacteria in a culture medium. The hydrophobicity of the bacterial cells was also changed due to the CHE treatment. The active compound of the CHE was characterised by thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and fourier transform infrared (FT-IR) analysis. HPLC spectrum showed a single peak and the FT-IR spectrum indicated the presence of an OH-group-containing compound in the extract. In conclusion the CHE could be used as a source for the isolation of antifouling compounds.
doi:10.1016/j.sjbs.2012.11.002
PMCID: PMC3730797  PMID: 23961225
Biofouling; Biofilm; Polyphenol; Extracellular polymeric substance; Adhesion
24.  Abiotic Factors Shape Microbial Diversity in Sonoran Desert Soils 
Applied and Environmental Microbiology  2012;78(21):7527-7537.
High-throughput, culture-independent surveys of bacterial and archaeal communities in soil have illuminated the importance of both edaphic and biotic influences on microbial diversity, yet few studies compare the relative importance of these factors. Here, we employ multiplexed pyrosequencing of the 16S rRNA gene to examine soil- and cactus-associated rhizosphere microbial communities of the Sonoran Desert and the artificial desert biome of the Biosphere2 research facility. The results of our replicate sampling approach show that microbial communities are shaped primarily by soil characteristics associated with geographic locations, while rhizosphere associations are secondary factors. We found little difference between rhizosphere communities of the ecologically similar saguaro (Carnegiea gigantea) and cardón (Pachycereus pringlei) cacti. Both rhizosphere and soil communities were dominated by the disproportionately abundant Crenarchaeota class Thermoprotei, which comprised 18.7% of 183,320 total pyrosequencing reads from a comparatively small number (1,337 or 3.7%) of the 36,162 total operational taxonomic units (OTUs). OTUs common to both soil and rhizosphere samples comprised the bulk of raw sequence reads, suggesting that the shared community of soil and rhizosphere microbes constitute common and abundant taxa, particularly in the bacterial phyla Proteobacteria, Actinobacteria, Planctomycetes, Firmicutes, Bacteroidetes, Chloroflexi, and Acidobacteria. The vast majority of OTUs, however, were rare and unique to either soil or rhizosphere communities and differed among locations dozens of kilometers apart. Several soil properties, particularly soil pH and carbon content, were significantly correlated with community diversity measurements. Our results highlight the importance of culture-independent approaches in surveying microbial communities of extreme environments.
doi:10.1128/AEM.01459-12
PMCID: PMC3485727  PMID: 22885757
25.  Microbial community structure in a biogas digester utilizing the marine energy crop Saccharina latissima 
3 Biotech  2012;3(5):407-414.
Seaweed is a highly attractive marine crop for the production of biofuels, due to its rapid growth rate as well as high polysaccharide and low lignin content. One appealing exploitation route is the production of biogas by anaerobic digestion. Interestingly, despite the compositional differences between seaweed and lignocellulosic biomass, available data indicate that conditions and inocula traditionally used for the latter may work well for seaweed. To gain more insight into the underlying microbial processes, we have generated 16S rRNA gene amplicon pyrosequencing data to comparatively describe microbial communities in biogas digesters containing either the seaweed Saccharina latissima or wheat straw. The seaweed digesters gave better biogas yield and a higher relative abundance of core group Methanosaeta-affiliated Archaea. Conversely, variation in biomass had only minor abundance effects towards dominant bacterial lineages and influenced only low-abundant bacterial OTUs. Affiliations between dominant archaeal and bacterial phylotypes described here and previously identified anaerobic digestion core groups indicate that trends are beginning to emerge within these newly explored microbial ecosystems, the understanding of which is currently impeded by limited published datasets.
doi:10.1007/s13205-012-0097-x
PMCID: PMC3781269
Biogas; Anaerobic digestion; Seaweed; Macroalgae; Methane

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