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1.  Hemogenic endocardium contributes to transient definitive hematopoiesis 
Nature communications  2013;4:1564.
Hematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial, and hematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a hemogenic organ akin to the dorsal aorta. Here we examine the hemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Hemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the hemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells expressing cardiac markers serve as a de novo source for transient definitive hematopoietic progenitors.
PMCID: PMC3612528  PMID: 23463007
2.  Complete Atrial-Specific Knockout of Sodium-Calcium Exchange Eliminates Sinoatrial Node Pacemaker Activity 
PLoS ONE  2013;8(11):e81633.
The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by “funny” current (If) through HCN4 channels (the “Membrane Clock“ hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two (“Coupled Clock”). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.
PMCID: PMC3836769  PMID: 24278453
3.  Scl represses cardiomyogenesis in prospective hemogenic endothelium and endocardium 
Cell  2012;150(3):590-605.
Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in hemogenic endothelium and preventing its misspecification to a cardiomyogenic fate. Scl−/− embryos activated a cardiac transcriptional program in yolk sac endothelium, leading to the emergence of CD31+Pdgfrα+ cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl−/− hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Sclfl/fl Rosa26Cre-ERT2 embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from endothelium. Scl−/− endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.
PMCID: PMC3624753  PMID: 22863011
4.  Migratory properties of pulmonary dendritic cells are determined by their developmental lineage 
Mucosal immunology  2012;6(4):678-691.
The chemokine receptor, CCR7, directs the migration of dendritic cells (DCs) from peripheral tissue to draining lymph nodes (LNs). However, it is unknown whether all pulmonary DCs possess migratory potential. Using novel Ccr7gfp reporter mice, we found that Ccr7 is expressed in CD103+ and a CD14med/lo subset of CD11bhi classical (c) DCs but not in monocyte-derived (mo) DCs, including Ly-6ChiCD11bhi inflammatory DCs and CD14hiCD11bhi DCs. Consequently, cDCs migrated to lung-draining LNs but moDCs did not. Mice lacking the chemokine receptor, CCR2, also lacked inflammatory DCs in the lung after lipopolysaccharide inhalation but retained normal levels of migratory DCs. Conversely, the lungs of fms-like tyrosine kinase 3 ligand (Flt3L)-deficient mice lacked cDCs but retained moDCs, which were functionally mature but did not express Ccr7 and were uniformly non-migratory. Thus, the migratory properties of pulmonary DCs are determined by their developmental lineage.
PMCID: PMC3652913  PMID: 23168837
5.  Pulmonary CD103+ dendritic cells prime Th2 responses to inhaled allergens 
Mucosal immunology  2011;5(1):53-65.
Allergic asthma stems largely from the actions of T helper 2 (Th2) cells, but the pathways that initiate Th2 responses to inhaled allergens are not fully understood. In the lung, there are two major subsets of dendritic cells (DCs), displaying CD11b or CD103. We found that after taking up inhaled ovalbumin in vivo, purified CD103+ DCs from the lung or lung-draining lymph nodes primed Th2 differentiation ex vivo. Th2 induction by CD103+ DCs was also seen when cockroach or house dust mite allergens were used. In contrast, CD11bhi DCs primed Th1 differentiation. Moreover, mice lacking CD103+ DCs displayed diminished Th2 priming to various inhaled allergens and did not develop asthma-like responses following subsequent allergen challenge. Low-level antigen presentation by CD103+ DCs was necessary, but not sufficient for Th2 priming. Together, these findings show that CD103+ DCs have a significant role in priming Th2 responses to inhaled allergens.
PMCID: PMC3697034  PMID: 22012243
6.  Cardiac origin of smooth muscle cells in the inflow tract 
Multipotent Isl1+ heart progenitors give rise to three major cardiovascular cell types; cardiac, smooth muscle, and endothelial cells, and play a pivotal role in lineage diversification during cardiogenesis. A critical question is pinpointing when this cardiac-vascular lineage decision is made, and how this plasticity serves to coordinate cardiac chamber and vessel growth. The posterior domain of the Isl1-positive second heart field contributes to the SLN-positive atrial myocardium and myocardial sleeves in the cardiac inflow tract, where myocardial and vascular smooth muscle layers form anatomical and functional continuity. Herein, using a new atrial specific SLN-Cre knockin mouse line, we report that an Isl1+/SLN+ transient cell population contributes to cardiac as well as smooth muscle cells at the heart-vessel junction in cardiac inflow tract. The Isl1+/SLN+ cells are capable of giving rise to cardiac and smooth muscle cells until late gestational stages. These data suggest that the cardiac and smooth muscle cells in the cardiac inflow tract share a common developmental origin.
PMCID: PMC3031779  PMID: 20974149
cardiogenesis; myogenic progenitor; smooth muscle; great vessel; plasticity
7.  IL-35 Production by ICOS+ Tregs Reverses Established IL-17-dependent Allergic Airways Disease 
Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined.
We sought to understand the cellular and molecular basis for suppression of established, IL-17-dependent allergic airways disease.
Mice were sensitized by airway instillations of ovalbumin (OVA) together with low levels of lipopolysaccharide. Leukocyte recruitment to the lung and AHR were assessed following daily challenges with aerosolized OVA. Flow cytometry and gene targeted mice were used to identify naturally-arising subsets of regulatory T cells (Tregs) and their cytokines required for the suppression of established allergic airway disease.
Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR declined, even though pulmonary levels of Th17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg subset expressing both Foxp3 and inducible co-stimulator (ICOS). These Tregs also expressed the regulatory cytokines, IL-10, TGF-beta and IL-35. Whereas IL-10 and TGF-beta were dispensable for suppression of airway hyperresponsiveness, IL-35 was required. Analysis of human ICOS+ Tregs revealed that they also selectively expressed IL-35.
IL-35 production by ICOS+ Tregs can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. The production of IL-35 by human ICOS+ Tregs suggests that targeting this pathway might be of therapeutic value for treating allergic asthma in humans.
PMCID: PMC3269135  PMID: 21906794
Asthma; airway hyperresponsiveness; AHR; IL-17; Th17; Th2; IL-35; ICOS; ovalbumin
8.  Atypical gaze patterns in children and adults with autism spectrum disorders dissociated from developmental changes in gaze behaviour 
Eye tracking has been used to investigate gaze behaviours in individuals with autism spectrum disorder (ASD). However, traditional analysis has yet to find behavioural characteristics shared by both children and adults with ASD. To distinguish core ASD gaze behaviours from those that change with development, we examined temporo-spatial gaze patterns in children and adults with and without ASD while they viewed video clips. We summarized the gaze patterns of 104 participants using multidimensional scaling so that participants with similar gaze patterns would cluster together in a two-dimensional plane. Control participants clustered in the centre, reflecting a standard gaze behaviour, whereas participants with ASD were distributed around the periphery. Moreover, children and adults were separated on the plane, thereby showing a clear effect of development on gaze behaviours. Post hoc frame-by-frame analyses revealed the following findings: (i) both ASD groups shifted their gaze away from a speaker earlier than the control groups; (ii) both ASD groups showed a particular preference for letters; and (iii) typical infants preferred to watch the mouth rather than the eyes during speech, a preference that reversed with development. These results highlight the importance of taking the effect of development into account when addressing gaze behaviours characteristic of ASD.
PMCID: PMC2982027  PMID: 20484237
eye tracking; eye movements; autism; development; mouth viewing; turn taking
9.  The Nitric Oxide-Responsive Regulator NsrR Controls ResDE-Dependent Gene Expression 
Journal of Bacteriology  2006;188(16):5878-5887.
The ResD-ResE signal transduction system is essential for aerobic and anaerobic respiration in Bacillus subtilis. ResDE-dependent gene expression is induced by oxygen limitation, but full induction under anaerobic conditions requires nitrite or nitric oxide (NO). Here we report that NsrR (formerly YhdE) is responsible for the NO-dependent up-regulation of the ResDE regulon. The null mutation of nsrR led to aerobic derepression of hmp (flavohemoglobin gene) partly in a ResDE-independent manner. In addition to its negative role in aerobic hmp expression, NsrR plays an important role under anaerobic conditions for regulation of ResDE-controlled genes, including hmp. ResDE-dependent gene expression was increased by the nsrR mutation in the absence of NO, but the expression was decreased by the mutation when NO was present. Consequently, B. subtilis cells lacking NsrR no longer sense and respond to NO (and nitrite) to up-regulate the ResDE regulon. Exposure to NO did not significantly change the cellular concentration of NsrR, suggesting that NO likely modulates the activity of NsrR. NsrR is similar to the recently described nitrite- or NO-sensitive transcription repressors present in various bacteria. NsrR likely has an Fe-S cluster, and interaction of NO with the Fe-S center is proposed to modulate NsrR activity.
PMCID: PMC1540067  PMID: 16885456
10.  Liver dysfunction among workers handling 5-nitro-o-toluidine 
Gut  2002;50(2):266-270.
Background: 5-Nitro-o-toluidine is an aromatic nitro amino compound. While other aromatic compounds are known to damage the human liver and are registered as toxic substances, toxicity information concerning 5-nitro-o-toluidine is lacking.
Aims: To investigate the hepatotoxicity of 5-nitro-o-toluidine.
Patients and methods: Of 15 workers in the same factory who handled 5-nitro-o-toluidine, three were hospitalised with symptoms of acute liver dysfunction. Suspecting a link between liver dysfunction and working conditions, we correlated workplace factors with clinical findings in all 15 workers.
Results: Blood biochemistry tests indicated liver damage in seven of 15 study subjects. Workers who handled 5-nitro-o-toluidine and nitrosyl sulphuric acid often loosened their respiratory protective equipment shortly after 5-nitro-o-toluidine powder had been dispersed into the air of the room. No potential hepatotoxins were present except for 5-nitro-o-toluidine. Six of the affected workers had handled 5-nitro-o-toluidine 12 to 20 times; the seventh worker had handled the powder three times; and the other eight workers without liver dysfunction had handled the material once or twice. No other significant differences in background were evident between the affected and unaffected workers, such as age, sex, or protective measures. Histological findings during recovery from liver damage were similar to those of acute viral hepatitis. None of the 15 subjects has demonstrated liver damage since the factory was closed.
Conclusions: A link between liver dysfunction and 5-nitro-o-toluidine exposure is suggested by greater severity of liver dysfunction associated with more episodes of handling.
PMCID: PMC1773104  PMID: 11788571
5-nitro-o-toluidine; liver toxicity; liver function; toxicity
11.  Interferon responsiveness in patients infected with hepatitis C virus 1b differs depending on viral subtype 
Gut  2001;49(2):263-267.
BACKGROUND—Genotype 1b of hepatitis C virus (HCV) comprises mainly three subtypes, each named for its geographic prevalence (worldwide, W; Japan, J; and not in Japan, NJ).
AIM—To characterise the newly identified subtypes of genotype 1b and to review factors associated with response to interferon (IFN) for each subtype.
PATIENTS—Chronic hepatitis patients (80 men and 41 women; mean age 48.5 years, range 20.7-69.3) with HCV genotype 1b (W type, n=41; J type, n=38) or genotype 2a (n=42) were treated according to the same IFN protocol. Forty four patients (36.4%) negative for serum HCV RNA six months after cessation of treatment were considered complete responders.
METHODS—Factors associated with complete response were investigated.
RESULTS—Genotype 2a patients had lower viral loads (odds ratio 0.11 (95% confidence intervals (CI) 0.049-0.256)) and a better IFN response (odds ratio 0.25 (95% CI 0.117-0.552)) than genotype 1b patients whereas W type and J type patients had similar viral loads and responses to IFN. IFN response in W type patients was associated with female sex (odds ratio 0.23 (95% CI 0.055-0.983)) and low viral load (odds ratio 84.00 (95% CI 14.04-502.6)) whereas response in J type patients was related to transfusion history (odds ratio 7.20 (95% CI 1.443-35.91)), low viral load (odds ratio 117.0 (95% CI 17.82-768.3)), and genetic mutation in the interferon sensitivity determining region of the virus (odds ratio 0.08 (95% CI 0.013-0.553)). Multivariate analysis found low viral load (odds ratio 64.19 (95% CI 14.66-281.06)) to be the only significant independent factor associated with IFN response.
CONCLUSIONS—Factors associated with IFN responsiveness in HCV infection differ with viral subtype.

Keywords: hepatitis C virus; genotype 1b; chronic hepatitis; interferon therapy; interferon sensitivity determining region
PMCID: PMC1728417  PMID: 11454804
12.  CFE-1, a Novel Plasmid-Encoded AmpC β-Lactamase with an ampR Gene Originating from Citrobacter freundii 
A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded β-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a β-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC β-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a β-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC β-lactamase, CFE-1, with an ampR gene derived from C. freundii.
PMCID: PMC375250  PMID: 15047515
13.  Transcriptional Activation by Bacillus subtilis ResD: Tandem Binding to Target Elements and Phosphorylation-Dependent and -Independent Transcriptional Activation 
Journal of Bacteriology  2004;186(7):2028-2037.
The expression of genes involved in nitrate respiration in Bacillus subtilis is regulated by the ResD-ResE two-component signal transduction system. The membrane-bound ResE sensor kinase perceives a redox-related signal(s) and phosphorylates the cognate response regulator ResD, which enables interaction of ResD with ResD-dependent promoters to activate transcription. Hydroxyl radical footprinting analysis revealed that ResD tandemly binds to the −41 to −83 region of hmp and the −46 to −92 region of nasD. In vitro runoff transcription experiments showed that ResD is necessary and sufficient to activate transcription of the ResDE regulon. Although phosphorylation of ResD by ResE kinase greatly stimulated transcription, unphosphorylated ResD, as well as ResD with a phosphorylation site (Asp57) mutation, was able to activate transcription at a low level. The D57A mutant was shown to retain the activity in vivo to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD itself, in addition to its activation through phosphorylation-mediated conformation change, senses oxygen limitation via an unknown mechanism leading to anaerobic gene activation.
PMCID: PMC374413  PMID: 15028686
14.  Multiple Pathways of Spx (YjbD) Proteolysis in Bacillus subtilis 
Journal of Bacteriology  2002;184(13):3664-3670.
ATP-dependent proteases degrade denatured or misfolded proteins and are recruited for the controlled removal of proteins that block activation of regulatory pathways. Among the ATP-dependent proteases, those of the Clp family are particularly important for the growth and development of Bacillus subtilis. Proteolytic subunit ClpP, together with regulatory ATPase subunit ClpC or ClpX, is required for the normal response to stress, for development of genetic competence, and for sporulation. The spx (formally yjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred by clpP and clpX. The level of Spx in wild-type cells grown in competence medium is low, and that in clpP mutants is high. This suggests that the Spx protein is a substrate for ClpP-containing proteases and that accumulation of Spx might be partly responsible for the observed pleiotropic phenotype resulting from the clpP mutation. In this study we examined, both in vivo and in vitro, which ClpP protease is responsible for degradation of Spx. Western blot analysis showed that Spx accumulated in clpX mutant to the same level as that observed in the clpP mutant. In contrast, a very low concentration of Spx was detected in a clpC mutant. An in vitro proteolysis experiment using purified proteins demonstrated that Spx was degraded by ClpCP but only in the presence of one of the ClpC adapter proteins, MecA or YpbH. However, ClpXP, either in the presence or in the absence of MecA and YpbH, was unable to degrade Spx. Transcription of spx, as measured by expression of spx-lacZ, was slightly increased by the clpX mutation. To exclude a possible effect of clpX and clpP on spx transcription, the spx gene was placed under the control of the IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible Pspac promoter. In this strain, Spx accumulated when ClpX or ClpP was absent, suggesting that ClpX and ClpP are required for degradation of Spx. Taken together, these results suggest that Spx is degraded by both ClpCP and ClpXP. The putative proteolysis by ClpXP might require another adapter protein. Spx probably is degraded by ClpCP under as yet unidentified conditions. This study suggests that the level of Spx is tightly controlled by two different ClpP proteases.
PMCID: PMC135134  PMID: 12057962
15.  Passive Immunization with Milk Produced from an Immunized Cow Prevents Oral Recolonization by Streptococcus mutans 
Cell surface protein antigen (PAc) and water-insoluble glucan-synthesizing enzyme (GTF-I) produced by cariogenic Streptococcus mutans are two major factors implicated in the colonization of the human oral cavity by this bacterium. We examined the effect of bovine milk, produced after immunization with a fusion protein of functional domains of these proteins, on the recolonization of S. mutans. To prepare immune milk, a pregnant Holstein cow was immunized with the fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I. After eight adult subjects received cetylpyridinium chloride (CPC) treatment, one subgroup (n = 4) rinsed their mouths with immune milk and a control group (n = 4) rinsed with nonimmune milk. S. mutans levels in saliva and dental plaque decreased after CPC treatment in both groups. Mouth rinsing with immune milk significantly inhibited recolonization of S. mutans in saliva and plaque. On the other hand, the numbers of S. mutans cells in saliva and plaque in the control group increased immediately after the CPC treatment and surpassed the baseline level 42 and 28 days, respectively, after the CPC treatment. The ratios of S. mutans to total streptococci in saliva and plaque in the group that received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling S. mutans in the human oral cavity.
PMCID: PMC96239  PMID: 11687453
16.  Protection against lethal bacterial infection in mice by monocyte-chemotactic and -activating factor. 
Infection and Immunity  1994;62(2):377-383.
Chemotactic factors regulate the recruitment of neutrophils, lymphocytes, or monocytes-macrophages to infectious and inflammatory sites. The purpose of this study was to determine whether monocyte-chemotactic and -activating factor (MCAF [MCP-1], a JE gene product) also influences the host defense mechanism against microbial infection. We evaluated the effect of recombinant human MCAF on the survival rate of mice systemically infected with Pseudomonas aeruginosa or Salmonella typhimurium. The administration of 2.5 micrograms of MCAF 6 h before infection completely protected the mice from lethal infection. Mice with cyclophosphamide-induced leukopenia exhibiting increased susceptibility to P. aeruginosa were also endowed with resistance by the same dose of MCAF. Administration of MCAF at -6 h was critical, since MCAF given either earlier or later than -6 h failed to rescue mice from lethal infection. The in vivo effect on the survival of mice paralleled the reduced recovery of viable P. aeruginosa or S. typhimurium from the peritoneal cavity, i.e., the number of recovered bacteria from the MCAF (2.5 micrograms per mouse)-treated mice was reduced to less than 2% of control mice for P. aeruginosa and 4% of control mice for S. typhimurium at 24 h. Since MCAF exhibited chemotaxis on murine macrophages as well as enhanced phagocytosis and killing of bacteria in vitro, the activation of macrophages, followed by the recruitment into the peritoneal cavity, is responsible for eliminating bacteria and thus enhancing the survival rate.
PMCID: PMC186118  PMID: 8300198
17.  Neutralization of microcystin shock in mice by tumor necrosis factor alpha antiserum. 
Microcystis aeruginosa is a common cyanobacterium in water blooms that appear widely in nutrient-rich, fresh, and brackish waters, and its toxic blooms cause the death of domestic animals. The administration of a crude toxic cell extract of M. aeruginosa K-139 to mice can produce tumor necrosis factor (TNF) and prompt severe physiological disturbances, especially liver damage, which can lead to death. The in vitro production of TNF-alpha by peritoneal macrophages was observed after stimulation with the cell extract or the purified toxin from K-139 cells. The expression of a TNF-alpha mRNA was also detected in spleen cells and peritoneal macrophages after stimulation with the cell extract. However, a previous injection of rabbit anti-murine TNF-alpha serum could prevent the liver damage to some extent and protect the mice from death. These findings indicate the involvement of TNF in microcystin shock.
PMCID: PMC182708  PMID: 2036020
18.  Classification of Human Coronary Atherosclerotic Plaques Ex-Vivo with T1, T2 and Ultra-short TE MRI 
JACC. Cardiovascular imaging  2013;6(4):466-474.
To determine whether the classification of human coronary atherosclerotic plaques with T1, T2 and ultrashort TE (UTE) MRI would correlate well with atherosclerotic plaque classification by histology.
MRI has been extensively used to classify carotid plaque but its ability to characterize coronary plaque remains unknown. In addition, the detection of plaque calcification by MRI remains challenging. Here we used T1, T2 and UTE MRI to evaluate atherosclerotic plaques in fixed post-mortem human coronary arteries. We hypothesized that the combination of T1, T2 and UTE MRI would allow both calcified and lipid-rich coronary plaques to be accurately detected.
28 plaques from human donor hearts with proven coronary artery disease were imaged at 9.4 Tesla with a T1 weighted 3D FLASH sequence (250 um resolution), a T2 weighted Rare sequence (in plane resolution 0.156mm), and an UTE sequence (300um resolution). Plaques showing selective hypointensity on T2 weighted MRI were classified as lipid-rich. Areas of hypointensity on the T1 weighted images but not the UTE images were classified as calcified. Hyperintensity on the T1 weighted and UTE images was classified as hemorrhage. Following MRI, histological characterization of the plaques was performed with a pentachrome stain and established AHA criteria.
MRI showed high sensitivity and specificity for the detection of calcification (100% and 90%) and lipid-rich necrotic cores (90% and 75%). Only two lipid-rich foci were missed by MRI, both of which were extremely small. Overall, MRI based classification of plaque was in complete agreement with the histological classification in 22/28 cases (weighted κ =0.6945, p<0.0001).
The utilization of UTE MRI allows plaque calcification in the coronary arteries to be robustly detected. High-resolution MRI with T1, T2 and UTE contrast enables accurate classification of human coronary atherosclerotic plaque.
PMCID: PMC3661771  PMID: 23498670
atherosclerosis; coronary artery; MRI; ultra-short TE; plaque classification
19.  Protein Dielectrophoresis: Advances, Challenges and Applications 
Electrophoresis  2013;34(7):10.1002/elps.201200482.
Protein dielectrophoresis (DEP) has the potential to play an important role as a manipulation, fractionation, pre-concentration and separation method in bioanalysis and as manipulation tool for nanotechnological applications. The first demonstrations of protein DEP have been reported almost twenty years ago. Since then various experimental realizations to manipulate proteins by DEP as well as more targeted applications employing protein DEP have been demonstrated. This review summarizes the experimental studies in the field of protein DEP trapping and focusing as well as specific applications in separation, molecular patterning, on bioprobes and biosensors. While a comprehensive theoretical model describing protein DEP is still lacking we also attempt to provide an overview of the factors influencing protein DEP and relate to currently available theoretical models. We further point out the variations in experimental conditions used in the past to study the somewhat 20 proteins as well as the implications of protein molecular structure to the DEP response.
PMCID: PMC3839426  PMID: 23400789
protein; dielectrophoresis; trapping; focusing; applications
20.  Variants in CPA1 are strongly associated with early-onset chronic pancreatitis 
Nature genetics  2013;45(10):1216-1220.
Chronic pancreatitis is an inflammatory disorder of the pancreas. We analyzed CPA1 encoding carboxypeptidase A1 in subjects with non-alcoholic chronic pancreatitis and controls in a German discovery cohort and three replication cohorts. Functionally impaired variants were present in 29/944 (3.1%) German patients and in 5/3,938 (0.1%) controls (odds ratio [OR] = 24.9; P = 1.5 × 10-16). The association was strongest in subjects aged ≤10 years (9.7%; OR = 84.0; P = 4.1 × 10-24). In the replication cohorts, defective CPA1 variants were observed in 8/600 (1.3%) patients and in 9/2,432 (0.4%) controls from Europe (P = 0.01), in 5/230 (2.2%) patients and 0/264 controls from India (P = 0.02), and in 5/247 (2.0%) patients but 0/341 controls from Japan (P = 0.013). The mechanism of increased pancreatitis risk by CPA1 variants may involve misfolding-induced endoplasmic reticulum stress rather than elevated trypsin activity as seen with other genetic risk factors.
PMCID: PMC3909499  PMID: 23955596
21.  Brain Tumor Initiating Cells Adapt to Restricted Nutrition through Preferential Glucose Uptake 
Nature neuroscience  2013;16(10):1373-1382.
Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) due to preferential BTIC survival and adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3 and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, TICs may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may instruct the tumor hierarchy and portend poor prognosis.
PMCID: PMC3930177  PMID: 23995067
22.  Radiotherapy-Induced Anti-Tumor Immunity Contributes to the Therapeutic Efficacy of Irradiation and Can Be Augmented by CTLA-4 Blockade in a Mouse Model 
PLoS ONE  2014;9(3):e92572.
There is growing evidence that tumor-specific immune responses play an important role in anti-cancer therapy, including radiotherapy. Using mouse tumor models we demonstrate that irradiation-induced anti-tumor immunity is essential for the therapeutic efficacy of irradiation and can be augmented by modulation of cytotoxic T lymphocyte (CTL) activity.
Methods and Materials
C57BL/6 mice, syngeneic EL4 lymphoma cells, and Lewis lung carcinoma (LL/C) cells were used. Cells were injected into the right femurs of mice. Ten days after inoculation, tumors were treated with 30 Gy of local X-ray irradiation and their growth was subsequently measured. The effect of irradiation on tumor growth delay (TGD) was defined as the time (in days) for tumors to grow to 500 mm3 in the treated group minus that of the untreated group. Cytokine production and serum antibodies were measured by ELISA and flow cytometry.
In the EL4 tumor model, tumors were locally controlled by X-ray irradiation and re-introduced EL4 cells were completely rejected. Mouse EL4-specific systemic immunity was confirmed by splenocyte cytokine production and detection of tumor-specific IgG1 antibodies. In the LL/C tumor model, X-ray irradiation also significantly delayed tumor growth (TGD: 15.4 days) and prolonged median survival time (MST) to 59 days (versus 28 days in the non-irradiated group). CD8(+) cell depletion using an anti-CD8 antibody significantly decreased the therapeutic efficacy of irradiation (TGD, 8.7 days; MST, 49 days). Next, we examined whether T cell modulation affected the efficacy of radiotherapy. An anti-CTLA-4 antibody significantly increased the anti-tumor activity of radiotherapy (TGD was prolonged from 13.1 to 19.5 days), while anti-FR4 and anti-GITR antibodies did not affect efficacy.
Our results indicate that tumor-specific immune responses play an important role in the therapeutic efficacy of irradiation. Immunomodulation, including CTLA-4 blockade, may be a promising treatment in combination with radiotherapy.
PMCID: PMC3970971  PMID: 24686897
23.  Effect of Silica Particle Size on Macrophage Inflammatory Responses 
PLoS ONE  2014;9(3):e92634.
Amorphous silica particles, such as nanoparticles (<100 nm diameter particles), are used in a wide variety of products, including pharmaceuticals, paints, cosmetics, and food. Nevertheless, the immunotoxicity of these particles and the relationship between silica particle size and pro-inflammatory activity are not fully understood. In this study, we addressed the relationship between the size of amorphous silica (particle dose, diameter, number, and surface area) and the inflammatory activity (macrophage phagocytosis, inflammasome activation, IL-1β secretion, cell death and lung inflammation). Irrespective of diameter size, silica particles were efficiently internalized by mouse bone marrow-derived macrophages via an actin cytoskeleton-dependent pathway, and induced caspase-1, but not caspase-11, activation. Of note, 30 nm-1000 nm diameter silica particles induced lysosomal destabilization, cell death, and IL-1β secretion at markedly higher levels than did 3000 nm-10000 nm silica particles. Consistent with in vitro results, intra-tracheal administration of 30 nm silica particles into mice caused more severe lung inflammation than that of 3000 nm silica particles, as assessed by measurement of pro-inflammatory cytokines and neutrophil infiltration in bronchoalveolar lavage fluid of mice, and by the micro-computed tomography analysis. Taken together, these results suggest that silica particle size impacts immune responses, with submicron amorphous silica particles inducing higher inflammatory responses than silica particles over 1000 nm in size, which is ascribed not only to their ability to induce caspase-1 activation but also to their cytotoxicity.
PMCID: PMC3969333  PMID: 24681489
24.  Singlet Molecular Oxygen Generation by Light-Activated DHN-Melanin of the Fungal Pathogen Mycosphaerella fijiensis in Black Sigatoka Disease of Bananas 
PLoS ONE  2014;9(3):e91616.
In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1Δg), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1Δg). A pigmented-strain generated more O2 (1Δg) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1Δg) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis.
PMCID: PMC3960117  PMID: 24646830
25.  Knockdown of GRP78 Promotes Apoptosis in Pancreatic Acinar Cells and Attenuates the Severity of Cerulein and LPS Induced Pancreatic Inflammation 
PLoS ONE  2014;9(3):e92389.
Acute pancreatitis (AP) is a potentially lethal disease characterized by inflammation and parenchymal cell death; also, the severity of AP correlates directly with necrosis and inversely with apoptosis. However, mechanisms of regulating cell death in AP remain unclear. The endoplasmic reticulum (ER) chaperone protein GRP78 has anti-apoptotic properties, in addition to modulating ER stress responses. This study used RNA interference (RNAi) approach to investigate the potential role of GRP78 in regulating apoptosis during AP. In vitro models of AP were successfully developed by treating AR42J cells with cerulein or cerulein plus lipoplysaccharide (LPS). There was more pancreatic inflammation and less apoptosis with the cerulein plus LPS treatment. Furthermore, knockdown of GRP78 expression markedly promoted apoptosis and reduced necrosis in pancreatic acinar cells. This was accomplished by enhancing the activation of caspases and inhibiting the activity of X-linked inhibitor of apoptosis protein (XIAP), as well as a receptor interacting protein kinase-1(RIPK1), which is a key mediator of necrosis. This attenuated the severity of pancreatic inflammation, especially after cerulein plus LPS treatment. In conclusion, these findings indicate that GRP78 plays an anti-apoptotic role in regulating the cell death response during AP. Therefore, GRP78 is a potential therapeutic target for AP.
PMCID: PMC3958537  PMID: 24643222

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