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1.  Cloning and expression in Escherichia coli of sdhA, the structural gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex. 
Journal of Bacteriology  1985;162(3):1180-1185.
Bacillus subtilis cytochrome b558 is a transmembrane protein which anchors succinate dehydrogenase (SDH) to the cytoplasmic membrane and is reduced by succinate. The structural gene for this cytochrome was cloned and expressed in Escherichia coli. Random BamHI or BglII fragments of B. subtilis 168 DNA were cloned in the BamHI site of plasmid pHV32. The derived plasmids were used to transform B. subtilis SDH mutants to chloramphenicol resistance by integration of the plasmid via DNA homology. Of some 3,000 transformants tested, 6 were SDH positive and had pHV32 integrated close to the sdh operon. Two plasmids, pKIM2 and pKIM4, with an insert of B. subtilis DNA of 5.7 and 3.4 kilobases, respectively, were generated by transforming E. coli with DNA from the SDH-positive transformants after cleavage with EcoRI or BglII and ligation. In E. coli carrying either of the two plasmids, about 4% of total membrane protein was B. subtilis cytochrome b558. E. coli (pKIM2) also contained antigen which reacted with antibodies specific for the flavoprotein and the iron-sulfur protein subunit of B. subtilis SDH. Enzymatically active, membrane-bound B. subtilis SDH could not be demonstrated in E. coli (pKIM2). The B. subtilis DNA insert in pKIM2 could transform B. subtilis sdhA (cytochrome b558), sdhB (flavoprotein), and sdhC (iron-sulfur protein) mutants to the wild type. The results suggest that pKIM2 carries the whole B. subtilis sdh operon. The data confirm the gene order and the proposed direction of transcription of the B. subtilis sdh operon. Most likely the sdh genes in E. coli(pKIM2) are controlled by their natural promoter.
PMCID: PMC215901  PMID: 2987185
2.  Divergence of nucleosome positioning between two closely related yeast species: genetic basis and functional consequences 
Inter-species hybrids can be used to dissect the relative contribution of cis and trans effects to the evolution of nucleosome positioning. Most (∼70%) differences in nucleosome positioning between two closely related yeast species are due to cis effects.Cis effects are primarily due to divergence of AT-rich nucleosome-disfavoring sequences, but are not associated with divergence of nucleosome-favoring sequences.Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis.Divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a neutral mode of evolution.
Phenotypic diversity is often due to changes in gene regulation, and recent studies have characterized extensive differences between the gene expression programs of closely related species (Khaitovich et al, 2006; Tirosh et al, 2009). However, very little is known about the mechanisms that drive this divergence. Here, we analyze the evolution of nucleosome positioning, by comparing the patterns of nucleosomes between two yeast species, as well as generating the allele-specific nucleosome profile in their hybrid. We ask two main questions: (1) what is the genetic basis of inter-species differences in nucleosome positioning? and (2) what is the regulatory function of these differences?
Generally speaking, we can classify the genetic basis of the divergence in nucleosome positioning into two mechanisms. First, mutations in the local DNA sequence may influence the ability to bind nucleosomes at this region; we refer to these as cis effects. Second, mutations may affect the activity of various proteins that alter nucleosome positioning either actively (e.g. chromatin-remodeling enzymes) or by simply competing with nucleosomes for binding to the same DNA sequence (e.g. transcription factors); we refer to these as trans effects.
To classify the observed inter-species differences into cis versus trans effects, we measured allele-specific nucleosome positions within the inter-specific hybrid of the two species (Wittkopp et al, 2004; Tirosh et al, 2009). The hybrid contains the alleles of both species; hence, cis effects, which involve mutations that discriminate between the two alleles, will be maintained in the hybrid so that nucleosome positioning will be different between the alleles coming from the different species. Trans effects, in contrast, will not discriminate between the two hybrid alleles from the different species, as these two alleles reside together at the same trans environment (hybrid nucleus) and are thus regulated by the same set of proteins—the combination of proteins from the two species. Using this approach, we found that ∼70% of the inter-species differences in nucleosome positioning are due to cis effects, whereas the rest is due to trans effects.
The local DNA sequence is indeed known to affect nucleosome positions, and many features of DNA sequences were proposed to influence nucleosome binding, either by rejecting nucleosomes, or by being favorable for nucleosome binding (Segal et al, 2006; Lee et al, 2007; Kaplan et al, 2009). We find, however, that nucleosome positions diverged primarily through changes in AT-rich sequences, which exclude nucleosomes, whereas mutations in sequences that correlate with high-nucleosome occupancy do not influence inter-species divergence.
Nucleosomes restrict the access of proteins to the DNA and may thus affect DNA-related processes such as transcription, recombination or replication. Indeed, promoters and regulatory sequences are often depleted of nucleosomes, and highly transcribed genes are associated with low occupancy of nucleosomes at their promoters (Lee et al, 2007). Several earlier studies also suggested that evolutionary divergence of gene expression is driven by changes in chromatin structure (Lee et al, 2006; Choi and Kim, 2008; Tirosh et al, 2008; Field et al, 2009). However, we find that nucleosome positions (or occupancy) at regulatory elements are largely conserved, and furthermore, that the inter-species differences in nucleosome positions do not correlate with gene expression differences. These results suggest that nucleosome positioning is not a central mechanism for evolutionary changes in gene regulation and that most of the observed changes may be due to neutral drift.
Does the apparent low influence of nucleosome positioning on gene expression divergence implies that nucleosome positions do not have a function in gene regulation? To address this, we examined two additional modes of gene regulation: transcriptional response to changes in growth conditions (glucose versus glycerol media), and the expression differences between different cell types (haploid versus diploid cells). Consistent with earlier studies, we found that the response to growth conditions is significantly, albeit weakly, associated with changes in nucleosome positioning. Interestingly, we also found a strikingly strong association between gene expression and nucleosomal changes in the two cell types. Taken together, these results suggest that nucleosome positioning is used preferentially for biological processes in which genes are turned on and off (e.g. different cell type), but less so during divergence of closely related species in which gradual changes accumulate over time.
Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genome-wide nucleosome positioning of two closely related yeast species and, by profiling their inter-specific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (∼70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in nucleosome-disfavoring AT-rich sequences, but are not associated with divergence of nucleosome-favoring sequences. Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis, and we provide evidence that nucleosome-free regions, but not the +1 nucleosome, serve as stable border elements. Surprisingly, although we find that differential nucleosome positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of nucleosome positioning.
PMCID: PMC2890324  PMID: 20461072
evolution; gene regulation; nucleosome positioning
3.  Urinary kidney injury molecule-1 is related to pathologic involvement in IgA nephropathy with normotension, normal renal function and mild proteinuria 
BMC Nephrology  2014;15:107.
IgA nephropathy (IgAN) may progress to renal failure for some patients without any clinical risk factors and it is not unusual to find severe pathologic damage in clinically mild IgAN. We therefore investigated whether urinary kidney injury molecule-1 (KIM-1) was related to pathologic involvement in clinically mild IgAN.
Urinary KIM-1/creatinine of 51 IgAN patients with normotension, normal renal function and proteinuria < 1.0 g/24 h were tested. Relationships between urinary KIM-1 and pathologic features were analyzed.
Eighteen of the 51 patients had elevated urinary KIM-1. The tubular atrophy/interstitial fibrosis was more severe in patients with elevated urinary KIM-1 than that in patients with normal urinary KIM-1 (T0/T1/T2, 13/5/0 vs. 33/0/0, P = 0.004). Proportion of glomeruli containing cresecents was higher in patients with elevated urinary KIM-1 than that in patients with normal urinary KIM-1 (50% vs. 18%, P = 0.026). Urinary KIM-1 correlated with the proportion of total crescents (R = 0.303, p = 0.031) and fibrous crescents (R = 0.456, p = 0.001), but did not correlate with the proportion of cellular crescents or fibrocellular crescents. Although the proportion of vascular lesions was higher in patients with elevated urinary KIM-1 (44.4%) than that in patients with normal urinary KIM-1 (18.1%), the difference was not significant (p = 0.057). There was no difference of the response to treatment between patients with and without elevated urinary KIM-1 during a short-term follow-up.
Urinary KIM-1 is a reflection of tubularinstitial injury. For patients with clinically mild IgAN, high urinary KIM-1 is related to relatively severe pathologic involvement on renal biopsy.
PMCID: PMC4094891  PMID: 24998891
KIM-1; IgA nephropathy; Oxford classification
4.  Integrative genome-scale metabolic analysis of Vibrio vulnificus for drug targeting and discovery 
Chromosome 1 of Vibrio vulnificus tends to contain larger portion of essential or housekeeping genes on the basis of the genomic analysis and gene knockout experiments performed in this study, while its chromosome 2 seems to have originated and evolved from a plasmid.The genome-scale metabolic network model of V. vulnificus was reconstructed based on databases and literature, and was used to identify 193 essential metabolites.Five essential metabolites finally selected after the filtering process are 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (AHHMP), D-glutamate (DGLU), 2,3-dihydrodipicolinate (DHDP), 1-deoxy-D-xylulose 5-phosphate (DX5P), and 4-aminobenzoate (PABA), which were predicted to be essential in V. vulnificus, absent in human, and are consumed by multiple reactions.Chemical analogs of the five essential metabolites were screened and a hit compound showing the minimal inhibitory concentration (MIC) of 2 μg/ml and the minimal bactericidal concentration (MBC) of 4 μg/ml against V. vulnificus was identified.
Discovering new antimicrobial targets and consequently new antimicrobials is important as drug resistance of pathogenic microorganisms is becoming an increasingly serious problem in human healthcare management (Fischbach and Walsh, 2009). There clearly exists a gap between genomic studies and drug discovery as the accumulation of knowledge on pathogens at genome level has not successfully transformed into the development of effective drugs (Mills, 2006; Payne et al, 2007). In this study, we dissected the genome of a microbial pathogen in detail, and subsequently developed a systems biological strategy of employing genome-scale metabolic modeling and simulation together with metabolite essentiality analysis for effective drug targeting and discovery. This strategy was used for identifying new drug targets in an opportunistic pathogen Vibrio vulnificus CMCP6 as a model.
V. vulnificus is a Gram-negative halophilic bacterium that is found in estuarine waters, brackish ponds, or coastal areas, and its Biotype 1 is an opportunistic human pathogen that can attack immune-compromised patients, and causes primary septicemia, necrotized wound infections, and gastroenteritis. We previously found that many metabolic genes were specifically induced in vivo, suggesting that specific metabolic pathways are essential for in vivo survival and virulence of this pathogen (Kim et al, 2003; Lee et al, 2007). These results motivated us to carry out systems biological analysis of the genome and the metabolic network for new drug target discovery.
V. vulnificus CMCP6 has two chromosomes. We first re-sequenced genomic regions assembled in low quality and low depth, and subsequently re-annotated the whole genome of V. vulnificus. Horizontal gene transfer was suspected to be responsible for the diversification of each chromosome of V. vulnificus, and the presence of metabolic genes was more biased to chromosome 1 than chromosome 2. Further studies on V. vulnificus genome revealed that chromosome 2 is more prone to diversification for better adaptation to the environment than its chromosome 1, while chromosome 1 tends to expand their genetic repertoire while maintaining the core genes at a constant level.
Next, a genome-scale metabolic network VvuMBEL943 was reconstructed based on literature, databases and experiments for systematic studies on the metabolism of this pathogen and prediction of drug targets. The VvuMBEL943 model is composed of 943 reactions and 765 metabolites, and covers 673 genes. The model was validated by comparing its simulated cell growth phenotype obtained by constraints-based flux analysis with the V. vulnificus-specific experimental data previously reported in the literature. In this study, constraints-based flux analysis is an optimization-based simulation method that calculates intracellular fluxes under the specific genetic and environmental condition (Kim et al, 2008). As a result, 17 growth phenotypes were correctly predicted out of 18 cases, which demonstrate the validity of VvuMBEL943.
The main objective of constructing VvuMBEL943 in this study is to predict potential drug targets by system-wide analysis of the metabolic network for the effective treatment of V. vulnificus. To achieve this goal, a set of drug target candidates was predicted by taking a metabolite-centric approach. Metabolite essentiality analysis is a concept recently introduced for the study of cellular robustness to complement conventional reaction or gene-centric approach (Kim et al, 2007b). Metabolite essentiality analysis observes changes in flux distribution by removing each metabolite from the in silico metabolic network. Hence, metabolite essentiality predicts essential metabolites whose absence causes cell death. By selecting essential metabolites, it is possible to directly screen only their structural analogs, which substantially reduces the number of chemical compounds to screen from the chemical compound library. As a result of implementing this approach, 193 metabolites were initially identified to be essential to the cell. These essential metabolites were then further filtered based on the predetermined criteria, mainly organism specificity and multiple connectivity associated with each metabolite, in order to reduce the number of initial target candidates towards identifying the most effective ones.
Five essential metabolites finally selected are 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (AHHMP), D-glutamate (DGLU), 2,3-dihydrodipicolinate (DHDP), 1-deoxy-D-xylulose 5-phosphate (DX5P), and 4-aminobenzoate (PABA). Enzymes that consume these essential metabolites were experimentally verified to be essential, which indeed demonstrates the essentiality of these five metabolites. On the basis of the structural information of these five essential metabolites, whole-cell screening assay was performed using their analogs for possible antibacterial discovery. We screened 352 chemical analogs of the essential metabolites selected from the chemical compound library, and found a hit compound 24837, which shows the minimal inhibitory concentration (MIC) of 2 μg/ml and minimal bactericidal concentration (MBC) of 4 μg/ml, showing good antibacterial activity without further structural modification. Although this study demonstrates a proof-of-concept, the approaches and their rationale taken here should serve as a general strategy for discovering novel antibiotics and drugs based on systems-level analysis of metabolic networks.
Although the genomes of many microbial pathogens have been studied to help identify effective drug targets and novel drugs, such efforts have not yet reached full fruition. In this study, we report a systems biological approach that efficiently utilizes genomic information for drug targeting and discovery, and apply this approach to the opportunistic pathogen Vibrio vulnificus CMCP6. First, we partially re-sequenced and fully re-annotated the V. vulnificus CMCP6 genome, and accordingly reconstructed its genome-scale metabolic network, VvuMBEL943. The validated network model was employed to systematically predict drug targets using the concept of metabolite essentiality, along with additional filtering criteria. Target genes encoding enzymes that interact with the five essential metabolites finally selected were experimentally validated. These five essential metabolites are critical to the survival of the cell, and hence were used to guide the cost-effective selection of chemical analogs, which were then screened for antimicrobial activity in a whole-cell assay. This approach is expected to help fill the existing gap between genomics and drug discovery.
PMCID: PMC3049409  PMID: 21245845
drug discovery; drug targeting; genome analysis; metabolic network; Vibrio vulnificus
5.  Effect of Renin-Angiotensin-Aldosterone System Inhibition, Dietary Sodium Restriction, and/or Diuretics on Urinary Kidney Injury Molecule 1 Excretion in Nondiabetic Proteinuric Kidney Disease: A Post Hoc Analysis of a Randomized Controlled Trial 
Tubulointerstitial damage plays an important role in chronic kidney disease (CKD) with proteinuria. Urinary kidney injury molecule 1 (KIM-1) reflects tubular KIM-1 and is considered a sensitive biomarker for early tubular damage. We hypothesized that a decrease in proteinuria by using therapeutic interventions is associated with decreased urinary KIM-1 levels.
Study Design
Post hoc analysis of a randomized, double-blind, placebo-controlled, crossover trial.
Setting & Participants
34 proteinuric patients without diabetes from our outpatient renal clinic.
Stepwise 6-week interventions of losartan, sodium restriction (low-sodium [LS] diet), their combination, losartan plus hydrochlorothiazide (HCT), and the latter plus an LS diet.
Outcomes & Measurements
Urinary excretion of KIM-1, total protein, and N-acetyl-β-D-glucosaminidase (NAG) as a positive control for tubular injury.
Mean baseline urine protein level was 3.8 ± 0.4 (SE) g/d, and KIM-1 level was 1,706 ± 498 ng/d (increased compared with healthy controls; 74 ng/d). KIM-1 level was decreased by using placebo/LS (1,201 ± 388 ng/d; P = 0.04), losartan/high sodium (1,184 ± 296 ng/d; P = 0.09), losartan/LS (921 ± 176 ng/d; P = 0.008), losartan/high sodium plus HCT (862 ± 151 ng/d; P = 0.008) and losartan/LS plus HCT (743 ± 170 ng/d; P = 0.001). The decrease in urinary KIM-1 levels paralleled the decrease in proteinuria (R = 0.523; P < 0.001), but not blood pressure or creatinine clearance. 16 patients reached target proteinuria with protein less than 1 g/d, whereas KIM-1 levels normalized in only 2 patients. Urinary NAG level was increased at baseline and significantly decreased during the treatment periods of combined losartan plus HCT only. The decrease in urinary NAG levels was not closely related to proteinuria.
Post hoc analysis.
Urinary KIM-1 level was increased in patients with nondiabetic CKD with proteinuria and decreased in parallel with proteinuria by using losartan, sodium restriction, their combination, losartan plus HCT, and the latter plus sodium restriction. These results are consistent with the hypothesis of amelioration of proteinuria-induced tubular damage. Long-term studies are warranted to evaluate whether targeting treatment on KIM-1 can improve outcomes in patients with CKD with proteinuria.
PMCID: PMC3298772  PMID: 18823687
Renin-angiotensin-aldosterone system; losartan; angiotensin II type 1 receptor blockade; proteinuria; interstitial renal damage; kidney injury molecule 1; N-acetyl-β-D-glucosaminidase; tubular damage marker; biomarker
6.  Regulation of Glucose Control in People with Type 2 Diabetes: A Review and Consensus 
Korean Diabetes Journal  2010;34(1):16-20.
A conference was convened by the Korean Diabetes Association and the Korean Endocrine Society on September 7, 2009 to discuss and organize the results of research on intensive glucose control for the prevention of cardiovascular disease in patients with type 2 diabetes. Professor Kyung Soo Park led the conference, and Professors Kwang Won Kim and Ho Young Son acted as chairmen. Professors Doo Man Kim, Tae Sun Park, and Bong Soo Cha reported on intensive glucose control and diabetic complications, including the UK Prospective Diabetes Study (UKPDS), Diabetes Control and Complication Trial (DCCT) research results, the recently published Action to Control Cardiovascular Risk in Diabetes (ACCORD), Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE), and Veterans Affairs Diabetes Trial (VADT) research, as well as meta-analyses. Professor Jeong-Taek Woo reported on the manuscript written by the committee for the Korean Diabetes Association which dealt with the treatment of diabetes mellitus. Professors Kyung Soo Ko, Joong Yeol Park, Hyun Shik Son, Moon-Kyu Lee, Dong-Won Byun, and Yoon-Sok Chung participated in the discussion and collected information for the manuscript from all of the participants. The aim of the debate was to determine how to establish target goals for intensive glucose control and how to individualize those goals. The participants concluded that there was no need to modify the recommendation of maintaining an HbA1c under 6.5%, the current blood glucose treatment goal that is recommended by the Korean Diabetes Association. In addition, individual target goals for glucose control were recommended depending on the situation of each patient. We report on the consensus statement from the meeting.
PMCID: PMC2879906  PMID: 20532015
7.  Degeneration of Leiomyoma in Patients Referred for Uterine Fibroid Embolization: Incidence, Imaging Features and Clinical Characteristics 
Yonsei Medical Journal  2012;54(1):215-219.
Imaging features and clinical characteristics of degenerated leiomyoma in patients referred for uterine fibroid embolization (UFE) were analyzed to assess the incidence of degenerated leiomyoma.
Materials and Methods
Patients referred for UFE between 2008 and 2009 were retrospectively analyzed (n=276). Patients ranged in age from 27 to 51 years (mean 38.0 years). All patients underwent screening MRI with contrast enhancement. Medical histories and clinical symptoms were evaluated.
Among the 276 patients who underwent MRI, 14 (5.1%) showed degenerated leiomyomas. Symptoms were abdominal pain (n=4, 26.7%), menorrhagia (n=5, 35.7%) and bulk-related symptoms (n=5, 35.7%) and no symptoms (n=5, 35.7%). Of the 14 patients with degenerated leiomyomas, 5 (42.9%) had a history of pregnancy in the past two years. For T1-weighted imaging (T1WI), a high signal intensity (SI) of the leiomyoma was the most common finding (n=9, 64.3%) and a hyperintense rim (n=4, 28.6%) was the second most common. On T2-weighted imaging (T2WI), a low SI of the leiomyoma was found in six patients (42.9%), a high SI in four (28.6%) and a heterogeneous SI in four (28.6%) patients. Conservative management was performed in 11 (78.6%) patients, surgery in 3 (21.4%) and uterine artery embolization in one (7.1%) patient.
The incidence of degeneration of leiomyoma in patients referred for UFE was 5.1%. Patients presented with variable clinical symptoms with or without a history of pregnancy. MR imaging showed a high SI on T1WI and various SIs on T2WI without contrast enhancement. An understanding of the degeneration of leiomyomata is essential when considering UFE.
PMCID: PMC3521269  PMID: 23225822
Uterine artery embolization; magnetic resonance imaging; degeneration; leiomyoma; uterus
8.  Correlating cone beam CT results with temporomandibular joint pain of osteoarthritic origin 
Dentomaxillofacial Radiology  2012;41(2):126-130.
The purpose of this study was to determine whether bony changes in temporomandibular joint (TMJ) osteoarthritis (OA) is correlated with pain and other clinical signs and symptoms.
Clinical data and cone beam CT (CBCT) images of 30 patients with TMJ OA were analysed. The criteria of Koyama et al (Koyama J, Nishiyama H, Hayashi T. Follow-up study of condylar bony changes using helical computed tomography in patients with temporomandibular disorder. Dentomaxillofac Radiol 2007; 36: 472–477.) and Ahmad et al [Ahmad M, Hollender L, Anderson Q, Kartha K, Ohrbach R, Truelove EL, et al. Research diagnostic criteria for temporomandibular disorders (RDC/TMD): development of image analysis criteria and examiner reliability for image analysis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 107: 844–860.] were used to classify the condyles observed on the CBCT. Clinical measures included self-reported pain, mandibular range of motion, TMJ sound, pain on palpation of the TMJ and masticatory muscles, and pain on jaw function. Generalized linear modelling was used to correlate the clinical and radiographic findings and Spearman's rho was used to correlate the two classification systems.
There was poor correlation between the maximum condyle change and pain rating (Koyama: r2 = 0.1443, p = 0.3995; Ahmad: r2 = 0.0273, p = 0.9490), maximum mouth opening (Koyama: r2 = 0.2910, p = 0.0629; Ahmad: r2 = 0.2626, p = 0.0951), protrusion (Koyama: r2 = 0.0875, p = 0.7001; Ahmad: r2 = 0.1658, p = 0.3612), right lateral motion (Koyama: r2 = 0.0394, p = 0.9093; Ahmad: r2 = 0.0866, p = 0.6877) and left lateral motion (Koyama: r2 = 0.0943, p = 0.6494; Ahmad: r2 = 0.1704, p = 0.3236). Strong correlation was observed between Koyama et al's and Ahmad et al's classifications for average (r = 0.9216, p < 0.001) and maximum (r = 0.7694; p < 0.0001) bony change.
There was poor correlation between condylar changes (as observed on CBCT images), pain and other clinical signs and symptoms in TMJ OA.
PMCID: PMC3520377  PMID: 22116122
cone beam computed tomography; temporomandibular joint; osteoarthritis; pain
9.  Urinary Kidney Injury Molecule 1 (KIM-1) and Interleukin 18 (IL-18) as Risk Markers for Heart Failure in Older Adults: The Health, Aging, and Body Composition (Health ABC) Study 
Kidney damage and reduced kidney function are potent risk factors for heart failure (HF), but existing studies are limited to assessing albuminuria or estimated glomerular filtration rate (eGFR). We evaluated the associations of urinary biomarkers of kidney tubular injury (interleukin 18 [IL-18] and kidney injury molecule 1 [KIM-1]) with future risk of HF.
Study Design
Retrospective cohort study.
Setting & Participants
2921 participants without HF in the Health, Aging, and Body Composition (Health ABC) cohort.
Ratios of urine KIM-1, IL-18, and albumin to creatinine (KIM-1:Cr, IL-18:Cr, and ACR, respectively).
Incident HF over a median follow-up of 12 years.
Median values of each marker at baseline were 812 (IQR, 497–1235) pg/mg for KIM-1:Cr, 31 (IQR, 19–56) pg/mg for IL-18:Cr, and 8 (IQR, 5–19) mg/g for ACR. 596 persons developed HF during follow-up. The top quartile of KIM-1:Cr was associated with risk of incident HF after adjustment for baseline eGFR, HF risk factors, and ACR (HR, 1.32; 95% CI, 1.02–1.70) in adjusted multivariate proportional hazards models. The top quartile of IL-18:Cr was also associated with HF in a model adjusted for risk factors and eGFR (HR, 1.35; 95% CI, 1.05–1.73), but was attenuated by adjustment for ACR (HR, 1.15; 95% CI, 0.89–1.48). The top quartile of ACR had a stronger adjusted association with HF (HR, 1.96; 95% CI, 1.53–2.51).
Generalizability to other populations is uncertain.
Higher urine concentrations of KIM-1 were independently associated with incident HF risk, although the associations of higher ACR were of stronger magnitude.
PMCID: PMC4069223  PMID: 24656453
IL-18; KIM-1; cystatin C; heart failure; CKD; risk marker; cardiovascular disease (CVD); albuminuria; kidney tubular injury
10.  Characteristics indicating adenomyosis coexisting with leiomyomas: a case–control study 
Human Reproduction (Oxford, England)  2010;25(5):1177-1182.
Adenomyosis is rarely diagnosed before hysterectomy and commonly coexists with uterine leiomyomas. The objective of this study was to identify distinct features of a concurrent diagnosis of adenomyosis in women with uterine leiomyomas.
We conducted a case–control study of women undergoing hysterectomy with a histologic diagnosis of both adenomyosis and leiomyomas and women with uterine leiomyomas but no adenomyosis. A retrospective medical record review of hospital and ambulatory records was performed to ascertain sociodemographic and anthropometric variables, as well as to confirm intraoperative and pathologic findings.
Our study sample comprised 255 patients, 85 women with adenomyosis and leiomyomas and 170 women with only leiomyomas. In multivariable logistic regression analyses, women with adenomyosis and leiomyomas were more likely to have more pelvic pain [odds ratio (OR) 3.4, 95% confidence interval (CI) 1.8–6.4], have less fibroid burden (OR per doubling in fibroid size 0.6, 95% CI 0.5–0.8), were more likely to be parous (OR 3.8, 95% CI 1.4–10.5) and have lower body mass index (OR per 5 unit increase in BMI 0.8, 95% CI 0.6–1.0) when compared with women with leiomyomas alone.
Women undergoing hysterectomy with both adenomyosis and leiomyomas have a number of different clinical features compared with women with only leiomyomas at the time of hysterectomy. Women with substantial pain despite a smaller fibroid burden may be more likely to have concomitant adenomyosis.
PMCID: PMC2854044  PMID: 20176591
adenomyosis; uterine leiomyomas; hysterectomy; epidemiology; pelvic pain
11.  Clinical presentations of gastric small gastrointestinal stromal tumors mimics functional dyspepsia symptoms 
World Journal of Gastroenterology : WJG  2014;20(33):11800-11807.
AIM: To explore whether clinical presentations of gastric small gastrointestinal tumors (GISTs) mimics gastrointestinal dyspepsia symptoms.
METHODS: The endosonographic data of 167 patients who underwent endoscopic submucosal dissection at the Tianjin Medical University General Hospital, China between 2009 and 2011 were analyzed. GISTs and leiomyomas had a similar intragastric distribution and similar locations within the gastric wall. Therefore, patients with GISTs were chosen as the study group and those with leiomyomas were chosen as the control group. Dyspepsia symptom questionnaires were used to investigate and compare the gastrointestinal symptoms of patients with GISTs and those with gastric leiomyomas before and after endoscopic submucosal dissection (ESD). The questionnaires evaluated symptoms such as epigastric pain, heartburn, regurgitation, epigastric discomfort, nausea and vomiting, abdominal bloating, and eructation. Symptoms were assessed using a four-point scoring scale.
RESULTS: GISTs were the most common gastric submucosal lesion (67 cases, 40.12%), followed by leiomyomas (38 cases, 22.75%). Both groups were similar in terms of gender distribution (P = 0.49), intragastric location (P = 0.525), and originating layer within the gastric wall (P = 0.449), but leiomyomas were more commonly found in the proximal fundus (P < 0.05). Overall, 94.2% of the patients with small GISTs and 93.5% of those with gastric leiomyomas experienced some dyspepsia; however, total symptom scores were significantly lower in the GIST group than in the leiomyoma group (1.34 ± 1.27 vs 2.20 ± 1.70, P < 0.05). Each component of the symptom score demonstrated a statistically significant improvement in the GIST patients after ESD (P < 0.05), including epigastric pain (0.80 ± 0.90 vs 0.13 ± 0.46), heartburn (0.63 ± 1.08 vs 0.13 ± 0.41), regurgitation (0.55 ± 0.87 vs 0.22 ± 0.57), epigastric discomfort (0.70 ± 0.98 vs 0.32 ± 0.47), nausea and vomiting (0.27 ± 0.62 vs 0.05 ± 0.21), abdominal bloating (0.70 ± 0.90 vs 0.27 ± 0.49), and eructation (0.36 ± 0.61 vs 0.21 ± 0.46). For leiomyoma patients, symptoms such as heartburn, nausea, vomiting, and eructation improved after treatment; however, these improvements were not statistically significant (P > 0.05). Thus, the pathophysiology of dyspepsia symptoms may be different between the two groups.
CONCLUSION: Symptoms of gastric small GISTs may mimic those of functional dyspepsia. An alternative diagnosis should be considered in patients with functional dyspepsia and treatment failure.
PMCID: PMC4155371  PMID: 25206285
Gastric small gastrointestinal stromal tumor; Gastric leiomyoma; Clinical presentation; Endoscopic ultrasonography
12.  Chikungunya Infection in India: Results of a Prospective Hospital Based Multi-Centric Study 
PLoS ONE  2012;7(2):e30025.
Chikungunya (CHIKV) has recently seen a re-emergence in India with high morbidity. However, the epidemiology and disease burden remain largely undetermined. A prospective multi-centric study was conducted to evaluate clinical, epidemiological and virological features of chikugunya infection in patients with acute febrile illness from various geographical regions of India.
Methods and Findings
A total of 540 patients with fever of up to 7days duration were enrolled at Karnataka Institute of Medical Sciences (KIMS), Karnataka (South); Sawai Man Singh Medical College (SMS) Rajasthan (West), and All India Institute of Medical Sciences (AIIMS) New Delhi (North) from June 2008 to May 2009. Serum specimens were screened for chikungunya infection concurrently through RT-PCR and serology (IgM). Phylogenetic analysis was performed using Bioedit and Mega2 programs. Chikungunya infection was detected in 25.37% patients by RT-PCR and/or IgM-ELISA. Highest cases were detected in south (49.36%) followed by west (16.28%) and north (0.56%) India. A difference in proportion of positives by RT-PCR/ELISA with regard to duration of fever was observed (p<0.05). Rashes, joint pain/swelling, abdominal pain and vomiting was frequently observed among chikungunya confirmed cases (p<0.05). Adults were affected more than children. Anti-CHIK antibodies (IgM) were detected for more than 60days of fever onset. Phylogenetic analysis based on E1 gene from KIMS patients (n = 15) revealed ∼99% homology clustering with Central/East African genotype. An amino acid change from lysine to glutamine at position 132 of E1 gene was frequently observed among strains infecting children.
The study documented re-emergence of chikungunya in high frequencies and severe morbidity in south and west India but rare in north. The study emphasizes the need for continuous surveillance for disease burden using multiple diagnostic tests and also warrants the need for an appropriate molecular diagnostic for early detection of chikungunya virus.
PMCID: PMC3281818  PMID: 22363413
13.  The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease 
The Journal of Experimental Medicine  2008;205(9):2005-2017.
MicroRNAs (miRNAs) are implicated in the differentiation and function of many cell types. We provide genetic and in vivo evidence that the two RNaseIII enzymes, Drosha and Dicer, do indeed function in the same pathway. These have previously been shown to mediate the stepwise maturation of miRNAs (Lee, Y., C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V.N. Kim. 2003. Nature. 425:415–419), and genetic ablation of either within the T cell compartment, or specifically within Foxp3+ regulatory T (T reg) cells, results in identical phenotypes. We found that miRNA biogenesis is indispensable for the function of T reg cells. Specific deletion of either Drosha or Dicer phenocopies mice lacking a functional Foxp3 gene or Foxp3+ cells, whereas deletion throughout the T cell compartment also results in spontaneous inflammatory disease, but later in life. Thus, miRNA-dependent regulation is critical for preventing spontaneous inflammation and autoimmunity.
PMCID: PMC2526196  PMID: 18725527
14.  Cytotoxic Activities of Leptospira interrogans Hemolysin SphH as a Pore-Forming Protein on Mammalian Cells  
Infection and Immunity  2002;70(1):315-322.
Leptospirosis is a spirochetal zoonosis that causes an acute febrile systemic illness in humans. Leptospira sp. hemolysins have been shown to be virulence factors for the pathogenesis of leptospirosis. Previously, we cloned a hemolysin SphH of Leptospira interrogans serovar lai, a homologue of L. borgpetersenii sphingomyelinase (SphA), from a genomic library (S. H. Lee, K. A. Kim, Y. K. Kim, I. W. Seong, M. J. Kim, and Y. J. Lee, Gene 254:19–28, 2000). Escherichia coli lysate harboring the sphH showed high hemolytic activities on sheep erythrocytes. However, it neither showed sphingomyelinase nor phospholipase activities, in contrast to SphA which was known to have sphingomyelinase activity. Interestingly, the SphH-mediated hemolysis on erythrocytes was osmotically protected by PEG 5000, suggesting that the SphH might have caused pore formation on the erythrocyte membrane. In the present study, we have prepared the Leptospira hemolysin SphH and investigated its hemolytic and cytotoxic activities on mammalian cells. SphH was shown to be a pore-forming protein on several mammalian cells: When treated with the SphH, the sheep erythrocyte membranes formed pores, which were morphologically confirmed by transmission electron microscopy. Furthermore, the SphH-mediated cytotoxicities on mammalian cells were demonstrated by the release of LDH and by inverted microscopic examinations. Finally, the immune serum against the full-length hemolysin could effectively neutralize the SphH-mediated hemolytic and cytotoxic activities. In conclusion, these results suggest that the virulence of Leptospira SphH was due to the pore formation on mammalian cell membranes.
PMCID: PMC127624  PMID: 11748197
15.  An excitatory amacrine cell detects object motion and provides feature-selective input to ganglion cells in the mouse retina 
eLife  null;4:e08025.
Retinal circuits detect salient features of the visual world and report them to the brain through spike trains of retinal ganglion cells. The most abundant ganglion cell type in mice, the so-called W3 ganglion cell, selectively responds to movements of small objects. Where and how object motion sensitivity arises in the retina is incompletely understood. In this study, we use 2-photon-guided patch-clamp recordings to characterize responses of vesicular glutamate transporter 3 (VGluT3)-expressing amacrine cells (ACs) to a broad set of visual stimuli. We find that these ACs are object motion sensitive and analyze the synaptic mechanisms underlying this computation. Anatomical circuit reconstructions suggest that VGluT3-expressing ACs form glutamatergic synapses with W3 ganglion cells, and targeted recordings show that the tuning of W3 ganglion cells' excitatory input matches that of VGluT3-expressing ACs' responses. Synaptic excitation of W3 ganglion cells is diminished, and responses to object motion are suppressed in mice lacking VGluT3. Object motion, thus, is first detected by VGluT3-expressing ACs, which provide feature-selective excitatory input to W3 ganglion cells.
eLife digest
Animals can use their eyes to detect moving objects, which helps them to avoid predators and other threats, and to spot potential prey or allies. Visual information from the eyes is sent to the brain, which processes the information to form a coherent picture of how the objects are moving. This processing has to be able to account for movements of the head, eyes, and body—which can cause the image of an object on the retina within the eye to move even if the object itself remains stationary.
Within the retina, light is converted into electrical signals by cells called rods and cones. A layer of cells called bipolar cells relay these signals to the ‘ganglion’ cells, which in turn pass them on to the brain. In mice, a type of ganglion cell called the W3 ganglion cell has been shown to respond selectively to small moving objects, but exactly how these cells acquire their motion sensitivity remained unclear.
Kim et al. now reveal that cells called amacrine cells, which regulate the transfer of signals from the bipolar cells to ganglion cells, supply the information needed for motion detection. The mouse eye contains up to 50 different types of amacrine cells. One of these—called the VG3-amacrine cell—increases its activity whenever an object moves relative to its background, but decreases its activity whenever the object and background move together. The overall effect is that the cells respond selectively to the presence of small moving objects.
Most amacrine cells regulate the transfer of signals within the retina by inhibiting the activity of ganglion cells. But, Kim et al. show that VG3-amacrine cells release a molecule called glutamate to activate W3 ganglion cells when a moving object is detected. These unusual and specialized cells are, thus, an essential component of a circuit in the nervous system that supports motion detection. It is possible that some other types of amacrine cells may also play specialized roles in the detection of other features in the visual world.
PMCID: PMC4467229  PMID: 25988808
retinal circuitry; amacrine cell; feature detection; VGluT3; mouse
16.  Role of a New Intimin/Invasin-Like Protein in Yersinia pestis Virulence 
Infection and Immunity  2012;80(10):3559-3569.
A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1+) Pgm− (pigmentation negative)/, KIM6(pCD1−) Pgm+, and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δilp had a 55-fold increase in the 50% lethal dose ([LD50] 1.64 × 104 CFU) compared to the parental wild-type strain LD50 (2.98 × 102 CFU). These findings identified Ilp as a novel virulence factor of Y. pestis.
PMCID: PMC3457552  PMID: 22851752
17.  Kidney injury molecule–1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells 
The Journal of Clinical Investigation  2008;118(5):1657-1668.
Following injury, the clearance of apoptotic and necrotic cells is necessary for mitigation and resolution of inflammation and tissue repair. In addition to macrophages, which are traditionally assigned to this task, neighboring epithelial cells in the affected tissue are postulated to contribute to this process. Kidney injury molecule–1 (KIM-1 or TIM-1) is an immunoglobulin superfamily cell-surface protein not expressed by cells of the myeloid lineage but highly upregulated on the surface of injured kidney epithelial cells. Here we demonstrate that injured kidney epithelial cells assumed attributes of endogenous phagocytes. Confocal images confirm internalization of apoptotic bodies within KIM-1–expressing epithelial cells after injury in rat kidney tubules in vivo. KIM-1 was directly responsible for phagocytosis in cultured primary rat tubule epithelial cells and also porcine and canine epithelial cell lines. KIM-1 was able to specifically recognize apoptotic cell surface-specific epitopes phosphatidylserine, and oxidized lipoproteins, expressed by apoptotic tubular epithelial cells. Thus, KIM-1 is the first nonmyeloid phosphatidylserine receptor identified to our knowledge that transforms epithelial cells into semiprofessional phagocytes.
PMCID: PMC2293335  PMID: 18414680
18.  Plasmid-determined cytotoxicity in Yersinia pestis and Yersinia pseudotuberculosis. 
Infection and Immunity  1986;51(3):788-794.
Yersinia pestis KIM5 was found to be cytotoxic for the IC21 and P388D1 mouse macrophage cell lines, as well as for resident peritoneal macrophages from C57BL/6 mice. Affected cells phagocytosed KIM5 inefficiently, became spherical, detached readily from culture dishes, and retained 51Cr poorly. The cytotoxic effect was dependent on the presence of the 75-kilobase plasmid pCD1. Because this plasmid also encodes the low calcium response (LCR), three Mu d1 insertion mutants previously shown to be LCR- and of reduced virulence in mice were examined for cytotoxicity; all were found to be atoxic. The insertions in these mutants lie within three distinct LCR loci (lcrB, C, and D). Like LCR, cytotoxicity was expressed only at 37 degrees C. Unlike LCR, it was not influenced by Ca2+ concentration, indicating that the V and W antigens are probably not involved. Yersinia pseudotuberculosis was found to have a similar plasmid-dependent cytotoxicity. Thus, biological activity observed as cytotoxicity in vitro may well be a common feature contributing to virulence of the yersiniae.
PMCID: PMC260967  PMID: 3949380
19.  Development of Guideline for Rating the Physical Impairment of Otolaryngologic Field 
Journal of Korean Medical Science  2009;24(Suppl 2):S258-S266.
We develop a guideline for rating the physical impairment of otolaryngologic fields. Assessment of hearing disturbance and tinnitus required physical examination, pure tone audiometry, speech audiometry, impedance audiometry, brainstem evoked response audiometry, Bekesy audiometry, otoacoustic emission test, and imaging examination. History taking, physical examination, and radiological examination for the vestibular organ and brain, righting reflex test, electronystagmography, and caloric test are taken for evaluation of balance disorder. Olfactory function tests include University of Pennsylvania Smell Identification test, Connecticut Chemosensory Clinical Research Center test, T and T olfactometry and Korean Version of Sniffin's Sticks test. Medical history and physical examination is mandatory to evaluatezseverity of respiration difficulty. Examinations include flexible fiberoptic nasopharyngoscope, bronchoscopy, simple soft-tissue radiography films of upper airway and high resolution computed tomography. Evaluation of mastication and swallowing are history taking, physical examination, examination for upper jaw, lower jaw, and temporomandibular joint, dental examination and radiological studies. Endoscopy and esophagography are also needed. Voice disorder is evaluated based on physical examination, oral pharynx and larynx endoscopy, larynx stroboscopy, hearing assessment, laryngeal electromyography, sound analysis test, aerodynamic test, electroglottography, and radiologic examination. Articulation disorder is assessed by picture consonant articulation test. These are position articulation test, Lee-Kim Korean articulation picture and speech intelligibility assessment.
PMCID: PMC2690073  PMID: 19503682
Hearing; Vestibule; Smell; Respiration; Voice; Mastication; Deglutition
20.  Early surgery for native valve infective endocarditis 
Critical Care  2013;17(1):304.
Expanded abstract
Kang DH, Kim YJ, Kim SH, Sun BJ, Kim DH, Yun SC, Song JM, Choo SJ, Chung CH, Song JK, Lee JW, Sohn DW: Early surgery versus conventional treatment for infective endocarditis. N Engl J Med 2012, 366: 2466-2473.
The timing and indications for surgical intervention to prevent systemic embolism in infective endocarditis (IE) remain controversial. This trial compares clinical outcomes of early surgery and conventional treatment in patients with IE.
To determine the effect of early surgery (<48 hours) to decrease the rate of death or embolic events as compared with conventional treatment for IE.
Prospective randomized trial.
Two academic medical centers in Korea.
Adult patients with left-sided, native-valve IE and a high risk of embolism.
Valve repair or replacement with removal of vegetation within 48 hours of random assignment versus no early surgery.
Composite primary endpoint of in-hospital death and embolic events occurring within 6 weeks after random assignment. Secondary endpoints, at 6 months, included death from any cause, embolic events, recurrence of IE, and repeat hospitalization due to the development of congestive heart failure.
Thirty-seven patients were assigned to the early-surgery group (<48 hours), whereas 39 were assigned to conventional therapy. Of the 39 randomly assigned to conventional therapy, 27 patients (77%) underwent surgery during the initial hospitalization and three during follow-up. One patient (3%) in the early-surgery group and nine (23%) in the conventional-treatment group reached the primary endpoint (hazard ratio (HR) 0.10, 95% confidence interval (CI) 0.01 to 0.82; P = 0.03). There was no significant difference in all-cause mortality at 6 months in the early-surgery and conventional-treatment groups (3% and 5%, respectively; HR 0.51, 95% CI 0.05 to 5.66; P = 0.59). The rates of the composite endpoint of death from any cause, embolic events, or recurrence of IE at 6 months were 3% in the early-surgery group and 28% in the conventional-treatment group (HR 0.08, 95% CI 0.01 to 0.65; P = 0.02).
Early surgery in patients with IE and large vegetations significantly reduced the composite endpoint of death from any cause and embolic events by effectively decreasing the risk of systemic embolism.
PMCID: PMC4057157  PMID: 23425501
21.  Genetic variance of Trichomonas vaginalis isolates by Southern hybridization 
In the present study, genomic DNAs were purified from Korean isolates (KT8, KT6, KT-Kim and KT-Lee) and foreign strains (CDC85, IR78 and NYH 286) of Trichomonas vaginalis, and hybridized with a probe based on the repetitive sequence cloned from T. vaginalis to observe the genetic differences. By Southern hybridization, all isolates of T. vaginalis except the NYH286 strain had 11 bands. Therefore all isolates examined were distinguishable into 3 groups according to their banding patterns; i) KT8, KT6 and KT-Kim isolates had 11 identical bands such as 1 kb, 1.2 kb, 1.6 kb, 1.9 kb, 2.3 kb, 2.7 kb, 3.2 kb, 3.4 kb, 3.8 kb, 4.9 kb and 6.0 kb. ii) The metronidazole-resistant IR78 strain had the same bands as KT-Lee isolate at bands of 1 kb, 1.2 kb, 1.6 kb, 1.8 kb, 2.1 kb, 2.5 kb, 2.7 kb, 2.9 kb, 3.4 kb, 5.0 kb and 6.0 kb. Bands of CDC85, metronidazole-resistant strain, were similar to those of IR78 and KT-Lee, except that 3.2 kb replaced 2.9 kb. iii) NYH286 particularly had 12 bands and band patterns were similar to IR78 with a few exceptions as follows: i) 6.2 kb in place of 6.0 kb, ii) 2.0 kb and 2.2 kb instead of 2.1 kb. Through the results obtained, genetic variance of T. vaginalis isolates was demonstrated by Southern hybridization.
PMCID: PMC2732933  PMID: 9755593
Trichomonas vaginalis; Southern hybridization; genetic diversity
22.  Differences in Levels of Secreted Locus of Enterocyte Effacement Proteins between Human Disease-Associated and Bovine Escherichia coli O157 
Infection and Immunity  2001;69(8):5107-5114.
Ongoing extensive epidemiological studies of verotoxin-carrying Escherichia coli O157 (stx+ eae+) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom. However, the incidence of disease in humans due to this pathogen is still very low. This study set out to investigate if there is a difference between strains isolated from human disease cases and those isolated from asymptomatic cattle which would account for the low disease incidence of such a ubiquitous organism. The work presented here has compared human disease strains from both sporadic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E. coli O157 strains from cattle. Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like toxin types 1, 2, and 2c; E. coli secreted protein genes espA, espB, and espP; the enterohemolysin gene; eae (intimin); ast (enteroaggregative E. coli stable toxin [EAST]); and genes for common E. coli adhesins. Strains were also phenotyped for hemolysin, EspP, Tir, and EspD expression as well as production of actin and cytoskeletal rearrangement associated with attaching and effacing (A/E) lesions on HeLa cells. The genotyping confirmed that there was little difference between the two groups, including carriage of stx2 and stx2c, which was similar in both sets. ast alleles were confirmed to all contain mutations that would prevent EAST expression. espP mutations were found only in cattle strains (5 of 30). Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media. EspD, as an indicator of LEE4 (espA, -B, and -D) expression, and Tir levels in supernatants were measured. Virtually all strains from both sources could produce EspD in Luria-Bertani broth, although at very different levels. Standard trichloroacetic acid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human strains. Addition of bovine serum albumin carrier protein before precipitation and enhanced detection techniques confirmed that EspD could be detected after growth in tissue culture medium for all strains, but levels from strains of human origin were on average 90-fold higher than those from strains of bovine origin. In general, levels of secretion also correlated with ability to form A/E lesions on HeLa cells, with only the high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype. This research shows significant differences between human- and bovine-derived E. coli O157 (stx+ eae+) strains and their production of certain LEE-encoded virulence factors. These data support the recent finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson, Proc. Natl. Acad. Sci. USA 96:13288–13293, 1999) proposing different E. coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors. Potentially only a subset of E. coli O157 isolates (stx+ eae+) in cattle may be capable of causing severe disease in humans.
PMCID: PMC98606  PMID: 11447192
23.  Growth hormone (GH) replacement in hypopituitary adults with GH deficiency evaluated by a utility-weighted quality of life index: a precursor to cost–utility analysis 
Clinical Endocrinology  2008;68(1):122-129.
To examine quality of life (QoL) measured by a utility-weighted index in GH-deficient adults on GH replacement and analyse the impact of demographic and clinical characteristics on changes in utilities during treatment.
Utilities for items in the QoL-Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDAutility) were estimated based on data obtained from the general population in England and Wales (E&W). These estimates were used to calculate QoL changes in GH-treated patients and compare these with normative population values.
A total of 894 KIMS patients (53% women) from E&W were followed for 1 to 6 years.
QoL-AGHDAutility at baseline and at the last reported visit, total QoL-AGHDAutility gain and QoL-AGHDAutility gain per year of follow-up.
QoL-AGHDAutility in patients before GH treatment differed from the expected population values [0·67 (SD 0·174) vs. 0·85 (SD 0·038), P < 0·0001], constituting a mean deficit of –0·19 (SD 0·168). There was a difference in the mean QoL-AGHDAutility deficit for men [–0·16 (SD 0·170)] and women [–0·21 (SD 0·162)] (P < 0·001). The main improvement occurred during the first year of treatment [reduction of a deficit to –0·07 (SD 0·163) (P < 0·001) in the total cohort]; however, patients’ utilities remained lower than those recorded for the general population during subsequent follow-up (P < 0·001). Despite an observed impact of age, primary aetiology, disease onset and comorbidities on QoL-AGHDAutility, all patients showed a similar beneficial response to treatment.
QoL-AGHDAutility efficiently monitors treatment effects in patients with GHD. The study confirmed the QoL-AGHDAutility deficit before treatment and a similar QoL-AGHDAutility gain observed after commencement of GH replacement in all patients.
PMCID: PMC2228397  PMID: 17803700
24.  Validation Study of Kim's Sham Needle by Measuring Facial Temperature: An N-of-1 Randomized Double-Blind Placebo-Controlled Clinical Trial 
Introduction. In 2008, Kim's sham needle was developed to improve the quality of double-blinded studies. The aim of this study is to validate Kim's sham needle by measuring facial temperature. Methods. We designed “N-of-1” trials involving 7 smokers. One session was composed of 2 stimulations separated by a 2 h washout period. Six sessions were applied daily for all subjects. Infrared thermal imaging was used to examine the effects of acupuncture (HT8, KI2) on facial temperature following smoking-induced decrease. Results. All subjects demonstrated decreased temperatures after sham needle treatment, but 5 of the 7 subjects showed increased temperatures after real needle treatment. 6 of the 7 subjects showed a significant difference (P < 0.05) between treatments with real and sham needles. Thus, the physiological stimulation of Kim's sham needle is different from that of a real needle, suggesting that Kim's sham needle is a potential inactive control intervention.
PMCID: PMC3310274  PMID: 22474506
25.  Co-authorship patterns and networks of Korean radiation oncologists 
Radiation Oncology Journal  2011;29(3):164-173.
This research aimed to analyze the patterns of co-authorship network among the Korean radiation oncologists and to identify attributing factors for the formation of networks.
Materials and Methods
A total of 1,447 articles including contents of 'Radiation Oncology' and 'Therapeutic Radiology' were searched from the KoreaMed database. The co-authorship was assorted by the author's full name, affiliation and specialties. UCINET 6.0 was used to figure out the author's network centrality and the cluster analysis, and KeyPlayer 1.44 program was used to get a result of key player index. Sociogram was analyzed with the Netdraw 2.090. The statistical comparison was performed by a t-test and ANOVA using SPSS 16.0 with p-value < 0.05 as the significant value.
The number of articles written by a radiation oncologist as the first author was 1,025 out of 1,447. The pattern of co-authorship was classified into five groups. For articles of which the first author was a radiation oncologist, the number of single-author articles (type-A) was 81; single-institution articles (type-B) was 687; and multiple-author articles (type-C) was 257. For the articles which radiation oncologists participated in as a co-author, the number of single-institution articles (type-D) was 280 while multiple-institution articles (type-E) were 142. There were 8,895 authors from 1,366 co-authored articles, thus the average number of authors per article was 6.51. It was 5.73 for type-B, 6.44 for type-C, 7.90 for type-D, and 7.67 for type-E (p = 0.000) in the average number of authors per article. The number of authors for articles from the hospitals published more than 100 articles was 7.23 while form others was 5.94 (p = 0.005). Its number was 5.94 and 7.16 for the articles published before and after 2001 (p = 0.000). The articles written by a radiation oncologist as the first author had 5.92 authors while others for 7.82 (p = 0.025). Its number was 5.57 and 7.71 for the Journal of the Korean Society for Therapeutic Radiology and Oncology and others (p = 0.000), respectively. Among the analysis, a significant difference in the average number of author per article was indicated. The out-degree centrality of network among authors was 4.26% ( while in-degree centrality was 1.31% ( The three significant nodes were classified and listed as following: Choi, Eun Kyung for 1991-1995, Kim, Dae Young for 1998-2001, Park, Won and Lee, Sang Wook for 2003-2010. Choi, Eun Kyung and Kim, Dae Young appeared in two cases, and ranked as the highest degree in centrality. In the key player analysis, Choi, Eun Kyung and Lee, Sang Wook appeared in two cases, and ranked as the highest. From the cluster analysis, Sungkyunkwan University, Seoul National University and Yonsei University revealed as the three large clusters when Ulsan University, Chonnam National University, and Korea Institute of Radiological & Medical Science as the medium clusters.
The Korean radiation oncologist's society shows a closed network with numerous relationships among the particular clusters, and the result indicates it is different from other institutions in the pattern of co-authorship formation of the major hospitals.
PMCID: PMC3429899  PMID: 22984667
Radiation Oncology; Co-authorship; Network

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