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1.  Revealing Maximal Diameter of Upper Limb Superficial Vein with an Elevated Environmental Temperature 
Ultrasonography is the primary tool for preoperative analysis of vein morphology for fistula creation in patients with end-stage renal disease. This study examines the effect of environmental temperature on the superficial vein size. Superficial veins of thirteen healthy volunteers were marked at three sites: cephalic vein in left lateral arm near cubital fossa, cephalic vein in left forearm at wrist, and basilic vein in left medial arm near cubital fossa. Mean diameters were recorded using ultrasound probe at 26°C and 43°C. Body temperature was increased using a Bair Hugger blanket. Mean values from the two temperatures were analyzed using paired sample t-test. All three superficial vein sites displayed statistically significant increase in diameter when the temperature was increased from 26°C to 43°C. Paired t-test showed p values of 0.001 for cephalic vein at wrist, 0.01 for cephalic vein near cubital fossa, and 0.01 for basilic vein near cubital fossa. This study proved that environmental temperature exerts a statistically significant effect on vein size measured by ultrasound during preoperative assessment for vascular access. Not to the extent of 43°C, the authors would recommend setting the room temperature higher during ultrasound vascular assessment to avoid underestimating the superficial vein size.
PMCID: PMC5002475  PMID: 27597987
2.  Potential impact of a bedside procedure service on training procedurally competent hospitalists in a community-based residency program 
The Society of Hospital Medicine has delineated procedures as one of the core competencies for hospitalists. Little is known about whether exposure to a medical procedure service (MPS) impacts the procedural certification rate in internal medicine trainees in a community hospital training program.
To determine whether or not exposure to an MPS would impact both the number of procedures performed and the rate of resultant certifications in a community hospital internal medicine training program.
Retrospective review.
Five cohorts of resident physicians and their procedure data were analyzed comparing months where residents were unexposed to the intervention (pre-MPS) to months where residents were exposed to the intervention (post-MPS). We calculated the average number of procedures performed per month for pre- versus post-MPS periods. For procedural certification, we compared two proportions: the number of certifications over the number of 6-month pre-MPS periods and the number of certifications over the number of 6-month post-MPS periods.
The study was conducted at a community-based academic medical center. Subjects included all internal medicine residents.
We found a statistically significant difference between the groups, with pre-MPS groups performing 4.3 procedures per month compared with post-MPS groups performing 6.7 procedures per month (p=0.0010). For certification rates, we found statistically significant differences in several categories – overall, paracentesis, femoral central lines, and jugular central lines.
This study demonstrated that resident exposure to an MPS statistically significantly increased the total number of procedures performed. This study also showed that overall certification rates were statistically significantly different between the pre- and post-MPS groups for several procedures.
PMCID: PMC4942516  PMID: 27406445
hospitalist training; internal medicine trainees; procedure service
3.  Further evidence for a parent-of-origin effect at the NOP9 locus on language-related phenotypes 
Specific language impairment (SLI) is a common neurodevelopmental disorder, observed in 5–10 % of children. Family and twin studies suggest a strong genetic component, but relatively few candidate genes have been reported to date. A recent genome-wide association study (GWAS) described the first statistically significant association specifically for a SLI cohort between a missense variant (rs4280164) in the NOP9 gene and language-related phenotypes under a parent-of-origin model. Replications of these findings are particularly challenging because the availability of parental DNA is required.
We used two independent family-based cohorts characterised with reading- and language-related traits: a longitudinal cohort (n = 106 informative families) including children with language and reading difficulties and a nuclear family cohort (n = 264 families) selected for dyslexia.
We observed association with language-related measures when modelling for parent-of-origin effects at the NOP9 locus in both cohorts: minimum P = 0.001 for phonological awareness with a paternal effect in the first cohort and minimum P = 0.0004 for irregular word reading with a maternal effect in the second cohort. Allelic and parental trends were not consistent when compared to the original study.
A parent-of-origin effect at this locus was detected in both cohorts, albeit with different trends. These findings contribute in interpreting the original GWAS report and support further investigations of the NOP9 locus and its role in language-related traits. A systematic evaluation of parent-of-origin effects in genetic association studies has the potential to reveal novel mechanisms underlying complex traits.
PMCID: PMC4908686  PMID: 27307794
Language impairment; Dyslexia; Genetic association; Parent-of-origin; Candidate gene
4.  Clinical Application of IUGA/ICS Classification System for Mesh Erosion 
Neurourology and urodynamics  2015;35(5):589-594.
Our aim was to assess the usability of the IUGA/ICS classification system for mesh erosion in a tertiary clinical practice and to determine if assigned classification is associated with patient symptoms, treatment, and outcome.
We retrospectively identified women who had mesh erosion after prolapse or incontinence surgery. Each erosion was classified using the IUGA/ICS category time site (CTS) system. Associations between classification and presenting symptom (asymptomatic, pain, bleeding, voiding, or defecatory dysfunction, infection, prolapse), treatment type, and outcome were evaluated with chi-squared test, student’s t-test, and univariate logistic regression.
We identified 74 subjects with mesh erosion; only 70% were classifiable. Asymptomatic patients (n = 19) (Category A) were more likely to be managed conservatively (P = 0.001). Symptomatic patients (n = 55) (Category B) were more likely to be managed surgically (P = 0.003). Other variables had no association with treatment. No variables were associated with outcome. Presenting symptom was associated with both treatment (P = 0.005) and outcome (P = 0.03). Asymptomatic subjects were more likely to have satisfactory outcome (P = 0.03). Urinary frequency and urgency were highly correlated with surgical management (P = 0.02).
One third of mesh erosions could not be retrospectively coded using the IUGA/ICS classification. The components of the system were not predictive of treatment nor outcome with exception of the Category A (asymptomatic) and Category B (symptomatic). Asymptomatic women with mesh erosion can be successfully managed with conservative measures. Use of a classification system may be enhanced if the system is simplified by limiting the number of variables to those associated with interventions and patient outcome.
PMCID: PMC4757498  PMID: 25874639
CTS classification system; IUGA/ICS classification system; mesh erosion; surgery
5.  Francisella philomiragia Bacteremia in a Patient with Acute Respiratory Insufficiency and Acute-on-Chronic Kidney Disease 
Journal of Clinical Microbiology  2015;53(12):3947-3950.
Francisella philomiragia is a very uncommon pathogen of humans. Diseases caused by it are protean and have been reported largely in near-drowning victims and those with chronic granulomatous disease. We present a case of F. philomiragia pneumonia with peripheral edema and bacteremia in a renal transplant patient and review the diverse reports of F. philomiragia infections.
PMCID: PMC4652090  PMID: 26400786
6.  KIF6 719Arg Genetic Variant and Risk for Thoracic Aortic Dissection 
AORTA Journal  2016;4(3):83-90.
Carriers of the 719Arg variant in KIF6, compared with noncarriers, have been reported to be at greater risk for coronary heart disease (CHD) in six prospective studies. Because CHD, thoracic aortic dissection, and nondissection thoracic aortic aneurysm share some risk factors and aspects of pathophysiology, we investigated whether carriers of the 719Arg variant also have greater odds of thoracic aortic dissection or nondissected thoracic aortic aneurysm than noncarriers.
We genotyped 140 thoracic aortic dissection cases, 497 nondissection thoracic aortic aneurysm cases, and 275 disease-free controls collected in the United States, Hungary, and Greece and investigated the association between KIF6 719Arg carrier status and thoracic aortic dissection, and between KIF6 719Arg carrier status and nondissection thoracic aortic aneurysm, using logistic regression models adjusted for age, sex, hypertension, smoking, and country.
The odds of aortic dissection were two-fold greater in KIF6 719Arg carriers compared with noncarriers (odds ratio (OR) 2.14, 95% confidence interval (CI) 1.18-3.9). To account for the potential of concomitant CHD to confound the association between the KIF6 719Arg and thoracic aortic dissection, we repeated the analysis after removing subjects with concomitant CHD; the estimates for association of KIF6 719Arg carrier status remained essentially the same (OR 2.04, 95% CI 1.11-3.77). In contrast, KIF6 719Arg carrier status was not associated with risk for nondissection thoracic aortic aneurysm.
We observed an association of the KIF6 719Arg genetic variant with thoracic aortic dissection in this multicenter case-control study. This association may enhance our management of patients with thoracic aortic disease.
PMCID: PMC5217719  PMID: 28097184
Aneurysm; Aorta; Dissection; KIF6; Biomarker
7.  Adenosine Receptors and Renal Ischemia Reperfusion Injury 
Acta physiologica (Oxford, England)  2014;213(1):222-231.
One of the frequent clinical complications that results in billions of dollars in health care costs annually in the United States is acute kidney injury (AKI). Ischemia reperfusion (IR) injury is a major cause AKI. Unfortunately, no effective treatment or preventive measure for AKI exists. With increased surgical complexity coupled with increasing number of elderly, the incidence of AKI is becoming more frequent. Adenosine is a metabolic breakdown product of adenosine triphosphate (ATP) and contributes to the regulation of multiple physiological events. Extracellular adenosine activates 4 subtypes of adenosine receptors (AR) including A1AR, A2AAR, A2BAR and A3AR. In the kidney, adenosine regulates glomerular filtration rate, vascular tone, renin release and is an integrative part of tubular glomerular feedback signal to the afferent arterioles. In addition, each AR subtype powerfully modulates renal IR injury. The A1AR activation protects against ischemic insult by reducing apoptosis, necrosis, and inflammation. Activation of A2AAR protects against renal injury by modulating leukocyte-mediated inflammation as well as directly reducing renal tubular inflammation. Activation of A2BAR acts via direct activation of renal parenchymal as well as renovascular receptors and is important in kidney preconditioning. Finally, activation of A3AR exacerbates renal damage following renal IR injury while A3AR antagonism attenuates renal damage following ischemic insult. Latest body of research suggests that kidney AR modulation may be a promising approach to treat ischemic AKI. This brief review focuses on the signaling pathways of adenosine in the kidney followed by the role for various AR modulations in protecting against ischemic AKI.
PMCID: PMC4270901  PMID: 25287331
Acute kidney injury; acute renal failure; apoptosis; inflammation; necrosis
8.  The Secretome of Hydrogel-Coembedded Endothelial Progenitor Cells and Mesenchymal Stem Cells Instructs Macrophage Polarization in Endotoxemia 
Endothelial progenitor cells (EPCs) were coembedded with renal mesenchymal stem cells (MSCs) to further ameliorate renal dysfunction. Their paracrine influence on cytokine/chemokine release and proinflammatory macrophages was examined. Hyaluronic acid-based hydrogel delivery of EPCs alone or codelivered with hypoxic preconditioned MSCs offered significant protection of renal and vascular function during endotoxemia, including reducing the circulating levels of cytokines/chemokines.
We previously reported the delivery of endothelial progenitor cells (EPCs) embedded in hyaluronic acid-based (HA)-hydrogels protects renal function during acute kidney injury (AKI) and promotes angiogenesis. We attempted to further ameliorate renal dysfunction by coembedding EPCs with renal mesenchymal stem cells (MSCs), while examining their paracrine influence on cytokine/chemokine release and proinflammatory macrophages. A live/dead assay determined whether EPC-MSC coculturing improved viability during lipopolysaccharide (LPS) treatment, and HA-hydrogel-embedded delivery of cells to LPS-induced AKI mice was assessed for effects on mean arterial pressure (MAP), renal blood flow (RBF), circulating cytokines/chemokines, serum creatinine, proteinuria, and angiogenesis (femoral ligation). Cytokine/chemokine release from embedded stem cells was examined, including effects on macrophage polarization and release of proinflammatory molecules. EPC-MSC coculturing improved stem cell viability during LPS exposure, an effect augmented by MSC hypoxic preconditioning. The delivery of coembedded EPCs with hypoxic preconditioned MSCs to AKI mice demonstrated additive improvement (compared with EPC delivery alone) in medullary RBF and proteinuria, with comparable effects on serum creatinine, MAP, and angiogenesis. Exposure of proinflammatory M1 macrophages to EPC-MSC conditioned medium changed their polarization to anti-inflammatory M2. Incubation of coembedded EPCs-MSCs with macrophages altered their release of cytokines/chemokines, including enhanced release of anti-inflammatory interleukin (IL)-4 and IL-10. EPC-MSC delivery to endotoxemic mice elevated the levels of circulating M2 macrophages and reduced the circulating cytokines/chemokines. In conclusion, coembedding EPCs-MSCs improved their resistance to stress, impelled macrophage polarization from M1 to M2 while altering their cytokine/chemokines release, reduced circulating cytokines/chemokines, and improved renal and vascular function when MSCs were hypoxically preconditioned.
This report provides insight into a new therapeutic approach for treatment of sepsis and provides a new and improved strategy using hydrogels for the delivery of stem cells to treat sepsis and, potentially, other injuries and/or diseases. The delivery of two different stem cell lines (endothelial progenitor cells and mesenchymal stem cells; delivered alone and together) embedded in a protective bioengineered scaffolding (hydrogel) offers many therapeutic benefits for the treatment of sepsis. This study shows how hydrogel-delivered stem cells elicit their effects and how hydrogel embedding enhances the therapeutic efficacy of delivered stem cells. Hydrogel-delivered stem cells influence the components of the overactive immune system during sepsis and work to counterbalance the release of many proinflammatory and prodamage substances from immune cells, thereby improving the associated vascular and kidney damage.
PMCID: PMC4479616  PMID: 25947337
Endothelial progenitor cells; Hyaluronic acid hydrogel; Acute kidney injury; Macrophage polarization; Mesenchymal stem cells; Lipopolysaccharide
9.  High mobility group box 1 is a novel deacetylation target of Sirtuin1 
Kidney international  2014;87(1):95-108.
High mobility group box 1 (HMGB1) undergoes acetylation, nuclear-to-cytoplasmic translocation and release from stressed kidneys, unleashing a signaling cascade of events leading to systemic inflammation. Here we tested whether the deacetylase activity of Sirtuin1 (SIRT1) participates in regulating nuclear retention of HMGB1 to ultimately modulate damage signaling initiated by HMGB1 secretion during stress. When immunoprecipitated acetylated HMGB1 was incubated with SIRT1, HMGB1 acetylation decreased by 57%. Proteomic analysis showed that SIRT1 deacetylates HMGB1 at four lysine residues (55, 88, 90 and 177) within the pro-inflammatory and nuclear localization signal domains of HMGB1. Genetic ablation or pharmacological inhibition of SIRT1 in endothelial cells increased HMGB1 acetylation and translocation. In vivo, deletion of SIRT1 reduced nuclear HMGB1 while increasing its acetylation and release into circulation during basal and ischemic conditions causing increased renal damage. Conversely, resveratrol pretreatment led to decreased HMGB1 acetylation, its nuclear retention, decreased systemic release and reduced tubular damage. Thus, a vicious cycle is set into motion in which the inflammation-induced repression of SIRT1 disables deacetylation of HMGB1, facilitates its nuclear-to-cytoplasmic translocation and systemic release, thereby maintaining inflammation.
PMCID: PMC4270955  PMID: 24940804
10.  Liver Enzymes in Children with beta-Thalassemia Major: Correlation with Iron Overload and Viral Hepatitis 
Beta Thalassemia is the most common chronic hemolytic anemia in Egypt (85.1%) with an estimated carrier rate of 9-10.2%. Injury to the liver, whether acute or chronic, eventually results in an increase in serum concentrations of Alanine transaminase (ALT) and Aspartate transaminase (AST).
Evaluating the potentiating effect of iron overload & viral hepatitis infection on the liver enzymes.
Eighty (80) thalassemia major patients were studied with respect to liver enzymes, ferritin, transferrin saturation, HBsAg, anti-HCV antibody and HCV-PCR for anti-HCV positive patients.
Fifty % of the patients were anti-HCV positive and 55% of them were HCV-PCR positive. Patients with elevated ALT and AST levels had significantly higher mean serum ferritin than those with normal levels. Anti-HCV positive patients had higher mean serum ferritin, serum ALT, AST and GGT levels and higher age and duration of blood transfusion than the negative group. HCV-PCR positive patients had higher mean serum ferritin and serum ALT and also higher age and duration of blood transfusion than the negative group.
Iron overload is a main leading cause of elevated liver enzymes, and presence of HCV infection is significantly related to the increased iron overload.
PMCID: PMC4877869  PMID: 27275237
Beta thalassemia; egyptian children; viral hepatitis; iron overload; liver enzymes
11.  Language impairment in a case of a complex chromosomal rearrangement with a breakpoint downstream of FOXP2 
We report on a young female, who presents with a severe speech and language disorder and a balanced de novo complex chromosomal rearrangement, likely to have resulted from a chromosome 7 pericentromeric inversion, followed by a chromosome 7 and 11 translocation.
Using molecular cytogenetics, we mapped the four breakpoints to 7p21.1-15.3 (chromosome position: 20,954,043-21,001,537, hg19), 7q31 (chromosome position: 114,528,369-114,556,605, hg19), 7q21.3 (chromosome position: 93,884,065-93,933,453, hg19) and 11p12 (chromosome position: 38,601,145-38,621,572, hg19). These regions contain only non-coding transcripts (ENSG00000232790 on 7p21.1 and TCONS_00013886, TCONS_00013887, TCONS_00014353, TCONS_00013888 on 7q21) indicating that no coding sequences are directly disrupted. The breakpoint on 7q31 mapped 200 kb downstream of FOXP2, a well-known language gene. No splice site or non-synonymous coding variants were found in the FOXP2 coding sequence. We were unable to detect any changes in the expression level of FOXP2 in fibroblast cells derived from the proband, although this may be the result of the low expression level of FOXP2 in these cells.
We conclude that the phenotype observed in this patient either arises from a subtle change in FOXP2 regulation due to the disruption of a downstream element controlling its expression, or from the direct disruption of non-coding RNAs.
Electronic supplementary material
The online version of this article (doi:10.1186/s13039-015-0148-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4460787  PMID: 26060509
Language impairment; Chromosomal rearrangement; FOXP2 regulation; Non-coding elements; Spanish
12.  Fruit and Vegetable Intake Assessed by Food Frequency Questionnaire and Plasma Carotenoids: A Validation Study in Adults 
Nutrients  2015;7(5):3240-3251.
Dietary validation studies of self-reported fruit and vegetable intake should ideally include measurement of plasma biomarkers of intake. The aim was to conduct a validation study of self-reported fruit and vegetable intakes in adults, using the Australian Eating Survey (AES) food frequency questionnaire (FFQ), against a range of plasma carotenoids. Dietary intakes were assessed using the semi-quantitative 120 item AES FFQ. Fasting plasma carotenoids (α- and β-carotene, lutein/zeaxanthin, lycopene and cryptoxanthin) were assessed using high performance liquid chromatography in a sample of 38 adult volunteers (66% female). Significant positive correlations were found between FFQ and plasma carotenoids for α-carotene, β-carotene and lutein/zeaxanthin (52%, 47%, 26%, p < 0.001, 0.003, 0.041; respectively) and relationships between plasma carotenoids (except lycopene) and weight status metrics (BMI, waist circumference, fat mass) were negative and highly significant. The results of the current study demonstrate that carotenoid intakes as assessed by the AES FFQ are significantly related to plasma concentrations of α-carotene, β-carotene and lutein/zeaxanthin, the carotenoids commonly found in fruit and vegetables. Lower levels of all plasma carotenoids, except lycopene, were found in individuals with higher BMI. We conclude that the AES can be used to measure fruit and vegetable intakes with confidence.
PMCID: PMC4446749  PMID: 25954899
diet assessment validation; food frequency questionnaire; fruit vegetable intake; plasma carotenoids
13.  Human Biosample Authentication Using the High-Throughput, Cost-Effective SNPtraceTM System 
PLoS ONE  2015;10(2):e0116218.
Cell lines are the foundation for much of the fundamental research into the mechanisms underlying normal biologic processes and disease mechanisms. It is estimated that 15%–35% of human cell lines are misidentified or contaminated, resulting in a huge waste of resources and publication of false or misleading data. Here we evaluate a panel of 96 single-nucleotide polymorphism (SNP) assays utilizing Fluidigm microfluidics technology for authentication and sex determination of human cell lines. The SNPtrace Panel was tested on 907 human cell lines. Pairwise comparison of these data show the SNPtrace Panel discriminated among identical, related and unrelated pairs of samples with a high degree of confidence, equivalent to short tandem repeat (STR) profiling. We also compared annotated sex calls with those determined by the SNPtrace Panel, STR and Illumina SNP arrays, revealing a high number of male samples are identified as female due to loss of the Y chromosome. Finally we assessed the sensitivity of the SNPtrace Panel to detect intra-human cross-contamination, resulting in detection of as little as 2% contaminating cell population. In conclusion, this study has generated a database of SNP fingerprints for 907 cell lines used in biomedical research and provides a reliable, fast, and economic alternative to STR profiling which can be applied to any human cell line or tissue sample.
PMCID: PMC4340925  PMID: 25714623
14.  The Comparative Validity and Reproducibility of a Diet Quality Index for Adults: The Australian Recommended Food Score 
Nutrients  2015;7(2):785-798.
Adult diet quality indices are shown to predict nutritional adequacy of dietary intake as well as all-cause morbidity and mortality. This study describes the reproducibility and validity of a food-based diet quality index, the Australian Recommended Food Score (ARFS). ARFS was developed to reflect alignment with the Australian Dietary Guidelines and is modelled on the US Recommended Food Score. Dietary intakes of 96 adult participants (31 male, 65 female) age 30 to 75 years were assessed in two rounds, five months apart. Diet was assessed using a 120-question semi-quantitative food frequency questionnaire (FFQ). The ARFS diet quality index was derived using a subset of 70 items from the full FFQ. Reproducibility of the ARFS between round one and round two was confirmed by the overall intraclass correlation coefficient of 0.87 (95% CI 0.83, 0.90), which compared favourably to that for the FFQ at 0.85 (95% CI 0.80, 0.89). ARFS was correlated with FFQ nutrient intakes, particularly fiber, vitamin A, beta-carotene and vitamin C (0.53, 95% CI 0.37–0.67), and with mineral intakes, particularly calcium, magnesium and potassium (0.32, 95% CI 0.23–0.40). ARFS is a suitable brief tool to evaluate diet quality in adults and reliably estimates a range of nutrient intakes.
PMCID: PMC4344560  PMID: 25625814
diet quality index; validation; comparative validity; reproducibility; food frequency questionnaire; intra-class correlation coefficient; dietary methods
15.  Occupational exposures, smoking and airway inflammation in refractory asthma 
BMC Pulmonary Medicine  2014;14:207.
The influence of occupation and ex/passive smoking on inflammatory phenotype is not well understood. The aim of this study was to examine the relationship between occupation, past smoking and current passive smoking and airway inflammation in a population of adults with refractory asthma.
Sixty-six participants with refractory asthma were characterised. Occupational exposure to asthma causing or worsening agents were identified with an asthma-specific job exposure matrix. Exposure to passive cigarette smoke was determined by questionnaire and exhaled carbon monoxide assessment. The carbon content of macrophages was assessed in a sub-group of participants.
Nineteen participants had smoked previously with low smoking pack years (median 1.7 years). Ex-smokers more commonly lived with a current smoker (26% vs. 9%, p = 0.11) and were more likely to allow smoking inside their home (26% vs. 4%, p = 0.02) compared to never smokers. Twenty participants had occupations with an identified exposure risk to an asthmagen; thirteen had exposures to irritants such as motor vehicle exhaust and environmental tobacco smoke. Sputum neutrophils were elevated in participants with asthma who had occupational exposures, particularly those who were diagnosed with asthma at a more than 30 years of age.
Sputum neutrophils are elevated in refractory asthma with exposure to occupational asthmagens. In addition to older age, exposure to both environmental and occupational particulate matter may contribute to the presence of neutrophilic asthma. This may help explain asthma heterogeneity and geographical variations in airway inflammatory phenotypes in asthma.
PMCID: PMC4391679  PMID: 25526871
Refractory asthma; Neutrophils; Occupational exposure
16.  Critical Glycosylated Residues in Exon Three of Erythrocyte Glycophorin A Engage Plasmodium falciparum EBA-175 and Define Receptor Specificity 
mBio  2014;5(5):e01606-14.
Erythrocyte invasion is an essential step in the pathogenesis of malaria. The erythrocyte binding-like (EBL) family of Plasmodium falciparum proteins recognizes glycophorins (Gp) on erythrocytes and plays a critical role in attachment during invasion. However, the molecular basis for specific receptor recognition by each parasite ligand has remained elusive, as is the case with the ligand/receptor pair P. falciparum EBA-175 (PfEBA-175)/GpA. This is due largely to difficulties in producing properly glycosylated and functional receptors. Here, we developed an expression system to produce recombinant glycosylated and functional GpA, as well as mutations and truncations. We identified the essential binding region and determinants for PfEBA-175 engagement, demonstrated that these determinants are required for the inhibition of parasite growth, and identified the glycans important in mediating the PfEBA-175–GpA interaction. The results suggest that PfEBA-175 engages multiple glycans of GpA encoded by exon 3 and that the presentation of glycans is likely required for high-avidity binding. The absence of exon 3 in GpB and GpE due to a splice site mutation confers specific recognition of GpA by PfEBA-175. We speculate that GpB and GpE may have arisen due to selective pressure to lose the PfEBA-175 binding site in GpA. The expression system described here has wider application for examining other EBL members important in parasite invasion, as well as additional pathogens that recognize glycophorins. The ability to define critical binding determinants in receptor-ligand interactions, as well as a system to genetically manipulate glycosylated receptors, opens new avenues for the design of interventions that disrupt parasite invasion.
Plasmodium falciparum uses distinct ligands that bind host cell receptors for invasion of red blood cells (RBCs) during malaria infection. A key entry pathway involves P. falciparum EBA-175 (PfEBA-175) recognizing glycophorin A (GpA) on RBCs. Despite knowledge of this protein-protein interaction, the complete mechanism for specific receptor engagement is not known. PfEBA-175 recognizes GpA but is unable to engage the related RBC receptor GpB or GpE. Understanding the necessary elements that enable PfEBA-175 to specifically recognize GpA is critical in developing specific and potent inhibitors of PfEBA-175 that disrupt host cell invasion and aid in malaria control. Here, we describe a novel system to produce and manipulate the host receptor GpA. Using this system, we probed the elements in GpA necessary for engagement and thus for host cell invasion. These studies have important implications for understanding how ligands and receptors interact and for the future development of malaria interventions.
PMCID: PMC4173783  PMID: 25205096
17.  Validity of the Australian Recommended Food Score as a diet quality index for Pre-schoolers 
Nutrition Journal  2014;13:87.
Diet quality tools provide researchers with brief methods to assess the nutrient adequacy of usual dietary intake. This study describes the development and validation of a pediatric diet quality index, the Australian Recommended Food Scores for Pre-schoolers (ARFS-P), for use with children aged two to five years.
The ARFS-P was derived from a 120-item food frequency questionnaire, with eight sub-scales, and was scored from zero to 73. Linear regressions were used to estimate the relationship between diet quality score and nutrient intakes, in 142 children (mean age 4 years) in rural localities in New South Wales, Australia.
Total ARFS-P and component scores were highly related to dietary intake of the majority of macronutrients and micronutrients including protein, β-carotene, vitamin C, vitamin A. Total ARFS-P was also positively related to total consumption of nutrient dense foods, such as fruits and vegetables, and negatively related to total consumption of discretionary choices, such as sugar sweetened drinks and packaged snacks.
ARFS-P is a valid measure that can be used to characterise nutrient intakes for children aged two to five years. Further research could assess the utility of the ARFS-P for monitoring of usual dietary intake over time or as part of clinical management.
PMCID: PMC4162955  PMID: 25178263
Diet quality index; Food frequency questionnaire; Pre-schoolers; Nutritional adequacy
18.  Dietary Intake Is Related to Multifactor Cardiovascular Risk Score in Obese Boys 
Healthcare  2014;2(3):282-298.
Cardiovascular disease (CVD) originates in childhood and early identification of risk factors provides an early intervention opportunity. The aim was to identify children at higher risk using a CVD risk score, developed from factors known to cluster in childhood. Risk was scored as very high (≥97.5th centile), high (≥95th), moderate (≥90th) or threshold (<90th) using normal pediatric reference ranges for 10 common biomedical risk factors. These were summed in a multifactor CVD risk score and applied to a sample of 285 observations from 136 overweight Australian children (41% male, aged 7–12 years). Strength of associations between CVD risk score and individual biomedical and dietary variables were assessed using univariate logistic regression. High waist circumference (Odds Ratio: 5.48 [95% CI: 2.60–11.55]), body mass index (OR: 3.22 [1.98–5.26]), serum insulin (OR: 3.37 [2.56–4.42]) and triglycerides (OR: 3.02 [2.22–4.12]) were all significantly related to CVD risk score. High intakes of total fat (OR: 4.44 [1.19–16.60]), sugar (OR: 2.82 [1.54–5.15]) and carbohydrate (OR 1.75 [1.11–2.77]) were significantly related to CVD risk score in boys only. This multifactor CVD risk score could be a useful tool for researchers to identify elevated risk in children. Further research is warranted to examine sex-specific dietary factors related to CVD risk in children.
PMCID: PMC4934591  PMID: 27429277
cardiovascular risk; childhood; obesity; diet; nutrition
19.  Multimeric Assembly of Host-Pathogen Adhesion Complexes Involved in Apicomplexan Invasion 
PLoS Pathogens  2014;10(6):e1004120.
PMCID: PMC4055764  PMID: 24945143
21.  Barriers to breast cancer screening among a sample of Egyptian females 
Breast cancer (BC) is usually diagnosed in late stages in countries with limited resources. Early detection of BC is likely to improve the outcome of the disease for women in these areas.
The aim of this study was to understand the possible personal, economic, and systems barriers to BC screening in a sample of Egyptian women.
Materials and Methods:
A cross-sectional study was conducted in family health centers representing the seven districts of Alexandria governorate, Egypt. A total of 612 women were randomly selected from the chosen centers.
In this sample of Egyptian women, the most frequently identified potential barriers to BC screening were the following: 81.8% would not seek care until they were ill, 77% were unwilling to have a mammogram until it was recommended by the doctor, 71.4% blamed the, lack of privacy, 69.2% thought that medical checkups were not worthwhile, and 64.6% blamed the cost of services. The study further revealed that women of lower education, women in the lower income category, women who did not do paid work, those who had poor knowledge of the risks of BC, and women with no family history of BC were more likely to perceive different screening barriers compared with their counterparts.
Many potential personal, economic, and health system barriers were identified. Addressing these barriers by increasing the awareness of BC and dealing with the misconceptions that the women have can help the policy makers to design more culturally relevant strategies to motivate women to utilize screening services.
PMCID: PMC4073560  PMID: 24987281
Breast cancer; barriers; Egypt; screening
22.  Genetic Variants in FBN-1 and Risk for Thoracic Aortic Aneurysm and Dissection 
PLoS ONE  2014;9(4):e91437.
A recent genome wide association study (GWAS) by LeMaire et al. found that two single nucleotide polymorphisms (SNPs), rs2118181 and rs10519177 in the FBN-1 gene (encoding Fibrillin-1), were associated with thoracic aortic dissection (TAD), non-dissecting thoracic aortic aneurysm (TAA), and thoracic aortic aneurysm or dissection (TAAD); the largest effect was observed for the association of rs2118181 with TAD. We investigated whether rs2118181 and rs10519177 were associated with TAD, TAA, and TAAD in the Yale study.
The genotypes of rs2118181 and rs10519177 were determined for participants in the Yale study: 637 TAAD cases (140 TAD, 497 TAA) and 275 controls from the United States, Hungary, and Greece. The association of the genotypes with TAD, TAA and TAAD were assessed using logistic regression models adjusted for sex, age, study center and hypertension.
Results and Conclusions
In the Yale study, rs2118181 was associated with TAD: compared with non-carriers, carriers of the risk allele had an unadjusted odds ratio for TAD of 1.80 (95% CI 1.15–2.80) and they had odds ratio for TAD of 1.87 (95% CI 1.09–3.20) after adjusting for sex, age, study center and hypertension. We did not find significant differences in aortic size, a potential confounder for TAD, between rs2118181 risk variant carriers and non-carriers: mean aortic size was 5.56 (95% CI: 5.37–5.73) for risk variant carriers (CC+CT) and was 5.48 (95% CI: 5.36–5.61) for noncarriers (TT) (p = 0.56). rs2118181 was not associated with TAA or TAAD. rs10519177 was not associated with TAD, TAA, or TAAD in the Yale study. Thus, the Yale study provided further support for the association of the FBN-1 rs2118181SNP with TAD.
PMCID: PMC3990573  PMID: 24743685
23.  The Cellular Basis for Biocide-Induced Fluorescein Hyperfluorescence in Mammalian Cell Culture 
PLoS ONE  2014;9(1):e84427.
Clinical examination of the ocular surface is commonly carried out after application of sodium fluorescein in both veterinary and medical practice by assessing the resulting ‘staining’. Although localized intensely stained regions of the cornea frequently occur after exposure to ‘adverse’ clinical stimuli, the cell biology underlying this staining is unknown, including whether intense fluorescein staining indicates the presence of damaged cells. Ocular exposure to certain contact lens multipurpose solutions (MPS) gives rise to intense fluorescein staining referred to as solution induced corneal staining (SICS), and we have made use of this phenomenon with Vero and L929 cell culture models to investigate the fundamental biology of fluorescein interactions with cells.
We found that all cells take up fluorescein, however a sub-population internalize much higher levels, giving rise to brightly staining ‘hyperfluorescent’ cells within the treated cultures, which contain fluorescein throughout the cell cytoplasm and nucleus. The numbers of these hyperfluorescent cells are significantly increased after exposure to MPS associated with SICS. Surprisingly, hyperfluorescent cells did not show higher levels of staining with propidium iodide, a marker of lysed cells. Consistently, treatment with the cytolytic toxin benzalkonium chloride resulted in almost all cells staining with propidium iodide, and the complete abolition of fluorescein hyperfluorescence. Finally we found that internalization of fluorescein and its loss from treated cells both require cellular activity, as both processes were halted after incubation at 4°C.
We conclude that fluorescein hyperfluorescence can be replicated in three diverse cell cultures, and is increased by MPS-treatment, as occurs clinically. The process involves the concentration of fluorescein by a sub-population of cells that are active, and does not occur in lysed cells. Our data suggest that corneal staining in the clinic reflects active living cells, and is not directly caused by dead cells being produced in response to adverse clinical stimuli.
PMCID: PMC3904830  PMID: 24489650
24.  Structural and Functional Basis for Inhibition of Erythrocyte Invasion by Antibodies that Target Plasmodium falciparum EBA-175 
PLoS Pathogens  2013;9(5):e1003390.
Disrupting erythrocyte invasion by Plasmodium falciparum is an attractive approach to combat malaria. P. falciparum EBA-175 (PfEBA-175) engages the host receptor Glycophorin A (GpA) during invasion and is a leading vaccine candidate. Antibodies that recognize PfEBA-175 can prevent parasite growth, although not all antibodies are inhibitory. Here, using x-ray crystallography, small-angle x-ray scattering and functional studies, we report the structural basis and mechanism for inhibition by two PfEBA-175 antibodies. Structures of each antibody in complex with the PfEBA-175 receptor binding domain reveal that the most potent inhibitory antibody, R217, engages critical GpA binding residues and the proposed dimer interface of PfEBA-175. A second weakly inhibitory antibody, R218, binds to an asparagine-rich surface loop. We show that the epitopes identified by structural studies are critical for antibody binding. Together, the structural and mapping studies reveal distinct mechanisms of action, with R217 directly preventing receptor binding while R218 allows for receptor binding. Using a direct receptor binding assay we show R217 directly blocks GpA engagement while R218 does not. Our studies elaborate on the complex interaction between PfEBA-175 and GpA and highlight new approaches to targeting the molecular mechanism of P. falciparum invasion of erythrocytes. The results suggest studies aiming to improve the efficacy of blood-stage vaccines, either by selecting single or combining multiple parasite antigens, should assess the antibody response to defined inhibitory epitopes as well as the response to the whole protein antigen. Finally, this work demonstrates the importance of identifying inhibitory-epitopes and avoiding decoy-epitopes in antibody-based therapies, vaccines and diagnostics.
Author Summary
Malaria is a devastating parasitic disease that kills one million people annually. The parasites invade and multiply within red blood cells, leading to the clinical symptoms of malaria. Therefore, preventing red blood cell, entry through vaccines is an attractive approach to controlling the disease. Although widespread efforts to develop a vaccine by identifying and combining critical parasite blood-stage proteins are underway, a protective vaccine for malaria has proved challenging. This is in part because, while parasite proteins have the ability to elicit antibodies that prevent red blood cell invasion, these antibodies are a small proportion compared to the total collection of ineffective antibodies produced. We show an antibody that prevents red blood cell invasion targets regions of the critical parasite protein PfEBA-175 required for red blood cell engagement. We also show that an antibody that does not prevent red blood cell invasion recognizes a region far removed from important functional segments of PfEBA-175. Our work demonstrates that identifying the regions targeted by antibodies, and the mechanisms by which antibodies that prevent invasion function, should drive future vaccine development and studies measuring the effectiveness of current vaccine combinations.
PMCID: PMC3662668  PMID: 23717209
25.  Treatment outcome in adults with chronic fatigue syndrome: a prospective study in England based on the CFS/ME National Outcomes Database 
Background: Chronic fatigue syndrome (CFS) is relatively common and disabling. Over 8000 patients attend adult services each year, yet little is known about the outcome of patients attending NHS services.
Aim: Investigate the outcome of patients with CFS and what factors predict outcome.
Design: Longitudinal patient cohort.
Methods: We used data from six CFS/ME (myalgic encephalomyelitis) specialist services to measure changes in fatigue (Chalder Fatigue Scale), physical function (SF-36), anxiety and depression (Hospital Anxiety and Depression Scale) and pain (visual analogue pain rating scale) between clinical assessment and 8–20 months of follow-up. We used multivariable linear regression to investigate baseline factors associated with outcomes at follow-up.
Results: Baseline data obtained at clinical assessment were available for 1643 patients, of whom 834 (51%) had complete follow-up data. There were improvements in fatigue [mean difference from assessment to outcome: −6.8; 95% confidence interval (CI) −7.4 to −6.2; P < 0.001]; physical function (4.4; 95% CI 3.0–5.8; P < 0.001), anxiety (−0.6; 95% CI −0.9 to −0.3; P < 0.001), depression (−1.6; 95% CI −1.9 to −1.4; P < 0.001) and pain (−5.3; 95% CI −7.0 to −3.6; P < 0.001). Worse fatigue, physical function and pain at clinical assessment predicted a worse outcome for fatigue at follow-up. Older age, increased pain and physical function at assessment were associated with poorer physical function at follow-up.
Conclusions: Patients who attend NHS specialist CFS/ME services can expect similar improvements in fatigue, anxiety and depression to participants receiving cognitive behavioural therapy and graded exercise therapy in a recent trial, but are likely to experience less improvement in physical function. Outcomes were predicted by fatigue, disability and pain at assessment.
PMCID: PMC3665909  PMID: 23538643

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