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1.  The effect of sex and irritable bowel syndrome on HPA axis response and peripheral glucocorticoid receptor expression 
Psychoneuroendocrinology  2016;69:67-76.
Background and aims
Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been reported in irritable bowel syndrome (IBS). Enhanced HPA axis response has been associated with reduced glucocorticoid receptor (GR) mediated negative feedback inhibition. We aimed to study the effects of IBS status, sex, or presence of early adverse life events (EAL) on the cortisol response to corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH), and on GR mRNA expression in peripheral blood mononuclear cells (PBMCs).
Rome III+ IBS patients and healthy controls underwent CRF (1 μg/kg ovine) and ACTH (250 μg) stimulation tests with serial plasma ACTH and cortisol levels measured (n = 116). GR mRNA levels were measured using quantitative PCR (n = 143). Area under the curve (AUC) and linear mixed effects models were used to compare ACTH and cortisol response measured across time between groups.
There were divergent effects of IBS on the cortisol response to ACTH by sex. In men, IBS was associated with an increased AUC (p = 0.009), but in women AUC was blunted in IBS (p = 0.006). Men also had reduced GR mRNA expression (p = 0.007). Cumulative exposure to EALs was associated with an increased HPA response. Lower GR mRNA was associated with increased pituitary HPA response and increased severity of overall symptoms and abdominal pain in IBS.
This study highlights the importance of considering sex in studies of IBS and the stress response in general. Our findings also provide support for PBMC GR mRNA expression as a peripheral marker of central HPA response.
PMCID: PMC4977028  PMID: 27038676
Irritable bowel syndrome; Hypothalamic pituitary-regulating; hormones; Adrenal cortex hormones; Glucocorticoids receptors; Sex differences
3.  High-grade renal injuries are often isolated in sports-related trauma 
Injury  2015;46(7):1245-1249.
Most high-grade renal injuries (American Association for Surgery of Trauma (AAST) grades III–V) result from motor vehicle collisions associated with numerous concomitant injuries. Sports-related blunt renal injury tends to have a different mechanism, a solitary blow to the flank. We hypothesized that high-grade renal injury is often isolated in sports-related renal trauma.
Material and methods
We identified patients with AAST grades III–V blunt renal injuries from four level 1 trauma centres across the United States between 1/2005 and 1/2014. Patients were divided into “Sport” or “Non-sport” related groups. Outcomes included rates of hypotension (systolic blood pressure <90 mm Hg), tachycardia (>110 bpm), concomitant abdominal injury, and procedural/surgical intervention between sports and non-sports related injury.
320 patients met study criteria. 18% (59) were sports-related injuries with the most common mechanisms being skiing, snowboarding and contact sports (25%, 25%, and 24%, respectively). Median age was 24 years for sports and 30 years for non-sports related renal injuries (p = 0.049). Males were more commonly involved in sports related injuries (85% vs. 72%, p = 0.011). Median injury severity score was lower for sports related injuries (10 vs. 27, p < 0.001). There was no difference in renal abbreviated injury scale scores. Sports related trauma was more likely to be isolated without other significant injury (69% vs. 39% (p < 0.001)). Haemodynamic instability was present in 40% and 51% of sports and non-sports renal injuries (p = 0.30). Sports injuries had lower transfusion (7% vs. 47%, p < 0.001) and lower mortality rates (0% vs. 6%, p = 0.004). There was no difference in renal-specific procedural interventions between the two groups (17% sports vs. 18% non-sports, p = 0.95).
High-grade sports-related blunt renal trauma is more likely to occur in isolation without other abdominal or thoracic injuries and clinicians must have a high suspicion of renal injury with significant blows to the flank during sports activities.
PMCID: PMC4896140  PMID: 25769197
Wounds; Non-penetrating; Abdominal injuries; Sports; Snow sports; Kidney
4.  Species specificity of symbiosis and secondary metabolism in ascidians 
The ISME Journal  2014;9(3):615-628.
Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these ‘chemical symbioses'. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=−0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and biotechnological studies.
PMCID: PMC4331574  PMID: 25171330
5.  Predictors of sperm recovery after cryopreservation in testicular cancer 
Asian Journal of Andrology  2015;18(1):35-38.
Our objective was to identify predictors of improved postthaw semen quality in men with testicular cancer banking sperm for fertility preservation. We reviewed 173 individual semen samples provided by 67 men with testicular germ cell tumor (TGCT) who cryopreserved sperm before gonadotoxic treatment between 1994 and 2010 at our tertiary university medical center. Our main outcomes measures were independent predictors for the greater postthaw total motile count (TMC) in men with TGCT. Men with NSGCT were more likely to be younger (P < 0.01) and had high cancer stage (II or III, P < 0.01) compared with men with seminoma. In our multiple regression model, NSGCT histology, use of density gradient purification, and fresh TMC > median fresh TMC each had increased odds of a postthaw TMC greater than median postthaw TMC. Interestingly, age, advanced cancer stage (II or III), rapid freezing protocol, and motility enhancer did not show increased odds of improved postthaw TMC in our models. In conclusion, men with TGCT or poor fresh TMC should consider preserving additional vials (at least 15 vials) before oncologic treatment. Density gradient purification should be routinely used to optimize postthaw TMC in men with TGCT. Larger, randomized studies evaluating cancer stage and various cryopreservation techniques are needed to assist in counseling men with TGCT regarding fertility preservation and optimizing cryosurvival.
PMCID: PMC4736354  PMID: 25999362
cryopreservation; fertility preservation; seminoma; testicular neoplasms
6.  Catecholaminergic Gene Polymorphisms Are Associated with GI Symptoms and Morphological Brain Changes in Irritable Bowel Syndrome 
PLoS ONE  2015;10(8):e0135910.
Genetic and environmental factors contribute to the pathophysiology of irritable bowel syndrome (IBS). In particular, early adverse life events (EALs) and the catecholaminergic system have been implicated.
To investigate whether catecholaminergic SNPs with or without interacting with EALs are associated with: 1) a diagnosis of IBS, 2) IBS symptoms and 3) morphological alterations in brain regions associated with somatosensory, viscerosensory, and interoceptive processes.
In 277 IBS and 382 healthy control subjects (HCs), 11 SNPs in genes of the catecholaminergic signaling pathway were genotyped. A subset (121 IBS, 209 HCs) underwent structural brain imaging (magnetic resonance imaging [MRI]). Logistic and linear regressions evaluated each SNP separately and their interactions with EALs in predicting IBS and GI symptom severity, respectively. General linear models determined grey matter (GM) alterations from the SNPs and EALs that were predictive of IBS.
1) Diagnosis: There were no statistically significant associations between the SNPs and IBS status with or without the interaction with EAL after adjusting for multiple comparisons. 2) Symptoms: GI symptom severity was associated with ADRA1D rs1556832 (P = 0.010). 3) Brain morphometry: In IBS, the homozygous genotype of the major ADRA1D allele was associated with GM increases in somatosensory regions (FDR q = 0.022), left precentral gyrus (q = 0.045), and right hippocampus (q = 0.009). In individuals with increasing sexual abuse scores, the ADRAβ2 SNP was associated with GM changes in the left posterior insula (q = 0.004) and left putamen volume (q = 0.029).
In IBS, catecholaminergic SNPs are associated with symptom severity and morphological changes in brain regions concerned with sensory processing and modulation and affect regulation. Thus, certain adrenergic receptor genes may facilitate or worsen IBS symptoms.
PMCID: PMC4546052  PMID: 26288143
7.  Genetic variants in interleukin genes are associated with breast cancer risk and survival in a genetically admixed population: the Breast Cancer Health Disparities Study 
Carcinogenesis  2014;35(8):1750-1759.
These data support the associations between IL genes and breast cancer risk and mortality in a large admixed population.
Interleukins (ILs) are key regulators of immune response. Genetic variation in IL genes may influence breast cancer risk and mortality given their role in cell growth, angiogenesis and regulation of inflammatory process. We examined 16 IL genes with breast cancer risk and mortality in an admixed population of Hispanic/Native American (NA) (2111 cases and 2597 controls) and non-Hispanic white (NHW) (1481 cases and 1585 controls) women. Adaptive Rank Truncated Product (ARTP) analysis was conducted to determine gene significance and lasso (least absolute shrinkage and selection operator) was used to identify potential gene by gene and gene by lifestyle interactions. The pathway was statistically significant for breast cancer risk overall (P ARTP = 0.0006), for women with low NA ancestry (P ARTP = 0.01), for premenopausal women (P ARTP = 0.02), for estrogen receptor (ER)+/progesterone receptor (PR)+ tumors (P ARTP = 0.03) and ER−/PR− tumors (P ARTP = 0.02). Eight of the 16 genes evaluated were associated with breast cancer risk (IL1A, IL1B, IL1RN, IL2, IL2RA, IL4, IL6 and IL10); four genes were associated with breast cancer risk among women with low NA ancestry (IL1B, IL6, IL6R and IL10), two were associated with breast cancer risk among women with high NA ancestry (IL2 and IL2RA) and four genes were associated with premenopausal breast cancer risk (IL1A, IL1B, IL2 and IL3). IL4, IL6R, IL8 and IL17A were associated with breast cancer-specific mortality. We confirmed associations with several functional polymorphisms previously associated with breast cancer risk and provide support that their combined effect influences the carcinogenic process.
PMCID: PMC4123645  PMID: 24670917
8.  Pathway Analysis Software: Annotation Errors and Solutions 
Molecular genetics and metabolism  2010;101(2-3):134-140.
Genetic databases contain a variety of annotation errors that often go unnoticed due to the large size of modern genetic data sets. Interpretation of these data sets requires bioinformatics tools that may contribute to this problem. While providing gene symbol annotations for identifiers (IDs) such as microarray probeset, RefSeq, GenBank and Entrez Gene is seemingly trivial, the accuracy is fundamental to any subsequent conclusions. We examine gene symbol annotations and results from three commercial pathway analysis software (PAS) packages: Ingenuity Pathways Analysis, GeneGO and Pathway Studio. We compare gene symbol annotations and canonical pathway results over time and among different input ID types. We find that PAS results can be affected by variation in gene symbol annotations across software releases and the input ID type analyzed. As a result, we offer suggestions for using commercial PAS and reporting microarray results to improve research quality. We propose a wiki type website to facilitate communication of bioinformatics software problems within the scientific community.
PMCID: PMC2950253  PMID: 20663702
annotation; gene identifiers; microarray; bioinformatics; systems biology
9.  Convergent Validity of the Patient Reported Outcomes Measurement Information System's (PROMIS) Physical Function Computerized Adaptive Test (PF-CAT) for the Knee and Shoulder Injury Sports Medicine Patient Population 
Orthopaedic Journal of Sports Medicine  2015;3(7 suppl2):2325967115S00147.
The Patient Reported Outcomes Measurement Instrument System (PROMIS) Physical Function Computer Adaptive Test (PF-CAT) has been developed by the National Institutes of Health as a means of providing validated outcomes scores. PF-CAT adapts to patient response by applying different validated questions based on prior responses to generate scores using between 4 to 12 questions, and has been validated in a variety of patient populations to assess physical function. The purpose of our study is to compare patient reported outcomes scores in a sports medicine (shoulder and knee injury) patient population by analyzing convergence validity of PF-CAT to IKDC, SST, ASES, and SANE scores. We also wanted to assess and compare time burden placed on patients in completing these outcomes scores.
All patient visits from April through September 2014 presenting with either knee or shoulder complaints were included from a university-based sports medicine clinic, during which both PF-CAT as well as SANE, SST, and ASES legacy outcomes scores for shoulder injury patients, and SANE and IKDC outcomes scores for knee injury patients were obtained via tablet during the initial part of each visit. A total of 415 shoulder and 450 knee clinical evaluations qualified for inclusion in the study. Spearman correlation was used to evaluate pair-wise agreement among outcomes scores. The total scores from each outcome test and the T-scores from the PF-CAT were used in this correlation analysis. For interpretation, results greater than 0.5-0.69 were considered to have fair correlation, while values greater than 0.7 were considered as having good correlation. Significance was assessed at the 0.05 level. Median and interquartile ranges were used to summarize scores. The minimum score (floor effects) and maximum score (ceiling effects) occurrences were tallied and reported as percentages. A Fishers exact test was used to test for a difference in the number of times floor and ceiling values occurred. The Wilcoxon signed rank test was used to test for differences in completion times (measured in seconds).
Good correlation was found between IKDC and PF-CAT scores (r=0.7511; p<0.001), and fair correlation was found between SST (r=0.6777; p<0.001) and ASES (r=0.6262; p<0.001). Percent of patients’ scores that hit floor and ceiling values was 15% on SST (p<0.001), 2.5% on ASES (ceiling only, p=0.001), and 0% for PF-CAT shoulder patients. In knee injury patients, 0% of IKDC and 0.44% of PF-CAT scores hit floor or ceiling values (p<0.514). Completion times for PF-CAT averaged 81 seconds versus 359 seconds for IKDC which was significant (p<0.001). Average completions times for shoulder patients completing PF-CAT, SST, and ASES scores was 94, 192, and 157 (349 combined) seconds respectively (p <0.001).
PF-CAT holds strong convergence correlation with IKDC outcome scores in a sports medicine knee injury population, and only fair convergence correlation with SST and ASES scores in a sports medicine shoulder injury population. In addition, PF-CAT avoided floor and ceiling effects equal to IKDC outcomes and better than SST and ASES outcomes. Finally, PF-CAT was completed in a shorter time period than IKDC scores as well as compared to SST and ASES times both separately and combined. The PROMIS PF-CAT is an acceptable outcomes tool to evaluate sports medicine knee injuries that places less time burden on patients to complete outcomes scores.
PMCID: PMC4901716
10.  Current Estimate of Down Syndrome Population Prevalence in the United States 
The Journal of pediatrics  2013;163(4):1163-1168.
To calculate a reliable estimate of the population prevalence of Down syndrome in the US.
Study design
The annual number of births of infants with Down syndrome were estimated by applying published birth prevalence rates of Down syndrome by maternal age to US data from the Centers for Disease Control and Prevention for the years for which births by maternal age were available (1940–2008). Death certificate data for persons with Down syndrome were available for the years 1968–2007. We estimated the number of people with Down syndrome on January 1, 2008, using a life table approach based on proportions of deaths by age. Monte Carlo sampling was used to create 90% uncertainty intervals (UIs) for our estimates.
We estimated the January 1, 2008, population prevalence of Down syndrome as approximately 250 700 (90% UI, 185 900–321 700) based on proportions of deaths by age from the most recent 2 years (2006–2007) of death certificate data. This estimate corresponds to a prevalence of 8.27 people with Down syndrome per 10 000 population (90% UI, 6.14–10.62).
Our estimate of Down syndrome prevalence is roughly 25%–40% lower than estimates based solely on current birth prevalence. The results presented here can be considered a starting point for facilitating policy and services planning for persons with Down syndrome.
PMCID: PMC4445685  PMID: 23885965
11.  Dynamics of HSPC Repopulation In Non-human Primates Revealed by A Decade-Long Clonal-Tracking Study 
Cell stem cell  2014;14(4):473-485.
In mice, clonal tracking of hematopoietic stem cells has revealed variations in repopulation characteristics. However, it is unclear whether similar properties apply in primates. Here, we examined this issue through tracking of thousands of hematopoietic stem and progenitor cells (HSPCs) in rhesus macaques for up to 12 years. Approximately half of the clones analyzed contributed to long-term repopulation (over 3–10 years) and likely represent self-renewing hematopoietic stem cells (HSCs), while the remainder contributed primarily for the first year. The long-lived clones could be further subdivided into functional groups contributing primarily to myeloid, lymphoid or both lineages. Over time, the 4–10% of clones with robust dual lineage contribution predominated in repopulation capacity. HSPCs expressing a CCR5 shRNA transgene behaved similarly to controls. Our study therefore documents HSPC behavior in a clinically-relevant model over a long time frame, and provides a substantial system-level dataset that is a reference point for future work.
PMCID: PMC4009343  PMID: 24702996
12.  Intralesional Injection of Mitomycin C at Transurethral Incision of Bladder Neck Contracture May Offer Limited Benefit: TURNS Study Group 
The Journal of urology  2014;193(2):587-592.
Injection of mitomycin C may increase the success of transurethral incision of the bladder neck for the treatment of bladder neck contracture. We evaluated the efficacy of mitomycin C injection across multiple institutions.
Materials and Methods
Data on all patients who underwent transurethral incision of the bladder neck with mitomycin C from 2009 to 2014 were retrospectively reviewed from 6 centers in the TURNS. Patients with at least 3 months of cystoscopic followup were included in the analysis.
A total of 66 patients underwent transurethral incision of the bladder neck with mitomycin C and 55 meeting the study inclusion criteria were analyzed. Mean ± SD patient age was 64 ± 7.6 years. Dilation or prior transurethral incision of the bladder neck failed in 80% (44 of 55) of patients. Overall 58% (32 of 55) of patients achieved resolution of bladder neck contracture after 1 transurethral incision of the bladder neck with mitomycin C at a median followup of 9.2 months (IQR 11.7). There were 23 patients who had recurrence at a median of 3.7 months (IQR 4.2), 15 who underwent repeat transurethral incision of the bladder neck with mitomycin C and 9 of 15 (60%) who were free of another recurrence at a median of 8.6 months (IQR 8.8), for an overall success rate of 75% (41 of 55). Incision with electrocautery (Collins knife) was predictive of success compared with cold knife incision (63% vs 50%, p=0.03). Four patients experienced serious adverse events related to mitomycin C and 3 needed or are planning cystectomy.
The efficacy of intralesional injection of mitomycin C at transurethral incision of the bladder neck was lower than previously reported and was associated with a 7% rate of serious adverse events.
PMCID: PMC4307389  PMID: 25200807
contracture; urinary bladder neck obstruction; urinary incontinence; mitomycin; administration and dosage
13.  Characterization of Symptoms in Irritable Bowel Syndrome with Mixed Bowel Habit Pattern 
Irritable bowel syndrome (IBS) with mixed bowel habits (IBS-M) is a heterogeneous subtype with varying symptoms of constipation and diarrhea, and has not been well characterized. We aimed to characterize gastrointestinal (GI) and non-GI symptoms in IBS-M patients from a U.S. community, and to compare them with IBS with constipation (IBS-C) and diarrhea (IBS-D).
Subjects answering community advertisements and meeting Rome III criteria for IBS completed symptom questionnaires.
Key Results
Of the initial 289 IBS patients identified, one-third (n=51, 32.5%) who met Rome III criteria for IBS-M endorsed having either loose stools or hard stools due to medication. These patients had more severe symptoms and longer duration of flares compared to the rest of the IBS-M group (p = 0.014, p = 0.005). Excluding IBS-M patients with medication-related extremes in stool form who could not be reclassified by medical history, 247 IBS patients were assessed. IBS-M was the most common (44.1%), followed by IBS-C (27.9%), IBS-D (26.3%), and IBS-U (unsubtyped, 1.6%). IBS-M shared symptoms with both IBS-C and IBS-D (p-value range: <0.001–0.002). IBS-M patients reported most bothersome symptoms more similarly to IBS-D, with the most common being irregular bowel habits (27.5%), bloating (26.6%), and abdominal pain (20.2%). There were no differences in non-GI symptoms between subtypes.
Conclusions & Inferences
IBS-M is a heterogeneous symptom group and thus requires that subclassification criteria be better defined. Use of laxative/anti-diarrheal medications adds to the diagnostic complexity in a potentially more severe subset of IBS-M and should be assessed for accurate subclassification.
PMCID: PMC3865067  PMID: 23991913
irritable bowel syndrome; diarrhea; constipation; mixed bowel habits; abdominal pain
14.  Deep Sequencing Reveals Low Incidence of Endogenous LINE-1 Retrotransposition in Human Induced Pluripotent Stem Cells 
PLoS ONE  2014;9(10):e108682.
Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3′ end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.
PMCID: PMC4188539  PMID: 25289675
15.  Autonomic Response to a Visceral Stressor is Dysregulated in Irritable Bowel Syndrome and Correlates with Duration of Disease 
Previous studies reported altered autonomic nervous system (ANS) responses in IBS at baseline and to colonic balloon distension. This study examined heart rate variability (HRV) and plasma catecholamines as an index of ANS responsiveness in IBS during flexible sigmoidoscopy (FS) and explored associations of HRV with clinical measures.
Rome III positive IBS patients and healthy controls completed questionnaires measuring GI and psychological symptoms. HRV measures were calculated using electrocardiogram (ECG) data at rest and during FS. Plasma catecholamines were measured before and after the FS. Linear mixed effects models were used to compare HRV with IBS status and IBS duration across six time points. Significance was assessed at the 0.05 level.
36 IBS patients (53% F, mean age 37.89) and 31 controls (58% F, mean age 37.26) participated. After adjusting for age, sex, BMI, and HAD anxiety, IBS patients had a non-significant lower cardiovagal tone (p=0.436) and higher cardiosympathetic balance (p=0.316) at rest. During FS, controls showed a transient increase in cardiosympathetic balance and decrease in cardiovagal tone. However, IBS patients had significantly less cardiosympathetic and cardiovagal responsiveness both leading up to (p=0.003, p=0.005) and following (p=0.001, p=0.001) this stimulus. Those with longer duration of disease had less cardiosympathetic (p=0.014) and cardiovagal (p=0.009) responsiveness than those with shorter duration. No differences in catecholamines between IBS and controls were found.
IBS demonstrated dysregulated ANS responses to a visceral stressor which could be related to disease duration. Therefore, autonomic dysregulation is an objective physiologic correlate of IBS.
PMCID: PMC3788031  PMID: 23822743
autonomic dysregulation; autonomic nervous system; blunting; duration of disease; heart rate variability; irritable bowel syndrome; physiologic correlate; plasma catecholamines; somatic stressor; visceral stressor
16.  High Frequency Nasal Ventilation for 21 Days Maintains Gas Exchange with Lower Respiratory Pressures and Promotes Alveolarization in Preterm Lambs 
Pediatric research  2013;75(4):507-516.
Short-term high-frequency nasal ventilation (HFNV) of preterm neonates provides acceptable gas exchange compared to endotracheal intubation and intermittent mandatory ventilation (IMV). Whether long-term HFNV will provide acceptable gas exchange is unknown. We hypothesized that HFNV for up to 21d would lead to acceptable gas exchange at lower inspired oxygen (O2) levels and airway pressures compared to intubation and IMV.
Preterm lambs were exposed to antenatal steroids, and treated with perinatal surfactant and postnatal caffeine. Lambs were intubated and resuscitated by IMV. At ~3h of age, half of the lambs were switched to non-invasive HFNV. Support was for 3d or 21d. By design, PaO2 and PaCO2 were not different between groups.
At 3d (n=5) and 21d (n=4) of HFNV, fractional inspired O2 (FiO2), peak inspiratory pressure, mean airway, intra-tracheal, and positive end-expiratory pressures, oxygenation index, and Alveolar-arterial gradient were significantly lower than matched periods of intubation and IMV. PaO2/FiO2 ratio was significantly higher at 3d and 21d of HFNV compared to matched intubation and IMV. HFNV led to better alveolarization at 3d and 21d.
Long-term HFNV provides acceptable gas exchange at lower inspired O2 levels and respiratory pressures compared to intubation and IMV.
PMCID: PMC3961520  PMID: 24378898
17.  Identification of a Functional TPH1 Polymorphism Associated with Irritable Bowel Syndrome Bowel Habit Subtypes 
Background & Aims
Alterations in 5-hydroxytryptamine (5-HT) signaling have been implicated as a factor contributing to the altered bowel habit of irritable bowel syndrome (IBS) patients. Tryptophan hydroxylase 1 (TPH1) is the rate–limiting enzyme in enterochromaffin cell 5-HT biosynthesis. We hypothesized that genetic variants affecting TPH1 gene expression might alter intestinal 5-HT bioavailability and subsequently the propensity for distinct bowel habit subtypes in IBS. In this study, we assessed the only common TPH1 proximal promoter variant (-347C/A; rs7130929) and its association with bowel habit predominance in IBS.
Electrophoretic mobility shift assays were performed to assess whether the -347C/A allele variant affects the DNA-binding of nuclear factors. Genotype distribution was determined for 422 IBS patients subtyped using Rome III criteria and for 495 healthy controls recruited from two university medical centers. Association with bowel habit was tested using a multinomial logistic regression model controlling for race, anxiety, depression, and study site.
Early growth response factor 1 (EGR-1) bound with higher affinity to a site comprising the minor A-allele of SNP -347C/A. TPH1 genotype frequencies did not differ between IBS patients and controls overall. The CC genotype was more prevalent in the IBS-D subtype (47%) than in the IBS-C (25%) and IBS-M (37%) subtypes (P=0.039) after adjusting for race and other covariates. Colonic biopsies from a small cohort of IBS patients from one center were tested for higher TPH1 mRNA expression in samples with CC compared to CA genotype, but the results did not reach statistical significance.
The TPH1 promoter SNP -347C/A differentially binds EGR1, correlates with IBS bowel habit subtypes and possibly colonic TPH1 expression consistent with its role in modulating intestinal 5-HT signaling.
PMCID: PMC4067697  PMID: 24060757
Egr1; Sp1; ZBP-89; 5HT; serotonin; SNP
18.  Association between Early Adverse Life Events and Irritable Bowel Syndrome 
Background & Aims
Although childhood and adult abuse are more prevalent among patients with irritable bowel syndrome (IBS) than healthy individuals (controls), other types of early adverse life events (EALs) have not been well characterized. We investigated whether different types of EALs, before an age of 18 years, are more prevalent among patients with IBS, and the effects of gender and non-gastrointestinal symptoms on the relationship between EALs and IBS.
EALs were evaluated in 294 IBS patients (79% women) and 435 controls (77% women) using the early trauma inventory self report form, which delineates sub-categories of general trauma and physical, emotional, and sexual abuse. Validated questionnaires assessed gastrointestinal, psychological, and somatic symptoms.
Compared to controls, IBS patients reported a higher prevalence of general trauma (78.5% vs 62.3%), physical punishment (60.6% vs 49.2%), emotional abuse (54.9% vs 27.0%), and sexual events (31.2% vs 17.9%) (all P’s <.001). These significant differences were mainly observed in women. Of the EAL domains, emotional abuse was the strongest predictor of IBS (P<.001). Eight of the 27 EAL items were significant (P<.001) and increased the odds of having IBS by 108%–305%. Although EAL and psychological variables were related, EALs had an independent association with IBS (P=.04).
Various types of EALs are associated with development of IBS—particularly among women. Psychological distress and somatic symptoms might contribute to this relationship. When appropriate, EALs and non-gastrointestinal symptoms should be assessed in IBS patients.
PMCID: PMC3311761  PMID: 22178460
ETI-SF; psychology; somatization; nervous system
19.  Serum and Colonic Mucosal Immune Markers in Irritable Bowel Syndrome 
Low-grade colonic mucosal inflammation has been postulated to have an important role in the pathophysiology of irritable bowel syndrome (IBS). The objectives of this study were (i) to identify serum and tissue-based immunological and neuroendocrine markers associated with mucosal inflammation in male (M) and female (F) patients with non-post-infectious IBS (non-PI-IBS) compared with healthy controls and (ii) to assess possible correlations of such markers with IBS symptoms.
Sigmoid mucosal biopsies were obtained from 45 Rome II positive IBS patients without a history of PI-IBS (26 F, 35.5% IBS-C, 33.3% IBS-D, 31.1% IBS-A/M) and 41 healthy controls (22 F) in order to measure immunological markers (serum cytokine levels, colonic mucosal mRNA levels of cytokines, mucosal immune cell counts) and neuroendocrine markers associated with mucosal inflammation (corticotropin releasing factor- and neurokinin (NK)-related ligands and receptors, enterochromaffin cells). Symptoms were measured using validated questionnaires.
Of all the serum and mucosal cytokines measured, only interleukin-10 (IL-10) mRNA expression showed a group difference, with female, but not male, patients showing lower levels compared with female controls (18.0 ± 2.9 vs. 29.5 ± 4.0, P = 0.006). Mucosal mRNA expression of NK-1 receptor was significantly lower (1.15 ± 0.19 vs. 2.66 ± 0.56, P = 0.008) in female, but not male, patients compared with healthy controls. No other significant differences were observed.
Immune cell counts and levels of cytokines and neuropeptides that are associated with inflammation were not significantly elevated in the colonic mucosa of non-PI-IBS patients, and did not correlate with symptoms. Thus, these findings do not support that colonic mucosal inflammation consistently has a primary role in these patients. However, the finding of decreased IL-10 mRNA expression may be a possible biomarker of IBS and warrants further investigation.
PMCID: PMC3297737  PMID: 22158028
20.  Methodology and software to detect viral integration site hot-spots 
BMC Bioinformatics  2011;12:367.
Modern gene therapy methods have limited control over where a therapeutic viral vector inserts into the host genome. Vector integration can activate local gene expression, which can cause cancer if the vector inserts near an oncogene. Viral integration hot-spots or 'common insertion sites' (CIS) are scrutinized to evaluate and predict patient safety. CIS are typically defined by a minimum density of insertions (such as 2-4 within a 30-100 kb region), which unfortunately depends on the total number of observed VIS. This is problematic for comparing hot-spot distributions across data sets and patients, where the VIS numbers may vary.
We develop two new methods for defining hot-spots that are relatively independent of data set size. Both methods operate on distributions of VIS across consecutive 1 Mb 'bins' of the genome. The first method 'z-threshold' tallies the number of VIS per bin, converts these counts to z-scores, and applies a threshold to define high density bins. The second method 'BCP' applies a Bayesian change-point model to the z-scores to define hot-spots. The novel hot-spot methods are compared with a conventional CIS method using simulated data sets and data sets from five published human studies, including the X-linked ALD (adrenoleukodystrophy), CGD (chronic granulomatous disease) and SCID-X1 (X-linked severe combined immunodeficiency) trials. The BCP analysis of the human X-linked ALD data for two patients separately (774 and 1627 VIS) and combined (2401 VIS) resulted in 5-6 hot-spots covering 0.17-0.251% of the genome and containing 5.56-7.74% of the total VIS. In comparison, the CIS analysis resulted in 12-110 hot-spots covering 0.018-0.246% of the genome and containing 5.81-22.7% of the VIS, corresponding to a greater number of hot-spots as the data set size increased. Our hot-spot methods enable one to evaluate the extent of VIS clustering, and formally compare data sets in terms of hot-spot overlap. Finally, we show that the BCP hot-spots from the repopulating samples coincide with greater gene and CpG island density than the median genome density.
The z-threshold and BCP methods are useful for comparing hot-spot patterns across data sets of disparate sizes. The methodology and software provided here should enable one to study hot-spot conservation across a variety of VIS data sets and evaluate vector safety for gene therapy trials.
PMCID: PMC3203353  PMID: 21914224
21.  Protein expression based multimarker analysis of breast cancer samples 
BMC Cancer  2011;11:230.
Tissue microarray (TMA) data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups.
We apply weighted correlation network analysis (WGCNA) to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%), moderate (22%) and high (50%) mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-β1, and TGF β receptor II) that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis.
We find that the rule-based grouping variable (referred to as WGCNA*) is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data), it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance). We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach.
We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes.
PMCID: PMC3142534  PMID: 21651811
Tissue microarray; breast cancer; tumor marker; prognostic marker; WGCNA
22.  High-Throughput, Sensitive Quantification of Repopulating Hematopoietic Stem Cell Clones ▿ †  
Journal of Virology  2010;84(22):11771-11780.
Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5′ and the 3′ vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.
PMCID: PMC2977897  PMID: 20844053
23.  Quantification of Cytokeratin 5 mRNA Expression in the Circulation of Healthy Human Subjects and after Lung Transplantation 
PLoS ONE  2009;4(6):e5925.
Circulating epithelial progenitor cells are important for repair of the airway epithelium in a mouse model of tracheal transplantation. We therefore hypothesized that circulating epithelial progenitor cells would also be present in normal human subjects and could be important for repair of the airway after lung injury. As lung transplantation is associated with lung injury, which is severe early on and exacerbated during episodes of infection and rejection, we hypothesized that circulating epithelial progenitor cell levels could predict clinical outcome following lung transplantation.
Methodology/Principal Findings
Quantitative Real Time PCR was performed to determine peripheral blood mRNA levels of cytokeratin 5, a previously characterized marker of circulating epithelial progenitor cells. Cytokeratin 5 levels were evaluated in healthy human subjects, in lung transplant recipients immediately post-transplant and serially thereafter, and in heart transplant recipients. All normal human subjects examined expressed cytokeratin 5 in their buffy coat in amounts that were not significantly influenced by age or gender. There was a profound, statistically significant decrease in cytokeratin 5 mRNA expression levels in lung transplant patients compared to healthy human subjects (p = 3.1×10−13) and to heart transplant recipients. There was a moderate negative correlation between improved circulating cytokeratin 5 mRNA levels in lung transplant recipients with recovering lung function, as measured by improved FEV1 values (rho = −0.39).
Levels of cytokeratin 5 mRNA, a proxy marker for circulating epithelial progenitor cells, inversely correlated with disease status in lung transplant recipients. It may therefore serve as a biomarker of the clinical outcome of lung transplant patients and potentially other patients with airway injury.
PMCID: PMC2691992  PMID: 19529775
24.  Integrated Weighted Gene Co-expression Network Analysis with an Application to Chronic Fatigue Syndrome 
BMC Systems Biology  2008;2:95.
Systems biologic approaches such as Weighted Gene Co-expression Network Analysis (WGCNA) can effectively integrate gene expression and trait data to identify pathways and candidate biomarkers. Here we show that the additional inclusion of genetic marker data allows one to characterize network relationships as causal or reactive in a chronic fatigue syndrome (CFS) data set.
We combine WGCNA with genetic marker data to identify a disease-related pathway and its causal drivers, an analysis which we refer to as "Integrated WGCNA" or IWGCNA. Specifically, we present the following IWGCNA approach: 1) construct a co-expression network, 2) identify trait-related modules within the network, 3) use a trait-related genetic marker to prioritize genes within the module, 4) apply an integrated gene screening strategy to identify candidate genes and 5) carry out causality testing to verify and/or prioritize results. By applying this strategy to a CFS data set consisting of microarray, SNP and clinical trait data, we identify a module of 299 highly correlated genes that is associated with CFS severity. Our integrated gene screening strategy results in 20 candidate genes. We show that our approach yields biologically interesting genes that function in the same pathway and are causal drivers for their parent module. We use a separate data set to replicate findings and use Ingenuity Pathways Analysis software to functionally annotate the candidate gene pathways.
We show how WGCNA can be combined with genetic marker data to identify disease-related pathways and the causal drivers within them. The systems genetics approach described here can easily be used to generate testable genetic hypotheses in other complex disease studies.
PMCID: PMC2625353  PMID: 18986552
25.  Merging microsatellite data: enhanced methodology and software to combine genotype data for linkage and association analysis 
BMC Bioinformatics  2008;9:317.
Correctly merged data sets that have been independently genotyped can increase statistical power in linkage and association studies. However, alleles from microsatellite data sets genotyped with different experimental protocols or platforms cannot be accurately matched using base-pair size information alone. In a previous publication we introduced a statistical model for merging microsatellite data by matching allele frequencies between data sets. These methods are implemented in our software MicroMerge version 1 (v1). While MicroMerge v1 output can be analyzed by some genetic analysis programs, many programs can not analyze alignments that do not match alleles one-to-one between data sets. A consequence of such alignments is that codominant genotypes must often be analyzed as phenotypes. In this paper we describe several extensions that are implemented in MicroMerge version 2 (v2).
Notably, MicroMerge v2 includes a new one-to-one alignment option that creates merged pedigree and locus files that can be handled by most genetic analysis software. Other features in MicroMerge v2 enhance the following aspects of control: 1) optimizing the algorithm for different merging scenarios, such as data sets with very different sample sizes or multiple data sets, 2) merging small data sets when a reliable set of allele frequencies are available, and 3) improving the quantity and 4) quality of merged data. We present results from simulated and real microsatellite genotype data sets, and conclude with an association analysis of three familial dyslipidemia (FD) study samples genotyped at different laboratories. Independent analysis of each FD data set did not yield consistent results, but analysis of the merged data sets identified strong association at locus D11S2002.
The MicroMerge v2 features will enable merging for a variety of genotype data sets, which in turn will facilitate meta-analyses for powering association analysis.
PMCID: PMC2515855  PMID: 18644149

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