The Respiratory Infections in Andean Peruvian Children (RESPIRA-PERU) study enrolled children who participated in a community-cluster randomized trial of improved stoves, solar water disinfection, and kitchen sinks  and children from additional Andean households. We quantified the burden of influenza-associated acute respiratory illness (ARI) in this household-based cohort.From May 2009 to September 2011, we conducted active weekly ARI surveillance in 892 children age <3 years, of whom 272 (30.5%) had participated in the IHIP trial. We collected nasal swabs during ARI, tested for influenza and other respiratory viruses by RT-PCR, and determined influenza incidence and risk factors using mixed-effects regression models.The overall incidence of influenza-associated ARI was 36.6/100 child-years; incidence of influenza A, B, and C was 20.5, 8.7, and 5.2/100 child-years, respectively. Influenza C was associated with fewer days of subjective fever (median 1 vs. 2) and malaise (median 0 vs. 2) compared to influenza A. Non-influenza ARI also resulted in fewer days of fever and malaise, and fewer healthcare visits than influenza A-associated ARI. Influenza incidence varied by calendar year (80% occurred in the 2010 season) and IHIP trial participation. Among households that participated in the IHIP trial, influenza-associated ARI incidence was significantly lower in intervention than in control households .Influenza burden is high among Andean children. ARI associated with influenza A and B had longer symptom duration and higher healthcare utilization than influenza C-associated ARI or non-influenza ARI. Environmental community interventions may reduce influenza morbidity. Influenza virus is a major cause of childhood morbidity, generating three to five million severe illnesses and up to 500,000 deaths annually worldwide. Over the past decade, the burden of influenza among children has been increasingly recognized in both developing Influenza is unique among viral respiratory pathogens in that illness can be prevented or attenuated by vaccination, and understanding the burden and temporal circulation of influenza is important for the implementation of vaccination policy. Using data from RESPIRA-PERU, we sought to determine the incidence of laboratory-confirmed influenza in Andean children, describe the clinical characteristics of these infections, and identify risk factors for influenza acquisition.The study protocol was approved by the Vanderbilt Institutional Review Board and by the ethics committee of the Instituto de Investigación Nutricional. Written informed consent was obtained from a parent or guardian of all children involved in this study.rd birthday, withdrawal of consent, loss to follow-up, death, or study end, whichever came first. Field-workers visited the home of each enrolled child weekly, interviewed the parent or guardian about ARI signs/symptoms during the preceding week, and assessed symptomatic children for World Health Organization-Integrated Management of Childhood Illness (WHO-IMCI) danger signs including: tachypnea, wheezing, retractions, grunting, nasal flaring, stridor, or cyanosis RESPIRA-PERU, a prospective household-based study of ARI conducted in the high-altitude (1500–4000 meters) province of San Marcos, Cajamarca, Peru has been previously described The Integrated Home-based Intervention Package (IHIP) trial was a community-cluster randomized controlled trial conducted in San Marcos Province from 2008–2010, that randomized 51 rural communities to either an intervention  or control arm  Workers collected nasal swabs (NS) for each ARI episode at the first household visit following onset of symptoms as previously described All statistical analyses were performed using Stata 12.1 .Crude influenza incidence was calculated as the number of influenza-positive ARI episodes, divided by the cumulative person-time under observation and at risk. Children were not considered at risk during an ARI or within 7 days of ARI resolution. Crude estimates were adjusted to account for recurrence of events within individuals (some children had more than one confirmed influenza episode) using zero-inflated negative binomial regression. Poisson regression was used to calculate incidence for each influenza type.The demographic and clinical features of influenza-associated ARI, other viral ARI, and ARI in which no virus was detected, were compared using medians and proportions. A mixed-effects logistic regression model was used to test for significant differences, accounting for the recurrence of ARI at the level of the individual child.Multivariable, mixed-effect Poisson regression modeling, accounting for correlation of influenza infection at the level of the individual child was used to evaluate potential risk factors for influenza illness. Only 279 of the 892 children enrolled in RESPIRA-PERU (31%) had received any influenza vaccination at any time prior to study end; vaccination status was therefore not included in the risk factor analysis. Because risk for influenza infection existed only when influenza was present in the community, the analysis was limited to months during which influenza was circulating, defined as any month during which more than one influenza-positive NS specimen was identified among study children. To minimize misclassification the analysis was further restricted to children already under observation at the start of each influenza season, since children entering observation during the influenza season may have experienced an influenza ARI prior to observation onset.The RESPIRA-PERU study enrolled 892 children from 810 households in 58 communities. These children experienced 4,475 symptomatic ARI episodes over the course of the study and NS specimens were collected for 3957 (88%) nd altitude quartile, and fewer in the 4th, compared to other ARI  showed 11 influenza ARI over 32.5 child-years among children from intervention households and 26 influenza ARI over 34.3 child-years among children from control households. In multivariable analysis including calendar year, age, and gender, children in IHIP intervention households had a RR for influenza illness of 0.40 (95% CI 0.20–0.82), compared to children from control households.Influenza is increasingly recognized as an important cause of severe respiratory infection in children worldwide The incidence of influenza ARI observed in RESPIRA-PERU (36.6/100 child-years) is higher than seen in recent household-based studies in other developing nations. A study in Dhaka, Bangladesh from 2004 to 2007 estimated an annual influenza risk of 10% among children <5 years of age, using viral culture to document influenza infection among children with fever or respiratory symptoms A study in Vietnam from 2007–2010 reported a seasonal risk of influenza infection of 41.8% among children aged 5–9 years (no similar data for younger children were reported), using a combination of serology and RT-PCR of NS samples Some important aspects of our study should be noted when comparing the results to other studies. RESPIRA-PERU was designed to capture all ARI episodes among cohort children and NS samples were collected only once per episode. Recurrence of fever or cough within 7 days of an antecedent ARI episode was considered an extension of the prior ARI, and not counted as a new episode. Thus, children were not considered at-risk for a new ARI during a symptomatic episode and for seven days thereafter, and this time not-at-risk was excluded from the person-time denominator. On average, children in RESPIRA-PERU experienced 6 ARI/year, lasting approximately five days each 2), yet the high incidence of influenza in our study compared to that observed in highly crowded Bangladesh Multiple aspects of the physical environment in which children in the RESPIRA-PERU cohort live may affect their susceptibility to respiratory infection. While living in a remote location might insulate households from circulating infections Previous studies have shown that Andean Peruvian children are at higher risk for hypoxia with ARI Only 4% of influenza-associated ARI in the RESPIRA-PERU study met WHO-IMCI criteria for severity, a proportion lower than for RSV, MPV, PIV 1–3, and even adenovirus The association between enrollment in the IHIP trial Our study has some important limitations. First, we did not employ serologic testing and NS samples were taken only during symptomatic ARI episodes. As discussed above, several serologic studies suggest that not all influenza infections are symptomatic enough to be detected by active ARI surveillance In conclusion, the incidence of influenza-associated illness among Peruvian Andean children during the 2009–2011 influenza seasons was high. Influenza type A and B infections resulted in more days with fever and malaise and a higher rate of care-seeking than ARI from which other viruses or no virus was detected. Given the low vaccination coverage among cohort children, a substantial amount of respiratory morbidity in Andean children might be averted by universal influenza vaccination. In addition, we found that a home-based intervention targeting home hygiene and indoor air pollution was associated with a substantial reduction in influenza ARI risk among children who participated in the IHIP trial. This suggests an additional role for environmental modifications in prevention of influenza morbidity in this setting.Table S1Influenza Incidence among Andean Peruvian Children 2009–2011 by Calendar Month.(DOCX)Click here for additional data file.


The aim of this study was to assess the effect of acute bilateral jugular vein compression on: (1) pial artery pulsation (cc-TQ); (2) cerebral blood flow velocity (CBFV); (3) peripheral blood pressure; and (4) possible relations between mentioned parameters.2 was measured using medical gas analyser. Bilateral jugular vein compression was achieved with the use of a sphygmomanometer held on the neck of the participant and pumped at the pressure of 40 mmHg, and was performed in the bend-over (BOPT) and swayed to the back  position.Experiments were performed on a group of 32 healthy 19–30 years old male subjects. cc-TQ and the subarachnoid width (sas-TQ) were measured using near-infrared transillumination/backscattering sounding (NIR-T/BSS), CBFV in the left anterior cerebral artery using transcranial Doppler, blood pressure was measured using Finapres, while end-tidal CO2 remained stable during BOPT and venous compression in both groups. Significant interdependence between changes in cc-TQ and PP after bilateral jugular vein compression in the initial position was found (r = −0.74).In the first group (n = 10) during BOPT, sas-TQ and pulse pressure (PP) decreased  and CBFV increased (+35.0%), while cc-TQ did not change (+1.91%). In the second group, in the initial position (n = 22) cc-TQ and CBFV increased , while sas-TQ and PP decreases were not statistically significant . End-tidal COAcute bilateral jugular venous insufficiency leads to hyperkinetic cerebral circulation characterised by augmented pial artery pulsation and CBFV and direct transmission of PP into the brain microcirculation. The Windkessel effect with impaired jugular outflow and more likely increased intracranial pressure is described. This study clarifies the potential mechanism linking jugular outflow insufficiency with arterial small vessel cerebral disease. A stable increase in cerebral venous pressure induces arteriolar vasodilation, which averages 6% to 12% of the control diameter in cats The amplitude of pial artery pulsation (cc-TQ) can be measured non-invasively using near-infrared transillumination/backscattering sounding (NIR-T/BSS), a new method based on infrared radiation (IR) that has been developed in the last decade by our team We hypothesised that impaired venous outflow would lead to pial artery dilation caused by a metabolic and/or autoregulatory mechanism due to decreased oxygen supply and/or increased intracranial pressure, respectively. Augmented pial artery compliance should result in increased cc-TQ and hyperdynamic brain circulation characterised by augmented cerebral blood flow velocity (CBFV). The investigated topic is not a purely theoretical concept, as increased cerebral venous blood volume has been recently reported during sympathetic activation  and gave written informed consent to participate in the study. The experimental protocol and the study were approved by the ethical committee of the Medical University of Gdansk (TKEBN/259). Subjects did not have any disorders and were not taking any medication. Before the experiment, each subject underwent a general and neurological examination. No nicotine, coffee, tea, cocoa or any methylxanthine-containing food or beverages were permitted for 8 hours before the tests. Additionally, prior to each test, the volunteers were asked to sit comfortably and rest for 30 minutes.Initial position: subject set comfortably for 10 minutes with head swayed to the back at 20° angle.BOPT: subject bends forwards at 45° angle (the trunk and the head) from the initial half-lying position on the back Then, for bilateral jugular vein compression participants were divided into two groups.Jugular veins were completely compressed 3 minutes after starting BOPT for 3 minutes.Subject returned to initial position.Subject returned to initial position 3 minutes after starting BOPT.Subject set comfortably in initial position for 3 minutes.Jugular veins were completely compressed for 3 minutes.The numerical values for statistical analysis were taken just before the end of each three-minute procedure. After bilateral jugular vein compression all subject were asked to sit comfortably in the initial position for 10 minutes.2.Bilateral jugular vein compression was achieved with the use of a sphygmomanometer held on the neck of the participant and pumped to a pressure of 40 mmHg. Blood pressure in jugular veins in the initial position should be close to zero, or even negative, so it can be assumed that compression was complete. The subjects were asked to breathe normally to avoid changes in PaCOTranscranial Doppler (TCD) measurement of cerebral blood flow velocity (CBFV) in the left anterior cerebral artery was performed with a Doppler ultrasound device TDS 4 . A pulse probe of 2 MHz was used and analysis of the results was carried out on a built-in IBM PC computer. To assure the best possible reproducibility of the recordings, the Doppler probe was mounted on the head with a special stabilising strip.Recording of changes in systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and HR were measured using Finapres . The Finapres sensor was mounted onto the middle finger of the non-dominant hand resting on the table. Beat-to-beat blood pressure was transferred to a computer console continuously displaying SAP, DAP and HR. Pulse pressure (PP) was calculated from the following equation: PP = SAP−DAP.2 was measured in mmHg with a medical gas analyser . The instrument was calibrated using a certified standard gas mixture before each experiment. End-tidal CO2 was recorded and printed continuously in real time. Values for subsequent analysis were read from the printouts and entered manually into a Statistics for Windows 8.0 database.End-tidal CORecording of changes in the amplitude of pial artery pulsation and in the width of SAS with NIR-T/BSS were performed with a head-mounted SAS 100 Monitor . The sensor unit consists of the emitter (E) and two photo-sensors located at different distances from the emitter. The NIR-T/BSS emitter is a near-infrared light-emitting diode (LED). The proximal sensor (PS) is located close to the emitter, while the distal sensor (DS) is located at a larger distance from the emitter. The stream of IR generated by the emitter penetrates the highly perfuse layer of the skin of the head, the skull bones and the SAS. The stream of radiation reflects from the surface of the brain and reaches the sensors, crossing the aforementioned layers of tissues in reverse order. Signals from the sensors undergo analogue-digital conversion in a specialised data acquisition system and are recorded on the microcomputer' s hard disk for subsequent analysis with on-line presentation on the computer monitor.Theoretical and practical foundations of the NIR-T/BSS method were provided in the model studies by our team Thus, in the transillumination quotient (TQ) we can identify three main components:constant or a non-pulsatile component, further referred to as the subarachnoid component (sas-TQ); its value depending on the width of the CSF-filled SAS,slow-variable pulsation, further referred to as the subcardiac component (scc-TQ); including, but not limited to, pulsation of a respiratory origin,fast-variable pulsation, further referred to as the cardiac component (cc-TQ); resulting from heart-generated arterial pulsation that is the cause of fast oscillations of the width of SAS.For further analysis, the first harmonic of the arterial pulsation-dependent oscillations of TQ was extracted through appropriate filtering, along with its modulation. Modulation of that harmonic is a fast-variable component (or cardiac component) of principal, second, and third harmonics of cardiac component waveform, respectively.2 responses were compared to baseline values. Correlation and regression analysis was performed to assess interdependences between SAP, DAP, PP, HR, CBFV, end-tidal CO2, sas-TQ and cc-TQ. All statistical calculations were performed using the Statistics for Windows 8.0 commercial package.W Shapiro-Wilk W, Mann-Whitney U and ANOVA tests were used to analyse the differences between average values. Changes in sas-TQ, cc-TQ, CBFV, SAP, DAP, PP, HR and end-tidal CO2 remained stable during BOPT and venous compression in both groups. Detailed descriptive statistics are provided in Both groups  were well-matched at baseline in terms of CBFV and BP. The BOPT test caused sas-TQ, cc-TQ, CBFV and PP decrease in both investigated groups (n = 10 and n = 22). However, acute bilateral jugular vein compression brought different results between the investigated groups. In the first group (n = 10) during BOPT sas-TQ and PP further decreased, CBFV increased while cc-TQ did not change . In the Correlation and regression analysis revealed significant interdependence between changes in cc-TQ and PP after bilateral jugular vein compression in the initial position  or in the abdominal-lying position, the brain floats forward toward the inner surface of the frontal bones and the SAS in the frontal region assumes its minimum value Bilateral jugular vein compression resulted in enhanced CBFV in both groups. We may assume that hampered jugular outflow results in decreased oxygen supply, which in turn lead to arteriole dilation and subsequent increase in cerebral blood flow etal. etal. Acute cerebral venous congestion produced significant cc-TQ increase in the initial position. Such a result is in line with the above-presented reasoning. In the initial position the SAS was insignificantly reduced by enhanced intracranial blood volume, and remained wide enough to allow for proper registration of the amplitude of pial artery pulsation . IncreasThe following study limitations should be taken into account. Only young, healthy males were investigated and acute jugular vein insufficiency was analysed. Therefore, any extrapolations to patients suffering from chronic cerebral venous insufficiency or normal pressure hydrocephalus should be viewed with caution. In our study the SAS decrease during bilateral jugular vein compression in the initial position was not statistically significant, which may suggest that even during bilateral jugular vein congestion the brain may be decompressed through the vertebral plexus In summary, acute bilateral jugular venous insufficiency leads to hyperkinetic cerebral circulation characterised by augmented pial artery pulsation and CBFV and direct transmission of PP into the brain microcirculation. From the mechanistic point of view, the Windkessel effect with impaired jugular outflow and most likely increased intracranial pressure is described. Impaired jugular outflow does not allow for dampening the pulsation energy and actually exaggerates the pulsatile flow, which in turn creates a hazardous environment for the brain microcirculation. Therefore, this study clarifies the potential mechanism linking jugular outflow insufficiency with small vessel arterial cerebral disease.


Staphylococcus aureus.High prevalence of co-colonization increases risk for colonization or infection by vancomycin-resistant  Staphylococcus aureus (MRSA) upon admission to the medical and surgical intensive care units (ICUs) of a tertiary-care facility between January 1, 2002, and December 31, 2003. Co-colonization was defined as a VRE-positive perirectal surveillance culture with an MRSA-positive anterior nares surveillance culture collected concurrently. Among 2,440 patients, 65 (2.7%) were co-colonized. Independent risk factors included age , admission to the medical ICU , male sex , and receiving antimicrobial drugs on a previous admission within 1 year . None of the co-colonized patients would have been identified with clinical cultures alone. We report a high prevalence of VRE/MRSA co-colonization upon admission to ICUs at a tertiary-care hospital.We assessed the prevalence, risk factors, and clinical outcomes of patients co-colonized with vancomycin-resistant enterococci (VRE) and methicillin-resistant  Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) cause nosocomial infections and are associated with increased rates of illness and death  isolates reported thus far have possessed the vanA gene, which confers resistance to vancomycin and is believed to have been acquired when an MRSA isolate conjugated with a co-colonizing VRE isolate  and surgical ICU (SICU) of the University of Maryland Medical Center (UMMC) between January 1, 2002, and December 31, 2003. UMMC is a tertiary-care facility in Baltimore, Maryland. The MICU is a 10-bed, private room unit providing care to patients who have acute or potentially life-threatening medical conditions including hematologic and other malignancies. The SICU is a 19-bed, private room unit providing care to adult patients with solid organ transplantation and abdominal, genitourinary, orthopedic, and otolaryngologic surgery.During the study period, routine surveillance cultures of the anterior nares for MRSA and perirectal area for VRE were obtained from patients within 48 hours of admission to both ICUs for infection control purposes. Cultures were obtained from an average of 80% of admitted patients. Patients from whom both cultures were not obtained upon ICU admission or had an ICU length of stay <5 hours were excluded. Patients may have had multiple admissions during the study period, and all eligible admissions were included in this analysis.–All data were abstracted from the UMMC central data repository that contains the patients' electronic medical records. The validity of these data was assessed by randomly sampling 10% of the patients' electronic data records and comparing them to the original paper medical records. The positive and negative predictive values of this assessment exceeded 99% for both validity measures, which was similar to values seen in previous studies with this same data source  and cultured in Baird Staphylococcus Enrichment broth . Presumptive S. aureus colonies were confirmed by positive catalase and Murex Staphaurex (Remel) reactions. MRSA were identified by growth on Mueller Hinton agar with 4% NaCl and 6 μg/mL oxacillin.All media and reagents were from BD Biosciences  unless otherwise noted. Enterococci were isolated by plating perirectal swabs on Columbia Modified CNA agar and examined at 24 and 48 hours. Presumptive enterococci colonies that were gram-positive cocci, catalase negative, and pyrrolidonyl-β-naphthylamide positive were plated on vancomycin screening agar and motility agar. Vancomycin-resistant, nonmotile enterococci were identified as VRE.Three risk factor analyses were conducted: 1) VRE/MRSA co-colonized patients were compared to all other ICU patients, 2) patients colonized with VRE alone were compared to patients not colonized with VRE, and 3) patients colonized with MRSA alone were compared to patients not colonized with MRSA. Patients co-colonized with MRSA or VRE were excluded for analyses 2 and 3 above that compared solitary VRE or MRSA colonization to noncolonized patients.t, chi-square, Fisher exact, and Wilcoxon rank sum tests were used for descriptive analyses to assess bivariable differences between groups. All variables that were significant (α = 0.1) in the bivariable analyses were included in the initial (full) multivariable logistic regression model. In each of the multivariable analyses performed, variables not significantly associated (α = 0.05) with the outcome (VRE/MRSA co-colonization and VRE or MRSA solitary colonization upon admission to the ICU) were removed from the model. Each of the removed variables was then reinserted into the model to assess if its presence altered the regression coefficient by >20%. If so, this risk factor was included in the final model. The resulting multivariable logistic regression model was considered the final model and was used to calculate odds ratios and 95% confidence intervals for the remaining risk factors.Student Differences in patients' clinical outcomes after assessing colonization status were also assessed. These variables included length of stay after the ICU admission culture, subsequent positive clinical cultures for VRE or MRSA, in-hospital death rate, and readmission to the index hospital or transfer to another healthcare facility. Subsequent positive clinical cultures were limited to sterile sites, and thus only blood, cerebrospinal fluid, or urine cultures were considered.During the 2-year cohort period , 3,090 patients were admitted to the MICU or SICU for a >5-hour stay. Of these, 2,440 patients (79.0%) had both anterior nares and perirectal admission cultures collected. Sixty-five patients (2.7%) were co-colonized with VRE and MRSA, 247 patients (10.1%) were colonized with VRE alone, and 175 patients (7.2%) were colonized with MRSA alone. Of the 57 MRSA/VRE co-colonized patients with perirectal samples available for additional analysis, 23 patients (40.4%) were perirectally colonized with MRSA in addition to perirectal VRE and MRSA nasal colonization. Results of the bivariable analyses for VRE/MRSA co-colonization are displayed in Independent risk factors identified with logistic regression for the different colonization states  were markedly different . Table 3–,Increasing VRSA prevalence in the healthcare setting could result in considerable illness and death - or VRSA-colonized or infected patients show that these organisms can be transmitted to close contacts or other hospitalized patients  of these patients were also colonized with MRSA (,Ray et al. assessed the prevalence of gastrointestinal Enterococcus faecalis has been more likely to be associated with conjugation events and subsequent VRSA colonization or infection compared with E. faecium (,E. faecium would be less likely to be involved in transmission of vancomycin resistance to MRSA compared with E. faecalis. Still, these data would have been useful and informative. A previous, hospitalwide study at UMMC suggested that among isolated VRE, the prevalence of E. faecium and E. faecalis were 87%, and 13%, respectively (A limitation of this study is that investigators were unable to determine the species of the VRE isolates. Historically, In summary, these data describe a high prevalence of patients co-colonized with VRE and MRSA on admission to an ICU at a tertiary-care hospital, none of whom would have been detected by clinical culture. Risk factors for VRE and MRSA co-colonization are also described. Given that many of these patients were discharged to other institutions, treating physicians and infection control personnel must be cognizant of the risks for VRSA colonization and infection and use appropriate precautions. Appropriate methods to rapidly detect co-colonized patients must be identified to suppress the emergence of VRSA; limit patient-to-patient transmission of MRSA, VRE, and VRSA; and prevent endemic VRSA colonization in healthcare institutions.


This study aimed to explore the association between obstructive sleep apnea (OSA) severity and pharyngeal parameters using sub-mental ultrasonography (US), and investigate the accuracy of US for identifying severe OSA patients.One hundred and five consecutive referrals for suspected OSA were enrolled. The diameters of the retro-glossal (RG) and retro-palatal (RP) regions were measured via sub-mental US upon expiration during tidal breathing, forced inspiration, and Müller maneuver (MM). Independent factors associated with severe OSA identified from two-thirds of randomly-selected patients (model-development group) were used to construct a model for predicting severe OSA. The accuracy of the model was validated in the remaining one-third of patients .Fifty severe OSA patients, 30 with mild-moderate OSA, and 25 without OSA were enrolled. Compared to non-OSA and mild-moderate OSA patients, those with severe OSA had narrower RP diameter in all three maneuvers. Using the prediction model constructed with changes of RP diameters at MM and neck circumference, the independent predictors of severe OSA in the model-development group had 100% sensitivity and 65% specificity.Sub-mental US can accurately discriminate the severity of OSA and be used to identify patients with severe OSA.NCT00674076ClinicalTrials.gov  Obstructive sleep apnea (OSA) is defined as the collapse of the upper airway during sleep, causing intermittent hypoxia and sleep fragmentation Currently, the diagnosis of OSA is based on clinical symptoms and episodes of apnea and hypopnea measured by polysomnography (PSG) A previous work has demonstrated that US can clearly demonstrate the vocal cords and air column of the upper airway where it can be used to identify patients at high risk for post-extubation stridor This study was performed at the Center of Sleep Disorder of National Taiwan University Hospital from January to June 2008. All Chinese patients aged ≥18 years-old and referred to the sleep laboratory for suspected OSA were eligible for the study. The exclusion criteria were refusal to participate; inability to perform the maneuvers; presence of congestive heart failure, chronic pulmonary disease demonstrated on pulmonary function testing, active neurologic event, active infection or surgery two weeks prior to screening; enrolment in other trials during the study period; other sleep disorders; ascites; and pregnancy. Clinical evaluation, anthropometric measurements, and US examination were immediately conducted upon enrolment whereas PSG was undertaken within three days after US.Two-thirds of the participants were randomly selected as the model-development group while the remaining one-third was the validation group. The allocation was based on a computer-generated random assignment. The Research Ethics Committee of National Taiwan University Hospital approved the study protocol and all participants provided written informed consent.Full-night PSG  was performed in the sleep laboratory according to a previous protocol The US was done by an independent operator (Shu CC) who was blinded to the PSG results. The US machines used was Aplio XV  with 5.0-MHz convex transducers in gray scale 2-dimensional mode. The detailed procedure was shown in Dynamic changes of transverse diameter of the retro-palatal (RP) and retro-glossal (RG) airspaces were determined in three maneuvers, namely, expiration during tidal breathing, forced inspiration, and the Müller maneuver (MM) . Before The intra- and inter-observer coefficients of variation (CV) were tested on eight healthy volunteers where US was done by two independent operators (Shu CC and Huang CT). The intra- and inter-observer CVs ranged from 2.3 (tongue thickness) to 9.1 (forced inspiration of RP diameter) and 3.0 (tongue thickness) to 10.4 (expiration of RG diameter), respectively . Moreovet test or one-way ANOVA, where appropriate, and the chi-square test for categorical variables. Factors correlated with AHI were identified by Pearson correlation. Multivariate logistic regression with forward conditional method was used to identify predictors for severe OSA. Thereafter, these predictors were used to estimate the patient's probability of severe OSA, which was then used to test the receiver operating characteristic (ROC) curve of diagnosing severe OSA. The optimal cut-off value of the ROC curve was defined as the one with the least (1 - sensitivity)2+ (1- specificity)2 in the model-development group p<0.05 was considered statistically significant. All statistical analyses were performed using the SPSS 13.0 .Inter-group differences for numerical variables were compared using student The trial profile was listed in The US parameters were shown in p<0.001), followed by RP diameter on MM , UAL (γ = 0.581), % RP shortening on MM , and BMI .Factors correlated with AHI included old age, male sex, body mass index (BMI), neck circumference, UAL, tongue thickness, RP diameter in three maneuvers, and RG diameter on forced inspiration and MM . Among tp = 0.008) and neck circumference  as independent predictors of severe OSA  (p<0.001). When the probability was>0.45, severe OSA was predicted with 100% sensitivity and 65% specificity.The ROC curve of the model-development group for identifying patients with severe OSA was shown in p<0.001) . When thIn this study, sub-mental US was used to measure the pharyngeal transverse diameter and nearby soft tissue in OSA patients. The pharyngeal variables, tongue thickness, and UAL had good correlation with OSA severity, especially RP diameter on MM. To predict severe OSA, % RP shortening during MM and neck circumference were used to construct a prediction model. The model was validated in an independent group where it had high sensitivity (100%) and fair specificity (65%).The prevalence rate of OSA is as high as 24% in middle-aged men and 9% in middle-aged women The US is also adequately sensitive for detecting dynamic changes of the pharyngeal airspace in three maneuvers, which means that like fiberoscopy and ultra-fast MRI, sub-mental US can demonstrate airway collapsibility and detect pharyngeal changes at apnea The tongue base and lateral pharyngeal wall measured by US are shown to have good correlations with AHI in OSA patients It has been demonstrated that the RP region is the primary site of upper airway narrowing in OSA patients in both Caucasians and Asians when compared to the RG region The present study has some limitations. First, a pharyngeal pressure monitor was not used to monitor the effects of the Müller maneuver. Although all enrolled patients cooperated and performed the maneuver well, its effect of MM might not be the same for all. Second, there was no MRI used to compare the anatomic markers detected by US.In conclusion, sub-mental US can accurately measure pharyngeal airspace and adjacent soft tissue, and can discriminate OSA severity. The % RP shortening during MM and neck circumference are independent factors for predicting severe OSA. Thus, sub-mental US may be a promising tool for screening severe OSA patients for early intervention.File S1Recruited population for examining intra- and inter-observer variation.(DOC)Click here for additional data file.Table S1Coefficients of variance of pharynx measured by ultrasonography.(DOC)Click here for additional data file.Table S2Correlations between clinical and ultrasonographic variables and apnea-hypopnea index in the study patients.(DOC)Click here for additional data file.


Combination of sulindac and PDTC lead to G0 arrest and massive apoptosis in co-treated cultures. Western blotting analysis argued for induction of the mitochondrial apoptotic pathway. These data demonstrate the synergistic cytotoxic effect of sulindac and PDTC on ovarian cancer cells through apoptosis and cell cycle arrest and prompt to test the efficacy of this combination in animal models.Sulindac, a non-steroidal anti-inflammatory drug, suppresses carcinogenesis and inhibits growth of tumor cells. Pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, has been also identified as a potential anti-neoplastic agent. We hypothesized that combination of sulindac and PDTC could result in augmentation of cytotoxicity against ovarian cancer cells. The effect of sulindac and PDTC was examined on several ovarian cancer lines. Tumor cell viability was assessed using the MTT assay. Annexin-V/PI staining was used to detect apoptosis, cell cycle distribution was analyzed in FACS, and expression of cellular proteins was detected by Western blotting. Incubation of OVA-14, OVP-10 and CAOV-1 ovarian cancer cells with sulindac and PDTC resulted in significantly greater inhibition of cell viability compared to either compound alone. In a model of OVA-14 cells it was evident that this effect was not related to the expression of COX enzymes since both active (sulindac sulfide) and inactive (sulindac)  Ovarian cancer is the most deadly malignancy of the female reproductive system with more than 70% of cases diagnosed at an advanced stage. Current treatment of this cancer includes cytoreductive surgery (tumor debulking) followed by platinum- and taxane-based therapy . Howeverin vitro experiments have all demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) appear to be effective in chemoprevention and possible treatment of various types of cancer . There are also attempts to use sulindac sulfone, known as exisulind, in combination treatments of various types of cancer  is a thiol-containing synthetic compound, which is known for its antioxidant, metal-chelating and strong NF-κB inhibitory properties ,27. Occaer cells –30. Intel models .In a previous study, we have demonstrated that sulindac and sulindac sulfide but not other NSAIDs such as acetylsalicylic acid and rofecoxib inhibited the growth of various ovarian cancer cells . We supp2 tissue flasks  at 37°C in a humidified atmosphere of 5% CO2 and were passaged two to three times weekly.The following ovarian cancer cell lines were used in the study: OVA-14  tumor, CAOV-1 , OVP-10 , MDAH 2774 (ATCC no. CRL-10303), SKOV-3 (ATCC no. HTB-77). The cells were grown in Dulbecco’s modified Eagle’s medium   or RPMI-1640  (OVP-10) supplemented with 10% heat-inactivated fetal calf serum  and antibiotic-antimycotic (Sigma). The cells were maintained in 25-cmSulindac was from Sigma and sulindac sulfide was purchased from Biomol Research Laboratories . The drugs were dissolved in dimethylsulfoxide  and stock solutions were prepared (200 mM) for further preparation. The cells in control cultures were incubated either without diluent or with 0.1% DMSO; no significant differences in cell growth were observed. Pyrrolidine dithiocarbamate (PDTC) was purchased from Sigma, with the stock solution (200 mM) prepared in distilled water.4/200 μl/well) with PDTC  and sulindac or sulindac sulfide , alone or in combination, for 24 h. At the end of the incubation, 25 μl of MTT (2.5 mg/ml) was added to each well for the last 4 h. Then the cells were centrifuged , supernatants were removed and the formazan product was dissolved in acid DMSO. The plates were read on an ELISA reader  using a 550 nm filter. The means and standard deviations were determined for triplicate samples. The cytotoxic effect was expressed as the relative viability and was calculated as follows: relative viability =  in double-treated cultures in comparison with cells incubated with sulindac alone, PDTC alone or untreated cultures  at the 4-h incubation timepoint (data not shown). In 24-h cultures, the process of apoptosis progressed and was especially exhibited in OVA-14 cells co-treated with sulindac and PDTC: 89% cells were found to be fully apoptotic, in comparison with 26% in sulindac cultures, 25% in PDTC cultures, and 8% in control cultures . To shedin vitro resulted in a synergistic cytotoxic effect against OVA-14 ovarian cancer cells (in vitro) and sulindac (inactive compound) synergized with PDTC. Moreover, OVA-14 cells did not express COX-1 and COX-2 enzymes in our preliminary experiments (data not shown).A combination approach of several therapeutic agents has become the standard treatment of ovarian cancer. In the present study, we demonstrated that the combined use of sulindac or sulindac sulfide and PDTC er cells . This efer cells . The prier cells . Increaser cells  or PDTC er cells . In our et al  in tumor tissue in a mouse model of colon cancer.Both sulindac and PDTC have been shown to inhibit the pro-survival NF-κB signaling pathway in different tumor models ,34–36. Het al  argue foin vitro studies have shown their antitumor efficacy in ovarian cancer models (COX inhibitors, including sulindac and its metabolites, have been recognized as promising agents in the prevention of neoplasia and in cancer treatment, especially when combined with standard therapy [ and clinr models ,13,19,23r models . Combinain vitro, these agents exert pleiotropic biological effects in vivo, beneficial from the therapeutic point of view. Upregulation of COX enzymes is a frequent feature of neoplasia and in ovarian cancers COXs have been implicated in platinum resistance and promotion of tumor progression (in vivo than those observed in ovarian cancer cell cultures in vitro. Thus, preclinical in vivo investigations of potential antitumor effect of PDTC and sulindac against ovarian cancers are worth continuing.Apart from the direct pro-apoptotic activity of sulindac and PDTC, which we and others observed gression ,12. Furtgression . Therefogression  and was gression . Recentlgression . Since Pgression ,40, its


To facilitate the clinical implementation of genomic medicine by next-generation sequencing, it will be critically important to obtain accurate and consistent variant calls on personal genomes. Multiple software tools for variant calling are available, but it is unclear how comparable these tools are or what their relative merits in real-world scenarios might be.We sequenced 15 exomes from four families using commercial kits (Illumina HiSeq 2000 platform and Agilent SureSelect version 2 capture kit), with approximately 120X mean coverage. We analyzed the raw data using near-default parameters with five different alignment and variant-calling pipelines . We additionally sequenced a single whole genome using the sequencing and analysis pipeline from Complete Genomics (CG), with 95% of the exome region being covered by 20 or more reads per base. Finally, we validated 919 single-nucleotide variations (SNVs) and 841 insertions and deletions (indels), including similar fractions of GATK-only, SOAP-only, and shared calls, on the MiSeq platform by amplicon sequencing with approximately 5000X mean coverage.SNV concordance between five Illumina pipelines across all 15 exomes was 57.4%, while 0.5 to 5.1% of variants were called as unique to each pipeline. Indel concordance was only 26.8% between three indel-calling pipelines, even after left-normalizing and intervalizing genomic coordinates by 20 base pairs. There were 11% of CG variants falling within targeted regions in exome sequencing that were not called by any of the Illumina-based exome analysis pipelines. Based on targeted amplicon sequencing on the MiSeq platform, 97.1%, 60.2%, and 99.1% of the GATK-only, SOAP-only and shared SNVs could be validated, but only 54.0%, 44.6%, and 78.1% of the GATK-only, SOAP-only and shared indels could be validated. Additionally, our analysis of two families , demonstrated additional accuracy gained in variant discovery by having access to genetic data from a multi-generational family.Our results suggest that more caution should be exercised in genomic medicine settings when analyzing individual genomes, including interpreting positive and negative findings with scrutiny, especially for indels. We advocate for renewed collection and sequencing of multi-generational families to increase the overall accuracy of whole genomes. Recent studies have substantiated the prevalence of rare mutations in the human genome ,2. WholeGiven the existence of multiple sequencing platforms and multiple data-analysis pipelines for next-generation sequencing, researchers and clinicians may be under the impression that these methods all work similarly to identify genetic variants from personal genomes. However, one group recently reported that when variants detected in the same sample by the 1000 genomes project (1 KGP) and the Complete Genomics (CG) platform were compared, 19% of the single-nucleotide variants (SNVs) derived were unique to one dataset . This isDespite these previous studies comparing technical platforms, there have not been many published systematic evaluations of a number of currently used bioinformatics pipelines when generating variant calls from the same set of raw sequence data. Additionally, despite the existence of many variant-calling software tools , their cTo address this issue, we carried out a study of 15 exomes and one whole genome from 15 research participants, analyzing the data with a range of different variant-calling pipelines using near-default parameters. Our results have significant implications for analyzing personal genomes from next-generation sequencing experiments.The collection and genomic analysis of the DNA were approved by the institutional review board at the University of Utah, and written informed consent was obtained from all study participants. Research was carried out in compliance with the Helsinki Declaration.The samples used in our study all came from families of human research participants ascertained in clinics at the University of Utah  to Complete Genomics (CG)  for sequencing. The whole-genome DNA was sequenced using nanoarray-based short-read sequencing by ligation technology , includiExome capture for all 15 samples was carried out using a commercially available in-solution method , following the manufacturer's guidelines. This method is designed to target all human exons, regions totaling approximately 44 Mb, covering 98.2% of the Consensus Coding Sequence (CCDS) database. For the capture, a DNA-shearing instrument  was used to randomly fragment the pure and high molecular weight genomic DNA samples , resulting in DNA fragments with a base-pair peak of 150 to 200 bp. Adaptors were then ligated to both ends of the resulting fragments. The adaptor-ligated templates were purified by magnetic beads , and fragments with an insert size of approximately 250 bp were excised. Extracted DNA was amplified by ligation-mediated (LM)-PCR, purified, and hybridized  for enrichment. Hybridized fragments bound to the strepavidin beads, whereas the unbound non-hybridized fragments were washed out after 24 hours of hybridization. Captured LM-PCR products were analyzed using a microfluidics-based platform  to estimate the magnitude of the enrichment. Paired-end sequencing was performed using a sequencing platform  with average read lengths of 90 bp. Raw image files were processed  for base-calling, using the default parameters. FASTQ files were produced from the pipeline for downstream sequence data analysis. A gender check was compatible with the known genders of the collected human participants.DNA samples were genotyped on the SNP arrays  with approximately 610,000 markers  at the Center for Applied Genomics . Total genomic DNA extracted from whole blood was used in the experiments. Standard data-normalization procedures and canonical genotype-clustering files provided by Illumina were used to process the genotyping signals. Concordance between SNPs from the arrays and SNPs from exome sequencing was determined by calculating the percentage of variants from exome sequencing and comparing this with the same genotype derived from the SNP arrays.http://sourceforge.net). The Picard tool was used to remove duplicate reads. GATK software tools  were used for improvement of alignments and genotype calling and refining with recommended parameters [Burrows-Wheeler aligner  was userameters . BAM filUsing the above BAM files, we used SAMtools (version 0.1.18) to generate genotype calls . The 'mpAdaptor and low-quality sequences were removed before mapping. Sequence reads identified from each individual were then aligned to human reference genome GRCh37 using SOAPaligner (version 2.21 ) with a P < 0.001 were reported. The GNUMAP pipeline cannot currently call indels.Diploid and monoploid SNVs for each individual were called using the GNUMAP pipeline (version 3.1.0 ). GNUMAPBWA  was usedhttp://www.r-project.org, and custom Perl scripts.Post-variant-calling analyses were performed using Golden Helix SVS  containing variants of interest in approximately the center of the amplicon. Primers were obtained in 96-well plate format, 10 μmol/L dilution each . All primers were first tested for PCR efficiency using a HAPMAP DNA sample  and DNA polymerase . k8101-49685 genomic DNA was used as template for the validation experiment. After quality-control steps using agarose gel, the product was purified  and pooled. Final PCR products were quantified , then library construction for the sequencer platform  was performed. Finally, before being loaded onto the MiSeq machine, the quality and quantity of the sample was verified using the Bioanalyzer (Agilent Technologies) and quantitative PCR .Validation variants were randomly selected from sets of particularly controversial variants, indels and SNVs unique to GATK, indels and SNVs unique to SOAP, and variants (both SNVs and indels) shared by these two pipelines. PCR primers were designed using the software program Primer 3 All of the data have been submitted to the Sequence Read Archive under project accession SRP019719, corresponding to the 15 exomes and the single whole genome analyzed during the course of our study  were prepared by exon capture , followed by sequencing on . On average, we obtained sequence coverage of approximately 120X  on targeted regions for these 15 samples. For all samples, sequence reads covered more than 80% of the targeted region with a depth of greater than 20 reads per base  revealed that the exome variant calls had moderate to high depth of coverage  were also within regions targeted for capture by the Agilent SureSelect version 2 kit. Of these, 2,085 (11%) were not called by any of the Illumina-based exome-analysis pipelines, despite computed high mappability scores for these variants  were contained within the union of all five Illumina pipelines , whereas 18,331 of these 35,653 putative Illumina SNVs were not called by CG, suggesting a high false positive rate in the union of the Illumina calls and/or conversely a high false-negative rate in the CG calls  were compared with the CG SNVs, only 64.4% 12507) of CG SNVs were contained within the concordant Illumina set, suggesting a high false-negative rate in this highly concordant Illumina set. Overall agreement decreased as the depth of coverage threshold value for Illumina calls increased, consistent with an increasing false-negative rate  and unique-to-pipeline indels were also compared to CG indel calls, and the concordance rates for each pipeline were similar to those of the unique-to-pipeline only variants , we used the orthogonal approach of PCR amplification of genomic DNA regions containing selected SNVs and indels, followed by pooled MiSeq sequencing. The PCR amplification (instead of exon capture), longer read lengths on the MiSeq platform, and the much higher depth of coverage provided a strong method of validation for SNVs and indels. A total of 1,140 SNVs found in sample k8101-49685 were selected for MiSeq validation; 760 of these SNVs were randomly selected from the set of SNVs that were unique to the GATK version 1.5 and SOAPsnp version 1.03 pipelines, 380 SNVs from each pipeline respectively. An additional 380 SNVs were randomly selected from the set of variants that were in agreement between GATK and SOAPsnp. After some analysis of the quality of the MiSeq data using FASTX, the MiSeq paired-end read data  was trimmed to 150 bp and then aligned with BWA version 0.6.2 to the human reference genome sequence GRCh37, and variants were called with GATK UnifiedGenotyper version 2.3-9.Of the 1,140 SNVs targeted for MiSeq validation, 919 (81.0%) were successfully amplified and sequenced, with an average read depth of 5,392. Validation rates for unique-to-GATK SNVs were high, with 306 of 315  being successfully validated. For unique-to-SOAPsnp, 174 of 289 SNVs (60.2%) were validated. SNVs that were called by both GATK and SOAP were validated in 312 of 315 instances (99.1%) . Of the 960 indels that were targeted for sequencing, 841 (83.5%) were successfully amplified and sequenced, with an average coverage of 4,866.Unique-to-GATK indels had a validation rate of 180 of 336 54.0%), being validated. The validation rate for unique-to-SOAPindel was found to be 44.6%, with 148 of 332 validating. For indels that were called by both SOAPindel and GATK, 132 of 169 (78.1%) were successfully validated  and 87.0%  respectively. However, for indel calls, the picture was quite different, with the GATK UnifiedGenotyper version 2.3-9 and GATK HaplotypeCaller version 2.3-9 modules showing an overall concordance with the GATK UnifiedGenotyper version 1.5 calls of 54.7%  and 54.6%  respectively  are shared among them in each exome, 21,146 on average for SNVs. Although all five pipelines converge on a relatively large number of SNVs, this still represents less than 60% of the total SNV call set determined by all five pipelines, and hence there still exists a considerable degree of variation between pipelines used, with near-default parameterization on the same raw sequencing data. This disagreement is likely to be the result of many factors including alignment methods, post-alignment data processing, parameterization efficacy of alignment and variant-calling algorithms, and the underlying models utilized by the variant-calling algorithm(s).SAMtools, SOAPsnp, and GATK use similar Bayesian methods to compute the posterior probability of the true genotype ,34-37, bdiscordant variants is expected to be low, but not zero. Indeed, for our comparison of SNVs called by SOAP, GATK, or both, MiSeq validation of unique-to-GATK and unique-to-SOAP variants demonstrated relatively high rates of validation, with 306 of 315 of the randomly selected SNVs from unique-to-GATK and 174 of 289 SNVs from unique-to-SOAPsnp being validated. Indels had lower, but still non-zero, validation rates, with 180 of 336 unique-to-GATK indels and 148 of 332 unique-to-SOAPindel indels being validated. Given that 'unique-to-pipeline' variants exist even in regions of relatively high sequence coverage, it is necessary to develop other approaches for including or excluding these variants from downstream analyses.Each variant-calling pipeline detects variants that others do not, and the accuracy of these In the realm of biomedical research, every variant call is a hypothesis to be tested in light of the overall research design. Missing even a single variant can mean the difference between discovering a disease-contributing mutation or not . For thiOne can minimize false positives by increasing stringency filters, but this automatically and correspondingly increases the false-negative rate. The intersection between multiple variant-calling pipelines will minimize the false-positive rate, but we have also shown that each pipeline uniquely identifies some true variants. Hence, clinicians and policy-makers should be aware that propagating forward the intersection of variant calls will result in a high false-negative rate with exome sequencing and WGS, and we discuss below the advantage brought to bear on this issue by studying families. Therefore, processing single sample datasets from one sequencing platform with multiple variant-calling pipelines should not be the long-term solution for generating variant calls with high sensitivity and specificity, and we discuss alternative approaches below.Although the focus of most variant-calling software has been on detecting SNVs, it is the case that large-scale structural copy number variants and small indels are known to also be a biologically relevant and prevalent form of genetic variation ,41. Indeet al. [In contrast to SNVs, few indel-calling tools have been developed, so current knowledge of the existing variation due to indels, as well as the clinical implications of indels, has lagged. In spite of the fact that indel detection is becoming an important aspect of structural-variant analysis , indel cet al.  designedet al.  and repoOur data demonstrates large discrepancies between indel-calling pipelines and suggests potentially high numbers of false positives and/or false negatives. Although putative false positives can be tested via modest resequencing efforts, false negatives and 'no-calls' require large-scale, often impractical, resequencing projects to discover them. Because of this limitation, few data exist on false-negative rates across pipelines, and inferring these rates from unique calls between pipelines is likely to be inadequate. Indel frequency, indel size, read length, and read depth are all known to affect the accuracy of indel-calling pipelines, and the performance of different pipelines also depends, in part, on experimental conditions . We showe novo variants from familial inherited variants may be more strongly limited by high false-negative rates in the parents than by high false-positive rates in the children. This can be significantly improved by having sequence data from one or more grandparents or other relatives. This finding is particularly salient for single-generation de novo studies, which attempt to characterize novel variants that are associated with genetic disease observed in the children of healthy parents [Our analysis of two families, one containing only two generations (parents and children) and the other containing three generations  demonstrates that the ability to accurately distinguish d parents -51. Whil parents -51, stud parents .The relative merits of WGS are expanding as both the cost of the technique decreases and as more scientists and clinicians use the technique in an increasing number of studies/analyses ,30,53,54de novo assembly, similarly to the SOAPindel pipeline used in this study, the new HaplotypeCaller from GATK potentially greatly improves upon its indel-calling accuracy. The more distinct differences observed between GATK indel calls by different versions reflects the fact that indel discovery is in the earlier stages of development, when large differences are often observed between and within pipelines, with accuracy potentially remaining relatively low. For example, in each pipeline, SNV calling relies on set algorithms, which are not dramatically changed in updated versions of the software. Therefore, we do not see great leaps in accuracy for SNVs with newer versions of GATK, despite the fact that we found in the current study that at least one other pipeline (SOAP) did uniquely discover some validated SNVs that were not discovered by any other pipeline or any version of BWA-GATK tested here, and vice versa. We also note that GATK discovered comparatively more unique SNVs not discovered by any other pipeline. One caveat is that we processed the MiSeq data with the newest BWA-GATK pipeline, so this might favor the exome variants previously called by GATK, but it is nonetheless the case that SOAP identified variants that GATK missed in the same exome data using near-default parameters.Many of the most recent advancements that have been made in variant discovery and sequencing analysis are those related to indel discovery and analysis. Indeed, newer versions of GATK have improved upon the false-negative and false-positive rates of their indel calls in both UnifiedGenotyper and with the newer HaplotypeCaller. By leveraging local It is important to note that our study did not examine somatic mutations in tumor samples, so our conclusions and our testing pipelines focus only on germline mutations from diploid genomes. We recognize that variant calling in cancer genomes represents similar but somewhat distinct challenges, and that software tools (such as SNVMix ) are typvice versa [de novo variants. We also note that the standardization of indel discovery and reporting in a way that allows more accurate comparison of indels between sequencing platforms, variant-calling pipelines, and most importantly between individuals in a population is a critical step that needs to be addressed before this functionally important class of variants can be comprehensively assessed in either a research or a clinical setting.We have shown that there remains significant discrepancy in SNV and indel calling between many of the currently available variant-calling pipelines when applied to the same set of Illumina sequence data under near-default software parameterizations, thus demonstrating fundamental methodological variation between these commonly used bioinformatics pipelines. In spite of this methodological variation between pipelines, there exists a set of robust calls that are shared between all pipelines even under lax parameterization. We have further shown that the relatively recent CG version 2.0 WGS pipeline detects a set of exon variants that are not detected by several variant-calling pipelines with an Illumina-based exon-capture sequencing strategy, even in regions of high mappability . Therefoce versa . We haveBAM: Binary alignment map; CG: Complete Genomics; CNV: Copy number variant; eir: Equivalent indel region; Indel: Small insertion/deletion; NGS: Next-generation sequencing; SAM: sequence alignment map; SNP: Single-nucleotide polymorphism; SNV: Single-nucleotide variant; VCF: Variant call format; WGS: Whole-genome sequencing.The authors declare that they have no competing interests.JO, TJ, WW, LT, PB, ZW and WEJ analyzed the exome and SNP genotyping data. TJ, GS, JH and colleagues at BGI performed all exome sequencing, Sanger sequencing and Sequenom genotyping. YW performed the PCR and MiSeq validation experiments. JO analyzed the MiSeq data and Complete Genomics data, and wrote the manuscript. GJL and KW conceived of the project, supervised all data analysis, and wrote and edited the manuscript. All authors reviewed and commented on the manuscript. All authors read and approved the final manuscript.The following additional data are available with the online version of this paper.Additional file Figure S1, Family pedigrees contained within the 15 sequenced exomes. Of the fifteen exomes that were sequenced, 14 were sequenced from families chosen for future disease discovery related work. Each sequenced individual (numbered) is displayed in the context of his or her constituent family pedigree.Figure S2, Fraction of target capture region covered versus coverage depth for 15 exomes. All exomes have at least 20 reads or more per base pair in > 80% or more of the 44 MB target region.Figure S3, Histograms of Illumina read depth at SNV coordinates. Read depth taken from each pipeline's independently aligned BAM file at genomic coordinates of SNVs called by each of the 5 alignment and variant calling pipelines. A) SOAPsnp, B) SNVer, C) SAMtools, D) GNUMAP and E) GATK, respectively. Frequency of read depths for all SNVs  as well as for SNVs having depths between 0 and 50  were plotted.Figure S4, SNV concordance measured at varying Illumina read depth threshold values. SNV concordance for a single exome, "k8101-49685", between five alignment and variant detection pipelines: GATK, SOAPsnp, SNVer, SAMtools, and GNUMAP. Concordance between each pipeline was determined by matching the genomic coordinate as well as the base pair change and zygosity for each detected SNV. Concordance was measured at varying Illumina read depth threshold values in each independently aligned BAM file, ranging from > 0 (no threshold) to > 30 reads.Figure S5, Histograms of illumina read depth at genomic coordinates of the unique to Complete Genomics SNV calls. Histograms of read depth taken from each of the five Illumina pipeline's independently aligned BAM file at genomic coordinates of SNVs that were found by Complete Genomics but not by any of the 5 Illumina pipelines: GATK, GNUMAP, SNVer, SAMtools and SOAPsnp, A, B, C, D and E respectively. All coordinates fell within the range of the Agilent SureSelect v.2 exons.Figure S6, SNV concordance for a single exome, "k8101-49685", between two sequencing pipelines: Illumina and Complete Genomics. For the Illumina sequencing, exons were captured using the Agilent SureSelect v.2 panel of capture probes. Complete Genomics SNVs consist of a subset of all SNVs called by CG that fell within the Agilent SureSelect v.2 exons. Concordance was determined by matching the genomic coordinates, base pair composition, and zygosity status for each detected SNV. Concordance was measured between CG SNVs and A) the union of all SNVs called by 5 variant calling pipelines  and B) only SNVs that all 5 Illumina pipelines collectively called .Figure S7, Cross-validation of illumina SNV calls using Complete Genomics SNV calls. SNVs called by each Illumina-data pipeline were cross-validated using SNVs called by Complete Genomics, an orthogonal sequencing technology, in sample "k8101-49685". The percentage of Illumina SNVs that were validated by CG sequencing was measured for variants having varying degrees of Illumina-data pipeline concordance. The same analysis was performed for variants that were considered novel (absent in dbSNP135).Figure S8, Average indel concordance among 15 exomes between three indel detecting pipelines: GATK, SAMtools and SOAPindel. Concordance was measured between raw, pre-standardized, indel calls. Indels were considered in agreement if the genomic coordinates, length and composition of indels matched between pipelinesFigure S9, Cross-validation of illumina indel calls using Complete Genomics indel calls. Indels called by each Illumina-data pipeline were cross-validated using indels called by Complete Genomics for sample "k8101-49685". The percentage of Illumina indels that were validated by CG sequencing was measured across varying degrees of Illumina pipeline concordance. The same analysis was done for novel indels (indels not found in dbSNP 135).Figure S10, A comparison between recent versions of various GATK variant calling modules. The similarity between SNV and indel calls made between two versions of GATK, v1.5 and v2.3-9, was measured. SNV and indel calls were made using both the UnifiedGenotyper and HaplotypeCaller modules on the same k8101-49685 participant sample. Pairwise comparisons were made between the GATK UnifiedGenotyper v1.5 and each of the GATK v2.3-9 modules .Table S1, Concordance rates with common SNPs genotyped on Illumina 610K genotyping chips.Additional file Additional file Figure S1-S7.Click here for fileCommand-line arguments for bio-informatics pipelines and instructions for accessing data analyzed in this paper.Click here for fileData production statistics.Click here for file


Both transcriptional control and microRNA (miRNA) control are critical regulatory mechanisms for cells to direct their destinies. At present, the combinatorial regulatory network composed of transcriptional regulations and post-transcriptional regulations is often constructed through a forward engineering strategy that is based solely on searching of transcriptional factor binding sites or miRNA seed regions in the putative target sequences. If the reverse engineering strategy is integrated with the forward engineering strategy, a more accurate and more specific combinatorial regulatory network will be obtained.In this work, utilizing both sequence-matching information and parallel expression datasets of miRNAs and mRNAs, we integrated forward engineering with reverse engineering strategies and as a result built a hypothetical combinatorial gene regulatory network in human cancer. The credibility of the regulatory relationships in the network was validated by random permutation procedures and supported by authoritative experimental evidence-based databases. The global and local architecture properties of the combinatorial regulatory network were explored, and the most important tumor-regulating miRNAs and TFs were highlighted from a topological point of view.By integrating the forward engineering and reverse engineering strategies, we manage to sketch a genome-scale combinatorial gene regulatory network in human cancer, which includes transcriptional regulations and miRNA regulations, allowing systematic study of cancer gene regulation. Our work establishes a pipeline that can be extended to reveal conditional combinatorial regulatory landscapes correlating to specific cellular contexts. Transcriptional factors (TF) and microRNAs (miRNAs) are important regulation factors to determine the expression levels of mRNAs and miRNAs . TFs actDatabases, such as TransFAC  on TF-toReverse engineering has been put into effect in inferring TF-controlled transcriptional regulation networks -13 as wePreviously, we integrated forward predicted gene regulation relationships with miRNA-perturbed gene expression datasets (MPGE datasets) and as a result elucidated miRNA-centered primary and secondary regulatory cascades in human cancer by using nonparametric test and linear regression modeling . ConfineIn the present work, we demonstrated an efficient integration of the forward-predicted candidate regulatory relationships with the NCI60 panel of parallel miRNA and mRNA expression datasets, giving rise to a genome-scale combinatorial network of transcriptional regulations and miRNA regulations in human cancer. The resultant combinatorial regulatory network makes a scaffold for systematic study of cancer gene regulation, and the demonstrated working pipeline can be extended to reveal conditional combinatorial regulatory landscapes in other cellular contexts.http://discover.nci.nih.gov/cellminer/), we downloaded the NCI-60 mRNA expression dataset assayed with the Affymetrix HG-U133 44 K probeset microarray [From the NCI-60 data source CellMiner , we obtained 118,408 human candidate miRNA-gene regulatory relationships involving 276 human microRNAs and 10,255 targets, which were a union of results from three forward predicting algorithms: PicTar [From the miRGen website  and TRED (http://rulai.cshl.edu/TRED/), which included 130,338 binary tuples involving 214 human TFs and 16,534 targets. For more information, please refer to our related work [A set of forward predicted TF-gene regulatory relationships were compiled by merging records from UCSC , its expression level , rt (a miRNA or a TF) which regulates the target t. ‘Intercept’ is a constant, and err, following a normal distribution with a zero mean, captures the variation of gene t's mRNA level that cannot be interpreted by its regulators.The simplicity of the linear regression model has led many groups to employ it to reveal the gene network from gene expression data (for a review please see ), and it1, α1 = 0.05) with their putative targets were disconnected with their putative targets. With regards to miRNA-to-target regulations, specially, we only kept the ones with negative regulation efficacy After regression of Equation 1, TFs and miRNAs without statistically significant relationship ; correspondingly, g, and The expression level  of a mRNA target blockage .m, Similarly, the expression level  of a miRNA target α1) in the pre-filtering step (Equation 1), and the stepwise linear regression was implemented to determine the ultimate regulators of a particular target. As we did for Equation 1, if a positive miRNA-to-target regulation efficacy appears in the final regression model of Equation 2, all regulations reserved in the model for the same target gene g were eliminated.In brief, expression levels of mRNAs (Equation 2) and miRNAs (Equation 3) were modeled by the regulators that passed the statistical test (p < =miRNAII.regression) is provided in the supplementary source codes , we culled a subset of regulatory relationships that were hopefully more plausible than the beginning set of forward-predicted regulatory relationships. These remaining regulator-target relationships make up a combinatorial network of transcriptional regulations and miRNA regulations in human cancer, which involves 3418 vertices and 5136 edges. The full information on the edges and vertices are included in Additional file To estimate the false discovery rate of our predicted regulatory relationships, we randomly permuted the expression values within each expression dataset and conducted the linear regression procedure  over the randomized miRNA/mRNA expression datasets for 100 times. The regulation edges in the combinatorial networks resulting from the randomized datasets were deemed as false discoveries. In this way, we estimated that the overall false discovery rate (FDR) of the regulation relationships remaining in the human cancer combinatorial gene regulatory network were 17.2% . While in real work we made use of all 59 expression data-points of a gene, in the jack-knife like evaluation procedure we excluded one data-point from the modeling  and used the fitted model (Equation 2/Equation 3) to predict the left-out data-point. For each target gene we had 59 iterations, and we calculated the Pearson correlation coefficient (PCC) between the measured expression values and the predicted ones. As a result, significantly higher PCCs were obtained with real expression data than with randomly permuted data , 197 overlapped the 8,388 frequently expressed genes of the NCI-60 mRNA expression dataset, of which 121 were found in the 3,259 genes remaining in the resultant regulatory network  , and fouhttp://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE6838) recording the gene expression changes in cells transfected with hsa-miR-106b, and 21 were among the top 5% down-regulated genes , and obtained 17 TF-TF pairs, 46 miR-miR pairs and 17 TF-miR pairs  and ‘feed-backward-loops’ (FBLs) by considering the connecting ways of directed regulations . PreviouOf the 18 motifs being surveyed, one FBL and four FFLs turned out to be significantly recurrent  to elucidate the miRNA-centered primary and secondary regulatory cascades in human cancer, which encompassed two types of mRNA regulation, miRNA-to-mRNA and TF-to-mRNA . By intrIn human cancer, miRNAs are presumed to preferentially couple its post-transcriptional inhibition with TF-initiated transcription in combinatorial regulatory circuits ,54. Our While the overall working pipeline turns out to be effective in this work, some specific steps need to be discussed. For example, the prefiltration step (Equation 1) is incurred primarily for reducing computation complexity of the following steps (Equation 2/Equation 3) or circumventing the n < p problem . Taking into account the forthcoming reduction in microarray or RNA-seq costs, the number of samples in future parallel expression datasets may be larger, which may greatly alleviate the pre-filtration pressure. For another example, doubts have been arising with regards to the stepwise (STEP) linear regression , and theTowards the ultimate goal of ‘differential networking’ analysis , we madeIn this work, we made an attempt to integrate the forward engineering and reverse engineering strategies and for the first time resulted in a global landscape of combinatorial gene regulatory network in a specific biological context (human cancer) that has a moderate false discovery rate and is enriched with confirmed regulations. The human cancer combinatory gene regulatory network is found to be a hierarchical scale-free network with MYC, hsa-miR-106b and has-let-7c being the most important regulators. From the network, 17 TF-TF pairs, 46 miR-miR pairs and 17 TF-miR pairs are identified as the significantly co-regulating regulator pairs, and four triple-vertex regulatory circuits (one FBL and three FFLs) turn out as significantly recurrent building motifs. We believe our work provides a scaffold combinatorial gene regulatory network allowing systematic study of cancer gene regulation, and that our pipeline can be extended to reveal conditional combinatorial regulatory landscapes correlating to specific cellular contexts.The authors’ declare that they have no competing interests.HY and KT contributed to the design and conception of the study, conducted computational experiments, analyzed and interpreted data and drafted the manuscript. YJW and JZM joined in the processing of data materials and wrote part of the computer codes. LX, YYL and YXL conceived of the project and participated in its design, helped to analyze and interpret the data and drafted the manuscript. All authors have read and approved the manuscript for publication. How we compiled TF-to-miRNA regulation relationships for intergenicmiRNAs.Click here for fileThe source codes/R functions for this study.Click here for fileTable S1The edges of the human cancer combinatorial gene regulation network.Click here for fileTable S2The vertices of the human cancer combinatorial gene regulation network.Click here for fileThe evaluation of predictability of our linear regression models.Click here for fileValidation of part of predicted hsa-miR-106b targets by a miRNA-transfection dataset GSE6838.Click here for fileTable S3. Significantly coordinating regulator pairs from the human cancer combinatorial regulatory network.Click here for file


Chest pain and other symptoms that may represent acute coronary syndromes (ACS) are common reasons for emergency department (ED) presentations, accounting for over six million visits annually in the United States [1]. Chest pain is the second most common ED presentation in the United States. Delays in diagnosis and inaccurate risk stratification of chest pain can result in serious morbidity and mortality from ACS, pulmonary embolism (PE), aortic dissection and other serious pathology. Because of the high morbidity, mortality, and liability issues associated with both recognized and unrecognized cardiovascular pathology, an aggressive approach to the evaluation of this patient group has become the standard of care. Clinical history, physical examination and electrocardiography have a limited diagnostic and prognostic role in the evaluation of possible ACS, PE, and aortic dissection, so clinicians continue to seek more accurate means of risk stratification. Recent advances in diagnostic imaging techniques particularly computed-tomography of the coronary arteries and aorta, have significantly improved our ability to diagnose life-threatening cardiovascular disease. In an era where health care utilization and cost are major considerations in how disease is managed, it is crucial to risk-stratify patients quickly and efficiently. Historically, biomarkers have played a significant role in the diagnosis and risk stratification of several cardiovascular disease states including myocardial infarction, congestive heart failure, and pulmonary embolus. Multiple biomarkers have shown early promise in answering questions of risk stratification and early diagnosis of cardiovascular pathology however many do not yet have wide clinical availability. The goal of this review will be to discuss these novel biomarkers and describe their potential role in direct patient care.  IScrutiny of new biomarkers must include validating analytical imprecision and detection limits, calibrator characterization, assay specificity and standardization, pre-analytical issues, and appropriate reference interval studies . An ideaIIInterest in inflammatory biomarkers is a result of the emerging appreciation of inflammation as a key part of the pathophysiologic sequence in the development of both coronary artery disease (CAD) and ACS. The role of C-reactive protein (CRP) in CAD has garnered significant attention over the past decade. Used as a marker of systemic inflammation, elevated serum levels of CRP have been associated with a wide range of detrimental outcomes, and are often clinically used to guide risk factors modification of stable coronary artery disease (CAD). After the publication of the JUPITER trial, the cardiology community has been split between the proponents of CRP as a mere serological biomarker, and those that argue for its pathophysiologic role in the development of coronary atherosclerosis . In eithThe prognostic value of CRP extends beyond presentations of ACS, as it correlates closely with worsened outcomes in patients presenting with an aortic dissection or with an abdominal aortic aneurysm , 11. OneInterleukins (IL) are major cytokines that may have clinical applications in the evaluation of potential ACS. IL-6 is a multifunctional cytokine and it plays a central role in inflammation and tissue injury. It acts as a surrogate marker of vessel wall inflammation that eventually leads to atherosclerotic plaque formation. However, there is limited evidence to evaluate the role of IL-6 as a screening tool for chest pain. Ridker and colleagues studied the plasma concentration of IL-6 and the risk of future MI in 202 healthy men during a 6-year follow-up . Median Similar to IL-6, elevated levels of serum IL-18 may be an independent predictor of higher cardiovascular mortality, especially among patients with either unstable or stable angina. There is evidence in animal models that IL-18 is associated with increased progression of atherosclerosis and worsened plaque instability. This may explain the results of a recent prospective study of 1229 patients with CAD. Median serum levels of IL-18 were significantly higher among patients who had a fatal cardiovascular event than among those who did not (p<0.0001); the hazard risk ratio remained almost unchanged even after adjusting for potential confounders . Serum IBalancing IL-18, Interleukin IL-10 is a cytokine that has anti-inflammatory and immunosuppressant properties. In the CAPTURE (c7E3 AntiPlatelet Therapy in Unstable Refractory angina) study, patients with elevated IL-10 levels had a decreased risk of death or nonfatal MI. Not surprisingly then, serum IL-18/IL-10 ratio appears to be an independent predictor of recurrent coronary events during long-term follow-up in patients presenting with ACS. In a study done to assess the long-term prognostic significance of admission interleukin-18 (IL-18)/interleukin-10 (IL-10) ratio for recurrent coronary events during a 1-year follow-up in patients presenting with an ACS, it was concluded that serum IL-18/IL-10 ratio is an independent predictor of recurrent coronary events during long-term follow-up in patients presenting with ACS . The stuLipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme mainly produced by monocytes and macrophages, generates potent pro-inflammatory products. The sub-endothelial oxidation of low-density lipoprotein (LDL) is viewed as a highly significant biological process that both initiates and accelerates arterial lesion development. Thus, preventing the generation of these mediators through inhibition of Lp-PLA2 is believed to retard atherosclerosis. Consistent with this notion is the recent observation that plasma levels of Lp-PLA2 represent an independent predictor of coronary heart disease and ischemic stroke. In the Rotterdam Study, a population-based follow-up study of 7983 subjects over 55 years old, there was a strong relation between Lp-PLA2 activity and coronary heart disease. Compared with the first quartile of Lp-PLA2 activity, multivariate-adjusted hazard ratios for coronary heart disease and ischemic stroke for the remaining quartiles were significant (P for trend< 0.05) . This stA case cohort study of 12,819 apparently healthy middle-aged men and women (the Atherosclerosis Risk in Communities study) examined the relation between Lp-PLA2, CRP, traditional risk factors, and risk for CAD events over a six-year period . Lp-PLA2The leukocyte enzyme myeloperoxidase (MPO) is secreted during acute inflammation and causes oxidation of lipoproteins that is linked with the presence of coronary disease. A case-control study of 158 patients with established CAD and 175 patients without CAD found that MPO levels were significantly greater in patients with CAD than in controls (P<0.001). Elevated levels of both leukocyte- and blood-MPO were associated with the presence of CAD. These findings support a potential role for MPO as an inflammatory marker in CAD and may have implications for early atherosclerosis diagnosis and risk assessment. More importantly, high levels of MPO may also predict the presence of acute coronary syndrome in patients with chest pain. In a prospective study conducted in 600 patients presenting with chest pain in the ED, plasma levels of MPO predicted the risk of MI even in patients that were negative for troponin T at baseline . In patiAtherosclerotic plaque inflammation is critical to the pathophysiology of ACS, and oxidized low-density lipoprotein (oxLDL) is thought to be a key mediator of this process -22. OxLDThe MIRACL study sought to define the relationship between oxidative biomarkers and CVD risk factors . The study measured oxLDL at baseline and 16 weeks of treatment with 80 mg Atorvastatin in over 2300 patients with ACS. In these patients, baseline levels of oxidative biomarkers varied according to specific CVD risk factors and were largely independent of inflammatory biomarkers. Atorvastatin administration led to a decrease in total oxidized phospholipids in ACS patients. Future studies are warranted to assess whether decrease in oxLDL levels correspond with improved cardiovascular outcomes.Microscopic study of atherosclerotic plaque (both coronary and aortic) in humans has demonstrated excess matrix degrading activity, suggesting that elevated levels of matrix metalloproteinase (MMP) might act as a surrogate marker of atherosclerotic plaque burden. Clinically however, elevated levels of MMP have demonstrated no utility in evaluation of ACS patients , though III.et al. demonstrated that the test had over 90% sensitivity for detecting heart failure in patients with acute dyspnea presenting to the ED when compared to a cardiologist who had access to the patient’s entire medical record [Brain natriuretic peptide (BNP) level measurement was initially studied as a tool for differentiating cardiac from non-cardiac causes of dyspnea. In a landmark 2002 study, Maisel l record . The util record . Based ol record . Of noteBNP has also demonstrated some utility as a prognostic marker of CHF; inpatient mortality is related to admission levels of BNP in a linear manner ,30. It mIn recent years, troponin has become the gold standard in detection of MI over its predecessors, CK-MB, CPK and AST. Its efficacy in diagnosis of MI has been well-validated. As test methods improved, the superiority of cTroponin I (cTnI) over T was established and as sensitivity for each assay increased, specificity for acute coronary syndrome has been called more and more into question , 33. A net al published a study not included in the analysis above, and studied this potential in a prospective cohort study of normotensive patients who were confirmed to have PE [et al [et al found a two-fold increase in adverse outcome for every standard deviation increase in hs-cTnT [Although the role of troponin in the identification of myocardial infarct has been clearly established, its use in detection and prognostication in the setting of PE is emerging as a potential tool in PE management. In a meta-analysis of 20 studies and 1985 patients, the investigators looked at the predictive value of troponin T and I on patients admitted to the hospital with PE . The pri have PE . Their rE [et al . Interes hs-cTnT . IVAfter clot formation, fibrinolysis of said clot leads to formation of fibrin split products, in particular D-dimer. The utility of this protein fragment has been well validated in the past for ruling out PE or DVT in patients with low or medium pretest probability. More recently, the role of D-dimer has been further established in the setting of aortic dissection as well as PE clot burden, location of PE and prognosis.et al. found that in patients with confirmed PE, D-dimer >3000 ng/mL are associated with increased death at 15 days, at 3 months, as well as a more centralized location of PE [In consecutive patients presenting to the ED, levels of D-dimer have been significantly associated with prognosis. Klok on of PE . A direcon of PE , 41.Interestingly, in hospitalized patients who are already hemodynamically stable, elevated D-dimer in PE does not have any prognostic value up to at least three months after diagnosis . These cet al. reported that patients presenting to the ED for chest pain with a D-dimer >0.8 g/mL and widened mediastinum had a sensitivity and specificity of 93.1% and 91.8%. [et al., patients presenting for suspected aortic dissection with D-Dimer values <0.9 ug/mL had a negative predictive value of 92% and for <0.1 umg/mL, 100% [PE is not the only life-threatening condition that can be ruled out with D-dimer. Emerging evidence suggests D-dimer can also greatly reduce post-test probability of aortic dissection as well. As intima is exposed, coagulation ensues with resultant liberation of fibrin split products. Hazui d 91.8%. . NegativmL, 100% . et al., 4018 pts from 18 prospective studies were evaluated for an association between fibrinogen and future vascular events [As inappropriate hemostasis can lead to coronary thrombosis unexpectedly, elevated levels of certain coagulation factors can be implicated in future thrombotic events. In a meta-analysis by Danesh r events . In patir events .As platelet activation is intimately related to thrombosis during ACS, it would stand to reason that markers of platelet activation might be useful in predicting outcomes after ACS. In the past, von Willbebrand’s Factor (vWF) has been studied as a marker of potential treatment selection and therapeutic target. Additionally, its potential has been explored as a prognosticator for adverse outcomes. et al. evaluated levels of vWF in consecutive patients presenting for ST elevation MI (STEMI) or Q-wave MI at admission and 24 hours later [Collet rs later . In thisrs later . It was In the ESSENCE trial, vWF was investigated along with several other factors in patients with unstable angina and q-wave infarction . The autet al. measured levels in patients presenting for severe UA or NSTEMI and demonstrated that sVCAM-1 was significantly elevated at presentation in those who later experienced major adverse cardiac events  during their hospitalization, which was independent of CRP level or classic risk factors [Similar to vWF, the potential for detection of disease and risk stratification in soluble vascular cell adhesion molecule-1 (svCAM-1) is related to the pathogenesis of thrombosis. Rallidis  factors . There wSoluble CD40 ligand (sCD40L) is involved in a variety of processes from atherogenesis to plaque destabilization and can be detected before and during MI. SCD40L is produced in a multitude of cells, most notably in endothelium, activated platelets and activated leukocytes. Although it is not yet a widely available lab test, preliminary evidence indicates potential for treatment selection, risk stratification and prognostication . et al., patients with angiographically confirmed coronary artery disease who presented with unstable angina were randomized to treatment with dalteparin or placebo 18-24 hours before angioplasty [In a trial by Heeschen ioplasty . The priet al. found similar results in patients presenting for suspected NSTEMI [Malarstig d NSTEMI . In thiset al. conducted a prospective, nested case-control study in which they studied 130 women who died of MI, stroke or other cardiovascular cause [et al., in which they sought to find associations with several biomarkers and adverse outcomes during and after acute coronary syndromes [Schonbeck ar cause . After byndromes . SCD40L Growth factors have been getting significantly more attention in recent years given the importance of angiogenesis in collateralization during progression of atherosclerosis. The most studied factors as pertains to coronary disease are vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and hepatocyte growth factor (HGF). et al. found that in patients presenting with ACS, VEGF levels > 300 ug/L were associated with a hazard ratio of 1.87 (CI 1.03-3.51) for MI and death during 6 months of follow up [VEGF produced in a variety of processes and in the setting of coronary disease has been used to predict future adverse events. Baldus ollow up . et al., serum was obtained from 547 patients admitted for ACS [et al. over a 6-month period, as well as a separate prospective trial by Apple et al. (despite the use of a lower PlGF level of 17 ng/L) [In a paper by Heeschen  for ACS . The rat17 ng/L) ,56. et al. found that in patients who presented with unstable angina, HGF levels > 4.7 ug/L were associated with a protective effect at 72 hours, 30 days and 6 months with HR of 0.29 (CI 0.16-0.53), 0.30 (CI 0.18-0.52) and 0.33 (CI 0.21-0.51), respectively [HGF, a potent angiogenic agent related to recovery after MI, may carry prognostic information as well. Heeschen ectively . This maVAs the number of cardiovascular biomarkers grows, it is critical to understand each one’s specific strengths and limitations. Furthermore, it is critical that all biomarkers are not used as stand-alone tests. They must be interpreted in their appropriate clinical context and do not replace other parts of the examination such as physical examination or imaging modalities. Despite the large number of existing and novel biomarkers for cardiovascular disease, no one marker has enough sensitivity and specificity to be evaluated in isolation. Therefore a multi-biomarker strategy is likely to be the most useful for clinical decision-making. In fact, most patients with ACS have multiple processes occurring simultaneously and these biomarkers can help detect distinct points in the pathway of development of ACS.