Increased intracranial pressure in a patient with Congenital Heart Defect and Ectodermal Dysplasia (CHDED): Extension of phenotype and review of literature.

Congenital heart defect (CHD) is a birth defect that affects the structure of the heart. Although CHD is often multifactorial, it can also be inherited as part of a Mendelian disorder such as in congenital heart defect and ectodermal dysplasia (CHDED). This disorder is caused by de novo variants in PRKD1. Here, we describe a patient with a novel de novo variant of PRKD1 with phenotypic features consistent with CHDED. Previously unreported features were noted including high intracranial pressure (ICP), partial anomalous pulmonary venous return (PAPVR), and bifid uvula. We suggest that these features may be associated with CHDED.

Current status of cardiopulmonary resuscitation training and automatic external defibrillator availability in high schools in Halifax, Nova Scotia, Canada.

Background: School-based cardiopulmonary resuscitation (CPR) training and early use of an automated external defibrillator (AED) have proven to increase the survival of victims of sudden cardiac arrest (SCA). This study aimed to determine the status of CPR training, availability of AEDs, and medical emergency response programs (MERPs) in high schools in Halifax Regional Municipality. Method: High school principals were asked to participate in a voluntary online survey comprising questions about demographics, AEDs availability, CPR training for staff and students, the existence of MERPs, and perceived barriers. Three autogenerated reminders followed the initial invitation. Results: Out of 51 schools, 21 (41%) responded, only 10% (2/21) and 33% (7/21) reported providing CPR training to students and staff, respectively. About 35% (7/20) of the schools reported having AEDs, but only 10% (2/20) have MERPs for SCA. All respondents reported in favor of AED availability in schools. The reported barriers to CPR training included limited financial resources (54%), perception of low priority (23%), and time constraints (23%). Respondents reported limited financial resources (85%) and the lack of trained staff to use (30%) as the main reasons for the unavailability of AEDs. Conclusion: This survey showed that all respondents overwhelmingly favour having access to AEDs. However, the availability of CPR and AED training for staff and students in schools remains inadequate. Emergency action plans have not been devised, and few schools have AED devices. More education and awareness are needed to ensure lifesaving equipment and practices in all Halifax Regional Municipality schools.

An epigenome-wide association study of total serum immunoglobulin E concentration.

Immunoglobulin E (IgE) is a central mediator of allergic (atopic) inflammation. Therapies directed against IgE can alleviate hay fever and allergic asthma. Genetic association studies have not yet identified novel therapeutic targets or pathways underlying IgE regulation. We therefore surveyed epigenetic associations between serum IgE concentrations and methylation at loci concentrated in CpG islands genome wide in 95 nuclear pedigrees, using DNA from peripheral blood leukocytes. We validated positive results in additional families and in subjects from the general population. Here we show replicated associations--with a meta-analysis false discovery rate less than 10(-4)--between IgE and low methylation at 36 loci. Genes annotated to these loci encode known eosinophil products, and also implicate phospholipid inflammatory mediators, specific transcription factors and mitochondrial proteins. We confirmed that methylation at these loci differed significantly in isolated eosinophils from subjects with and without asthma and high IgE levels. The top three loci accounted for 13% of IgE variation in the primary subject panel, explaining the tenfold higher variance found compared with that derived from large single-nucleotide polymorphism genome-wide association studies. This study identifies novel therapeutic targets and biomarkers for patient stratification for allergic diseases.

Female and male perspectives on male partner roles in expanded carrier screening.

PURPOSE: To explore facilitators and barriers for male partner follow through carrier screening (CS) after their female partners were identified as carriers, from both male and female perspectives. METHODS: Participants were either females identified as a carrier through CS (512 participants) or males who had CS (125 participants). Participants were recruited via e-mails with survey links. The survey explored factors surrounding decisions to pursue CS or not. RESULTS: Males who attended the females' CS appointment were more likely to have CS (OR: 2.07). More male partners of females identified as carriers of severe or profound conditions pursued CS (82.0%) than male partners of females who were carriers for moderate conditions (50.0%). Logistic factors were more impactful for males who pursued CS. Females whose male partners did not test endorsed personal belief factors as most impactful, reporting the perceived low risk (75.0%) and his low concern for the specific condition (65.5%) were the top reasons their partners did not test. CONCLUSION: Many factors impact how male partners appraise reproductive risk from CS and make decisions regarding their own screening. Advising that male partners attend CS appointments may increase the likelihood of follow through CS. Thorough and repeated risk counseling is indicated.

Preparing for CBME: How often are faculty observing residents?

BACKGROUND: The Royal College of Physicians and Surgeons of Canada officially launched 'Competence by Design' in July 2017, moving from time-based to outcomes-based training. Transitioning to competency-based medical education (CBME) necessitates change in resident assessment. A greater frequency of resident observation will likely be required to adequately assess whether entrustable professional activities have been achieved. PURPOSE: Characterize faculty and resident experiences of direct observation in a single paediatric residency program, pre-CBME implementation. Qualitatively describe participants' perceived barriers and incentives to participating in direct observation. METHODS: Surveys were sent to paediatric residents and faculty asking for demographics, the frequency of resident observation during an average 4-week rotation, perceived ideal frequency of observation, and factors influencing observation frequency. Descriptive data were analyzed. Institutional research ethics board approval was received. RESULTS: The response rate was 54% (34/68 faculty and 16/25 residents). When asked the MAXIMUM frequency FACULTY observed a resident take a history, perform a physical examination, or deliver a plan, the median faculty reply was 1, 2, and 3, for outpatient settings and 0, 1, and 2, for inpatient settings. The median RESIDENT reply was 2, 4, and 10 for outpatient settings and 1, 2, and 20 for inpatient settings. When asked the MINIMUM frequency for each domain, the median FACULTY and RESIDENT reply was 0, except for delivering a plan in the inpatient setting. Faculty reported observing seniors delivering the plan more frequently than junior residents. Faculty and resident median replies for how frequently residents should be observed for each domain were the same, three to four, three to four, and five to six times. Four per cent of faculty reported regularly scheduling observations, and 77% of residents regularly ask to be observed. The most common barriers to observation were too many patients to see and both faculty and residents were seeing patients at the same time. Most faculty and resident responders felt that observation frequency could be improved if scheduled at the start of the rotation; faculty were provided a better tool for assessment; and if residents asked to be observed. CONCLUSIONS: This study provides baseline data on how infrequent faculty observation is occurring and at a frequency lower than what faculty and residents feel is necessary. The time needed for observation competes with clinical service demands, but better scheduling strategies and assessment tools may help.

Systematic design and comparison of expanded carrier screening panels.

PurposeThe recent growth in pan-ethnic expanded carrier screening (ECS) has raised questions about how such panels might be designed and evaluated systematically. Design principles for ECS panels might improve clinical detection of at-risk couples and facilitate objective discussions of panel choice.MethodsGuided by medical-society statements, we propose a method for the design of ECS panels that aims to maximize the aggregate and per-disease sensitivity and specificity across a range of Mendelian disorders considered serious by a systematic classification scheme. We evaluated this method retrospectively using results from 474,644 de-identified carrier screens. We then constructed several idealized panels to highlight strengths and limitations of different ECS methodologies.ResultsBased on modeled fetal risks for "severe" and "profound" diseases, a commercially available ECS panel (Counsyl) is expected to detect 183 affected conceptuses per 100,000 US births. A screen's sensitivity is greatly impacted by two factors: (i) the methodology used (e.g., full-exon sequencing finds more affected conceptuses than targeted genotyping) and (ii) the detection rate of the screen for diseases with high prevalence and complex molecular genetics (e.g., fragile X syndrome).ConclusionThe described approaches enable principled, quantitative evaluation of which diseases and methodologies are appropriate for pan-ethnic expanded carrier screening.

Clinical Utility of Expanded Carrier Screening: Reproductive Behaviors of At-Risk Couples.

Expanded carrier screening (ECS) analyzes dozens or hundreds of recessive genes to determine reproductive risk. Data on the clinical utility of screening conditions beyond professional guidelines are scarce. Individuals underwent ECS for up to 110 genes. Five-hundred thirty-seven at-risk couples (ARC), those in which both partners carry the same recessive disease, were invited to participate in a retrospective IRB-approved survey of their reproductive decision making after receiving ECS results. Sixty-four eligible ARC completed the survey. Of 45 respondents screened preconceptionally, 62% (n = 28) planned IVF with PGD or prenatal diagnosis (PNDx) in future pregnancies. Twenty-nine percent (n = 13) were not planning to alter reproductive decisions. The remaining 9% (n = 4) of responses were unclear. Of 19 pregnant respondents, 42% (n = 8) elected PNDx, 11% (n = 2) planned amniocentesis but miscarried, and 47% (n = 9) considered the condition insufficiently severe to warrant invasive testing. Of the 8 pregnancies that underwent PNDx, 5 were unaffected and 3 were affected. Two of 3 affected pregnancies were terminated. Disease severity was found to have significant association (p = 0.000145) with changes in decision making, whereas guideline status of diseases, controlled for severity, was not (p = 0.284). Most ARC altered reproductive planning, demonstrating the clinical utility of ECS. Severity of conditions factored into decision making.

Pulse oximetry screening in newborns to enhance detection of critical congenital heart disease.

Pulse oximetry screening is safe, noninvasive, easy to perform and proven to enhance detection of critical congenital heart disease in newborns. However, this test has yet to be adopted as routine practice in Canada. The present practice point highlights essential details and recommendations for screening, which research has shown to be highly specific, with low false-positive rates. Optimal screening for critical congenital heart disease should include prenatal ultrasound, physical examination and pulse oximetry screening. Screening should be performed between 24 hours and 36 hours postbirth, using the infant's right hand and either foot to minimize false-positive results. Newborns with abnormal results should undergo a thorough evaluation by the most responsible health care provider. When a cardiac diagnosis cannot be excluded, referral to a paediatric cardiologist for consultation and echocardiogram is advised.

Predicting DNA methylation level across human tissues.

Differences in methylation across tissues are critical to cell differentiation and are key to understanding the role of epigenetics in complex diseases. In this investigation, we found that locus-specific methylation differences between tissues are highly consistent across individuals. We developed a novel statistical model to predict locus-specific methylation in target tissue based on methylation in surrogate tissue. The method was evaluated in publicly available data and in two studies using the latest IlluminaBeadChips: a childhood asthma study with methylation measured in both peripheral blood leukocytes (PBL) and lymphoblastoid cell lines; and a study of postoperative atrial fibrillation with methylation in PBL, atrium and artery. We found that our method can greatly improve accuracy of cross-tissue prediction at CpG sites that are variable in the target tissue [R(2) increases from 0.38 (original R(2) between tissues) to 0.89 for PBL-to-artery prediction; from 0.39 to 0.95 for PBL-to-atrium; and from 0.81 to 0.98 for lymphoblastoid cell line-to-PBL based on cross-validation, and confirmed using cross-study prediction]. An extended model with multiple CpGs further improved performance. Our results suggest that large-scale epidemiology studies using easy-to-access surrogate tissues (e.g. blood) could be recalibrated to improve understanding of epigenetics in hard-to-access tissues (e.g. atrium) and might enable non-invasive disease screening using epigenetic profiles.

Genetic Counselors in Startup Companies: Redefining the Genetic Counselor Role.

Genetic counselors (GCs) have recently begun moving into non-clinic based roles in increasing numbers. A relatively new role for GCs is working for startup companies. Startups are newly established companies in the phase of developing and researching new scalable businesses. This article explores the experiences of four GCs working at different startup companies and aims to provide resources for GCs interested in learning more about these types of roles. The article describes startup culture, including a relatively flat organizational structure, quick product iterations, and flexibility, among other unique cultural characteristics. Financial considerations are described, including how to understand and evaluate a company's financial status, along with a brief explanation of alternate forms of compensation including stock options and equity. Specifically, the article details the uncertainties and rewards of working in a fast-paced startup environment that affords opportunities to try new roles and use the genetic counseling skill set in new ways. This article aims to aid GCs in determining whether a startup environment would be a good fit, learning how to evaluate a specific startup, and understanding how to market themselves for positions at startups.

Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans.

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.

Electrocardiogram interpretation by Canadian general paediatricians: Examining practice, accuracy and confidence.

BACKGROUND: Paediatric electrocardiograms (ECGs) are ordered and interpreted by general paediatricians; however, no previous studies have evaluated the accuracy of their ECG interpretations. OBJECTIVE: To determine general paediatricians' practice and opinions regarding ECG use, accuracy of their interpretation of paediatric ECGs, and the relationship between accuracy and self-perceived confidence. METHODS: In the present cross-sectional study, Canadian general paediatricians were asked to complete a questionnaire and interpret 18 paediatric ECGs. The questionnaire assessed characteristics of ECG use, self-perceived confidence and opinions regarding ECG use in general paediatric practice. For the ECGs provided, respondents were asked whether the ECG was normal or abnormal, what abnormality the ECG demonstrated and how confident they were in this interpretation. RESULTS: ECG interpretation was performed by 124 general paediatricians. General paediatricians frequently use ECGs in their practice and regard this investigation as useful in patient assessment. The mean (± SD) accuracy of identifying ECGs as normal or abnormal, and identifying the specific abnormality was 80±12% and 56±20%, respectively. The sensitivity and specificity of identifying abnormal ECGs were 80% (95% CI 78% to 82%) and 79% (95% CI 75% to 83%), respectively. Correct ECG interpretation for isolated rhythm disturbances (73%) was significantly better than for abnormalities in axis (25%), chamber hypertrophy (41%) and ECG intervals (49%) (P<0.001). Overall confidence in ECG interpretation correlated with and was the only significant predictor of interpretation accuracy (r=0.396, P<0.001). CONCLUSION: General paediatricians were adept at detecting abnormal ECGs, but were less able to identify the abnormalities. Further education in ECG interpretation may be important for this population.

HISTORIQUE: Ce sont des pédiatres généralistes qui demandent et interprètent les électrocardiogrammes (ECG) en pédiatrie, mais aucune étude n'a porté sur l'exactitude de leur interprétation. OBJECTIF: Déterminer la pratique et les avis des pédiatres généralistes en matière d'utilisation des ECG et de l'exactitude des ECG en pédiatrie et établir le lien entre la précision et l'autoperception de la confiance. MÉTHODOLOGIE: Dans la présente étude transversale, les pédiatres généralistes canadiens ont été invités à remplir un questionnaire et à interpréter 18 ECG en pédiatrie. Le questionnaire visait à évaluer les caractéristiques liées à l'utilisation des ECG, l'autoperception de la confiance et les avis relatifs à l'utilisation des ECG en pédiatrie générale. Les répondants étaient invités à préciser si les ECG four-nis étaient normaux ou anormaux, les anomalies démontrées et leur confiance quant à leur interprétation. RÉSULTATS: Cent vingt-quatre pédiatres généralistes ont interprété les ECG. Les pédiatres généralistes utilisent souvent les ECG dans leur pratique et les considèrent comme utiles dans l'évaluation des patients. L'exactitude moyenne dans l'identification des ECG comme normaux ou anormaux et dans la détermination de l'anomalie précise correspondait à 80±12 % et à 56±20 %, respectivement. La sensibilité et la spécificité de l'identification des ECG anormaux s'établissaient à 80 % (95 % IC 78 % à 82 %) et à 79 % (95 % IC 75 % à 83 %), respectivement. La bonne interprétation des ECG révélant des troubles isolés du rythme cardiaque (73 %) était considérablement plus élevée que celle des anomalies de l'axe (25 %), de l'hypertrophie ventriculaire (41 %) et des intervalles d'ECG (49 %) (P<0,001). Dans l'ensemble, la confiance à l'égard de l'interprétation des ECG était corrélée avec l'exactitude des interprétations et en était le seul prédicteur important (r=0,396, P<0,001). CONCLUSION: Les pédiatres généralistes décelaient bien les ECG anormaux, mais réussissaient moins bien à déterminer les anomalies exactes. Il serait peut-être important de leur fournir un perfectionnement dans l'interprétation des ECG.

Dietary exposure to polyaromatic hydrocarbons of the Hong Kong population.

Polyaromatic hydrocarbons (PAHs) are ubiquitous in the environment and food. The Joint FAO/WHO Expert Committee on Food Additives concluded 13 individual PAHs are carcinogenic and genotoxic in vitro and in vivo. Food is recognized as the main source of exposure to PAHs for adult non-smokers, which contributed to more than 90% of total exposure. In this study, 300 food samples were collected in Hong Kong, analysed the levels of 16 European Union priority PAHs, the dietary exposure to these PAHs by the local adult population from these food items, and the associated health risk. The most predominant detectable PAH was chrysene (CHR) (14.4%), followed by benzo[c]fluorene (11.2%), benzo[a]anthracene (BaA) (10.6%) and benzo[b]fluoranthene (BbFA) (7.8%). The dietary exposures for average consumers of benzo[a]pyrene (BaP) and PAH4 (sum of BaP, CHR, BaA and BbFA) were 0.13-0.90 and 1.4-4.2 ng/kg bw/day respectively for lower and upper bound approaches. Cereal and its products contributed more than 50% to BaP and PAH4 for average consumers in a lower-bound approach. The margin of exposure (MOE) approach was used to assess the health risks of consumers. The calculated MOE values for both BaP and PAH4 of the average and high consumers (90th percentile) were >50,000, indicating a low concern for the health of the Hong Kong population.

MAP3K8 is a potential therapeutic target in airway epithelial inflammation.

BACKGROUND: We have previously discovered clusters of sequentially negative and positive modulators of acute inflammation during cytokine stimulation in epithelial cells and identified potential targets for therapy within these clusters. MAP3K8 is a druggable kinase that we found to be a hub of a principal interaction network. We describe here the results of MAP3K8 knockdown in the A549 lung cancer cell line, the BEAS-2B epithelial cell line and normal human bronchial epithelial (NHBE) cells following IL-1ß stimulation. We analysed signalling transduction and global gene expression after IL-1ß stimulation with and without MAP3K8 knockdown, quantifying levels of the inflammatory cytokines IL-6, IL-8 and RANTES levels by qPCRs and/or by ELISAs. We also examined potential small molecule inhibitors for MAP3K8 in the same models. RESULTS: IL-1ß significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1ß stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1ß stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1ß stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected < 0.01 respectively) demonstrated a distinct altered temporal response to IL-1ß following suppression of MAP3K8. However, putative MAP3K8 inhibitors including Tpl2-1, Tpl2-2 and GSK2222867A only showed inhibition of IL-6 and IL-8 production at a high dose. CONCLUSIONS: These results confirm that MAP3K8 is a key mediator of the early inflammatory response and that it is a potential target in inflammatory diseases. However, current tool compounds do not effectively inhibit its effects.

Fluctuation and dissipation at a quantum critical point.

In nonrelativistic field theories, quantum fluctuations give rise to dissipative behavior even at zero temperature. Here we use holographic methods to explore the dissipative dynamics of massive particles coupled to quantum critical theories. We present analytic expressions for correlation functions and response functions. The behavior changes qualitatively as the dynamical exponent passes through z=2. In particular, for z>2, the long-time dynamics of the particle is independent of its inertial mass.

Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma.

Asthma is caused by a combination of poorly understood genetic and environmental factors. We have systematically mapped the effects of single nucleotide polymorphisms (SNPs) on the presence of childhood onset asthma by genome-wide association. We characterized more than 317,000 SNPs in DNA from 994 patients with childhood onset asthma and 1,243 non-asthmatics, using family and case-referent panels. Here we show multiple markers on chromosome 17q21 to be strongly and reproducibly associated with childhood onset asthma in family and case-referent panels with a combined P value of P < 10(-12). In independent replication studies the 17q21 locus showed strong association with diagnosis of childhood asthma in 2,320 subjects from a cohort of German children (P = 0.0003) and in 3,301 subjects from the British 1958 Birth Cohort (P = 0.0005). We systematically evaluated the relationships between markers of the 17q21 locus and transcript levels of genes in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines from children in the asthma family panel used in our association study. The SNPs associated with childhood asthma were consistently and strongly associated (P < 10(-22)) in cis with transcript levels of ORMDL3, a member of a gene family that encodes transmembrane proteins anchored in the endoplasmic reticulum. The results indicate that genetic variants regulating ORMDL3 expression are determinants of susceptibility to childhood asthma.

Impact of mobile phone use on accidental falls risk in young adult pedestrians.

Background: Mobile phone use is known to be a distraction to pedestrians, increasing their likelihood of crossing into oncoming traffic or colliding with other people. However, the effect of using a mobile phone to text while walking on gait stability and accidental falls in young adults remains inconclusive. This study uses a 70 cm low friction slip hazard and the threat of hazard to investigate the effects of texting while walking on gait stability, the ability to recover balance after a slip hazard and accidental falls. Methods: Fifty healthy young adults performed six walking tasks, and one seated texting task in random order. The walks were conducted over a 10-m walkway. Four progressive hazard levels were used: 1) Seated; 2) Normal Walk (walking across the walkway with no threat of a slip); 3) Threat (walking with the threat of a slip); and 4) Slip (walking with an actual 70 cm slip hazard). The three walking conditions were repeated twice with and without the mobile phone texting dual-task. Gait kinematics and trunk posture were recorded using wearable sensors attached to the head, trunk, pelvis and feet. Study outcomes were analyzed using repeated measures analysis of variance with significance set to P≤.05. Results: Mobile phone use significantly impaired postural balance recovery when slipping, as demonstrated by increased trunk sway. Mobile phone use negatively impacted gait stability as demonstrated by increased step time variability and decreased harmonic ratios. Increased hazard levels also led to reduced texting accuracy. Conclusions: Using a mobile phone to text while walking may compete with locomotor tasks, threat assessment and postural balance control mechanisms, which leads to an increased risk of accidental falls in young adults. Pedestrians should therefore be discouraged through new educational and technology-based initiatives (for example a "texting lock" on detection of walking) from texting while walking on roadside footpaths and other environments where substantial hazards to safety exist.

Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA.

Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA). This study was designed to quantitate the effect the cationic/amino-alkyl nucleotide base modification had on the kinetic and thermodynamic properties of OPNA-DNA hybridization using surface plasmon resonance and UV thermal melt studies. Kinetic studies reveal a favorable 10-30 fold increase in affinity for a single cationic modification on the base of an adenine, cytosine, or guanidine OPNA sequence compared to the nonmodified PNA strand. The increase in affinity is correlated directly with a favorable decrease in the dissociation rate constant and increase in the association rate constant. Introducing additional amino-alkyl base modifications further favors a decrease in the dissociation rate (3-10-fold per amino-alkyl). The thermodynamics driving the OPNA hybridization is promoted by an additional favorable -80 kJ/mol enthalpy of binding for a single amino-alkyl modification compared to the PNA strand. This increase in enthalpy is consistent with an ion-ion interaction with the DNA strand. These kinetic and thermodynamic hybridization studies reveal for the first time that this type of cationic/amino-alkyl base-modified PNA has favorable hybridization properties suitable for development as an antisense oligomer.

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water.

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.