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1.
Diabetes Care ; 46(9): 1652-1658, 2023 09 01.
Article in English | MEDLINE | ID: mdl-37478323

ABSTRACT

OBJECTIVE: Meals are a consistent challenge to glycemic control in type 1 diabetes (T1D). Our objective was to assess the glycemic impact of meal anticipation within a fully automated insulin delivery (AID) system among adults with T1D. RESEARCH DESIGN AND METHODS: We report the results of a randomized crossover clinical trial comparing three modalities of AID systems: hybrid closed loop (HCL), full closed loop (FCL), and full closed loop with meal anticipation (FCL+). Modalities were tested during three supervised 24-h admissions, where breakfast, lunch, and dinner were consumed per participant's home schedule, at a fixed time, and with a 1.5-h delay, respectively. Primary outcome was the percent time in range 70-180 mg/dL (TIR) during the breakfast postprandial period for FCL+ versus FCL. RESULTS: Thirty-five adults with T1D (age 44.5 ± 15.4 years; HbA1c 6.7 ± 0.9%; n = 23 women and n = 12 men) were randomly assigned. TIR for the 5-h period after breakfast was 75 ± 23%, 58 ± 21%, and 63 ± 19% for HCL, FCL, and FCL+, respectively, with no significant difference between FCL+ and FCL. For the 2 h before dinner, time below range (TBR) was similar for FCL and FCL+. For the 5-h period after dinner, TIR was similar for FCL+ and FCL (71 ± 34% vs. 72 ± 29%; P = 1.0), whereas TBR was reduced in FCL+ (median 0% [0-0%] vs. 0% [0-0.8%]; P = 0.03). Overall, 24-h control for HCL, FCL, and FCL+ was 86 ± 10%, 77 ± 11%, and 77 ± 12%, respectively. CONCLUSIONS: Although postprandial control remained optimal with hybrid AID, both fully AID solutions offered overall TIR >70% with similar or lower exposure to hypoglycemia. Anticipation did not significantly improve postprandial control in AID systems but also did not increase hypoglycemic risk when meals were delayed.


Subject(s)
Diabetes Mellitus, Type 1 , Insulin , Male , Humans , Adult , Female , Middle Aged , Insulin/therapeutic use , Diabetes Mellitus, Type 1/drug therapy , Blood Glucose , Hypoglycemic Agents/therapeutic use , Meals , Insulin, Regular, Human/therapeutic use , Insulin Infusion Systems , Cross-Over Studies
2.
J Interv Card Electrophysiol ; 64(2): 323-331, 2022 Aug.
Article in English | MEDLINE | ID: mdl-33826085

ABSTRACT

PURPOSE: Early recurrence of atrial tachyarrhythmia (ER) is predictive of late recurrence of atrial tachyarrhythmia (LR) after first-time atrial fibrillation (AF) ablation, but the association in patients undergoing repeat AF ablation is unknown. We aim to determine the incidence and prognostic significance of ER after repeat ablation. METHODS: A total of 259 consecutive patients (mean age 64 years, 75.3% male) undergoing repeat AF ablation with complete follow-up data were included at a single institution from 2010 to 2015. ER and LR were defined as atrial tachyarrhythmia (AF, atrial flutter or atrial tachycardia) > 30 s within the 3-month blanking period (BP) and after the 3-month BP, respectively. RESULTS: ER occurred in 79/259 (30.5%), and LR occurred in 138/259 (53%) at a median follow-up of 1221 (IQR: 523-1712) days. Four-year freedom from LR was 22% and 56% in patients with and without ER, respectively (p < 0.001). After multivariate adjustment, ER was strongly associated with LR, cardioversion post BP, and repeat ablation, but not associated with hospitalization. Compared to those with no ER, there was a higher risk of LR when ER occurred within the first month of the BP [month 1: hazard ratio (HR) 2.32, confidence interval (CI) 1.57-3.74, p < 0.001; month 2: HR 2.01, CI 1.13-3.83, p = 0.02; month 3: HR 1.46, CI 0.5-3.36, p = 0.37], however the prediction of LR based on timing within the BP was poor (area under curve 0.64). CONCLUSION: Following repeat AF ablation, ER is strongly associated with LR, cardioversion post BP, and repeat ablation.


Subject(s)
Atrial Fibrillation , Catheter Ablation , Pulmonary Veins , Atrial Fibrillation/epidemiology , Catheter Ablation/adverse effects , Female , Humans , Male , Middle Aged , Pulmonary Veins/surgery , Recurrence , Tachycardia/surgery , Treatment Outcome
3.
Diabetes Care ; 2021 Aug 15.
Article in English | MEDLINE | ID: mdl-34400480

ABSTRACT

OBJECTIVE: Meals are a major hurdle to glycemic control in type 1 diabetes (T1D). Our objective was to test a fully automated closed-loop control (CLC) system in the absence of announcement of carbohydrate ingestion among adolescents with T1D, who are known to commonly omit meal announcement. RESEARCH DESIGN AND METHODS: Eighteen adolescents with T1D (age 15.6 ± 1.7 years; HbA1c 7.4 ± 1.5%; 9 females/9 males) participated in a randomized crossover clinical trial comparing our legacy hybrid CLC system (Unified Safety System Virginia [USS]-Virginia) with a novel fully automated CLC system (RocketAP) during two 46-h supervised admissions (each with one announced and one unannounced dinner), following 2 weeks of data collection. Primary outcome was the percentage time-in-range 70-180 mg/dL (TIR) following the unannounced meal, with secondary outcomes related to additional continuous glucose monitoring-based metrics. RESULTS: Both TIR and time-in-tight-range 70-140 mg/dL (TTR) were significantly higher using RocketAP than using USS-Virginia during the 6 h following the unannounced meal (83% [interquartile range 64-93] vs. 53% [40-71]; P = 0.004 and 49% [41-59] vs. 27% [22-36]; P = 0.002, respectively), primarily driven by reduced time-above-range (TAR >180 mg/dL: 17% [1.3-34] vs. 47% [28-60]), with no increase in time-below-range (TBR <70 mg/dL: 0% median for both). RocketAP also improved control following the announced meal (mean difference TBR: -0.7%, TIR: +7%, TTR: +6%), overall (TIR: +5%, TAR: -5%, TTR: +8%), and overnight (TIR: +7%, TTR: +19%, TAR: -5%). RocketAP delivered less insulin overall (78 ± 23 units vs. 85 ± 20 units, P = 0.01). CONCLUSIONS: A new fully automated CLC system with automatic prandial dosing was proven to be safe and feasible and outperformed our legacy USS-Virginia in an adolescent population with and without meal announcement.

4.
PLoS One ; 16(6): e0253266, 2021.
Article in English | MEDLINE | ID: mdl-34166392

ABSTRACT

PURPOSE: To evaluate if specific AADs prescribed in the blanking period (BP) after catheter ablation of atrial fibrillation (AF) may be associated with reduced risk of early recurrence (ER) and/or late recurrence (LR) of atrial arrhythmias. METHODS: A total of 478 patients undergoing first-time ablation at a single institution were included. Outcomes were: ER, LR, discontinuation of AAD less than 90 days post-ablation, and second ablation. ER was defined as AF, atrial flutter (AFL), or atrial tachycardia (AT) > 30 seconds within BP. LR was defined as AF/AFL/AT > 30 seconds after BP. RESULTS: Of 478 patients, 14.9% were prescribed no AAD, 26.4% propafenone/flecainide, 34.5% sotalol/dofetilide, 10.7% dronedarone, and 13.6% amiodarone. Patients prescribed amiodarone were more likely to have persistent AF, hypertension, diabetes, and other comorbidities. In unadjusted analyses, there were no differences between groups in relation to ER (log rank P = 0.171), discontinuation of AAD before ninety days post-ablation (log rank P = 0.235), or freedom from second ablation (log rank P = 0.147). After multivariable adjustment, patients prescribed amiodarone or dronedarone were more likely to experience LR than those prescribed no AAD [Adjusted Hazard Ratio (AHR) 1.83, 95% CI 1.10-3.04, p = 0.02; AHR 1.79, 95% CI 1.05-3.05, p = 0.03, respectively]. CONCLUSION: Following first-time catheter ablation, there were no differences between specific AAD prescription and risk of ER, while those prescribed amiodarone or dronedarone in the BP were more likely to experience LR than those prescribed no AAD, which may represent an association due to confounding by indication.


Subject(s)
Anti-Arrhythmia Agents/administration & dosage , Atrial Fibrillation/therapy , Catheter Ablation , Drug Prescriptions , Registries , Aged , Atrial Fibrillation/physiopathology , Female , Humans , Male , Middle Aged , Recurrence
5.
Diabetes Technol Ther ; 23(4): 277-285, 2021 04.
Article in English | MEDLINE | ID: mdl-33270531

ABSTRACT

Objective: Physical activity is a major challenge to glycemic control for people with type 1 diabetes. Moderate-intensity exercise often leads to steep decreases in blood glucose and hypoglycemia that closed-loop control systems have so far failed to protect against, despite improving glycemic control overall. Research Design and Methods: Fifteen adults with type 1 diabetes (42 ± 13.5 years old; hemoglobin A1c 6.6% ± 1.0%; 10F/5M) participated in a randomized crossover clinical trial comparing two hybrid closed-loop (HCL) systems, a state-of-the-art hybrid model predictive controller and a modified system designed to anticipate and detect unannounced exercise (APEX), during two 32-h supervised admissions with 45 min of planned moderate activity, following 4 weeks of data collection. Primary outcome was the number of hypoglycemic episodes during exercise. Continuous glucose monitor (CGM)-based metrics and hypoglycemia are also reported across the entire admissions. Results: The APEX system reduced hypoglycemic episodes overall (9 vs. 33; P = 0.02), during exercise (5 vs. 13; P = 0.04), and in the 4 h following (2 vs. 11; P = 0.02). Overall CGM median percent time <70 mg/dL decreased as well (0.3% vs. 1.6%; P = 0.004). This protection was obtained with no significant increase in time >180 mg/dL (18.5% vs. 16.6%, P = 0.15). Overnight control was notable for both systems with no hypoglycemia, median percent in time 70-180 mg/dL at 100% and median percent time 70-140 mg/dL at ∼96% for both. Conclusions: A new closed-loop system capable of anticipating and detecting exercise was proven to be safe and feasible and outperformed a state-of-the-art HCL, reducing participants' exposure to hypoglycemia during and after moderate-intensity physical activity. ClinicalTrials.gov NCT03859401.


Subject(s)
Diabetes Mellitus, Type 1 , Hypoglycemia , Pancreas, Artificial , Adult , Blood Glucose , Cross-Over Studies , Diabetes Mellitus, Type 1/drug therapy , Exercise , Humans , Hypoglycemia/prevention & control , Hypoglycemic Agents/therapeutic use , Insulin/therapeutic use , Insulin Infusion Systems , Middle Aged
6.
Am J Cardiol ; 142: 66-73, 2021 03 01.
Article in English | MEDLINE | ID: mdl-33290688

ABSTRACT

Catheter ablation improves clinical outcomes in atrial fibrillation (AF) patients with heart failure (HF) with reduced ejection fraction (HFrEF). However, the role of catheter ablation in HF with a preserved ejection fraction (HFpEF) is less clear. We performed a literature search and systematic review of studies that compared AF recurrence at one year after catheter ablation of AF in patients with HFpEF versus those with HFrEF. Risk ratio (RR; where a RR <1.0 favors the HFpEF group) and mean difference (MD; where MD <0 favors the HFpEF group) 95% confidence intervals (CI) were measured for dichotomous and continuous variables, respectively. Six studies with a total of 1,505 patients were included, of which 764 (51%) had HFpEF and 741 (49%) had HFrEF. Patients with HFpEF experienced similar recurrence of AF 1 year after ablation on or off antiarrhythmic drugs compared with those with HFrEF (RR 1.01; 95% CI 0.76, 1.35). Fluoroscopy time was significantly shorter in the HFpEF group (MD -5.42; 95% CI -8.51, -2.34), but there was no significant difference in procedure time (MD 1.74; 95% CI -11.89, 15.37) or periprocedural adverse events between groups (RR 0.84; 95% CI 0.54,1.32). There was no significant difference in hospitalizations between groups (MD 1.18; 95% CI 0.90, 1.55), but HFpEF patients experienced significantly less mortality (MD 0.41; 95% CI 0.18, 0.94). In conclusion, based on the results of this meta-analysis, catheter ablation of AF in patients with HFpEF appears as safe and efficacious in maintaining sinus rhythm as in those with HFrEF.


Subject(s)
Atrial Fibrillation/surgery , Catheter Ablation , Heart Failure/physiopathology , Stroke Volume , Atrial Fibrillation/complications , Heart Failure/complications , Hospitalization/statistics & numerical data , Humans , Mortality , Operative Time , Postoperative Complications/epidemiology , Recurrence , Treatment Outcome
7.
Am J Cardiol ; 136: 62-70, 2020 12 01.
Article in English | MEDLINE | ID: mdl-32941815

ABSTRACT

Catheter ablation improves outcomes in atrial fibrillation (AF) patients with heart failure (HF) with reduced ejection fraction (HFrEF). We sought to evaluate the efficacy and safety of catheter ablation of AF in HF patients with a preserved ejection fraction (HFpEF). We performed a retrospective study of all patients who underwent de novo radiofrequency catheter ablation enrolled in the UC San Diego AF Ablation Registry. The primary outcome was recurrence of all atrial arrhythmias on or off antiarrhythmic drugs (AAD). Of 547 total patients, 51 (9.3%) had HFpEF, 40 (7.3%) had HFrEF, and 456 (83.4%) were without HF. There was no difference in recurrence of atrial arrhythmias on or off AAD (Adjusted Hazard Ratio [AHR] 1.92 [95% CI 0.97 to 3.83] for HFpEF vs HFrEF and AHR 0.90 [95% CI 0.59 to 1.39] for HFpEF vs no HF) or off AAD (AHR 1.96 [95% CI 0.99 to 3.90] for HFpEF vs HFrEF and AHR 1.14 [95% CI 0.74 to 1.77] for HFpEF vs no HF). There was also no difference in rates of all-cause hospitalizations (AHR 1.80 [95% CI 0.97 to 3.33] for HFpEF vs HFrEF and AHR 2.05 [95% CI 1.30 to 3.23] for HFpEF vs no HF) or rates of all-cause mortality (AHR 0.53 [95% CI 0.05 to 6.11] for HFpEF vs HFrEF and AHR 2.46 [95% CI 0.34 to 17.92] for HFpEF vs no HF). There were no significant differences in AAD use (p = 0.176) or procedural complications between groups (p = 0.980). In conclusion, there were no significant differences in arrhythmia-free survival between patients with HFpEF and HFrEF that underwent catheter ablation of AF.


Subject(s)
Atrial Fibrillation/complications , Atrial Fibrillation/surgery , Catheter Ablation , Heart Failure/complications , Heart Failure/physiopathology , Stroke Volume , Aged , Cohort Studies , Female , Humans , Male , Middle Aged , Retrospective Studies , Treatment Outcome
8.
Diabetes Care ; 43(4): 799-805, 2020 04.
Article in English | MEDLINE | ID: mdl-32144167

ABSTRACT

OBJECTIVE: Insulin dosing in type 1 diabetes (T1D) is oftentimes complicated by fluctuating insulin requirements driven by metabolic and psychobehavioral factors impacting individuals' insulin sensitivity (IS). In this context, smart bolus calculators that automatically tailor prandial insulin dosing to the metabolic state of a person can improve glucose management in T1D. RESEARCH DESIGN AND METHODS: Fifteen adults with T1D using continuous glucose monitors (CGMs) and insulin pumps completed two 24-h admissions in a hotel setting. During the admissions, participants engaged in an early afternoon 45-min aerobic exercise session, after which they received a standardized dinner meal. The dinner bolus was computed using a standard bolus calculator or smart bolus calculator informed by real-time IS estimates. Glucose control was assessed in the 4 h following dinner using CGMs and was compared between the two admissions. RESULTS: The IS-informed bolus calculator allowed for a reduction in postprandial hypoglycemia as quantified by the low blood glucose index (2.02 vs. 3.31, P = 0.006) and percent time <70 mg/dL (8.48% vs. 15.18%, P = 0.049), without increasing hyperglycemia (high blood glucose index: 3.13 vs. 2.09, P = 0.075; percent time >180 mg/dL: 13.24% vs. 10.42%, P = 0.5; percent time >250 mg/dL: 2.08% vs. 1.19%, P = 0.317). In addition, the number of hypoglycemia rescue treatments was reduced from 12 to 7 with the use of the system. CONCLUSIONS: The study shows that the proposed IS-informed bolus calculator is safe and feasible in adults with T1D, appropriately reducing postprandial hypoglycemia following an exercise-induced IS increase.


Subject(s)
Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/drug therapy , Drug Dosage Calculations , Exercise/physiology , Hypoglycemia/prevention & control , Insulin Infusion Systems , Insulin Resistance/physiology , Insulin/administration & dosage , Adult , Blood Glucose/drug effects , Blood Glucose/metabolism , Blood Glucose Self-Monitoring/instrumentation , Blood Glucose Self-Monitoring/methods , Diabetes Mellitus, Type 1/physiopathology , Equipment Design , Female , Humans , Hyperglycemia/blood , Hypoglycemia/blood , Hypoglycemia/chemically induced , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/adverse effects , Insulin/adverse effects , Male , Meals , Middle Aged , Postprandial Period/drug effects , Young Adult
9.
Curr Opin Cardiol ; 35(3): 260-270, 2020 05.
Article in English | MEDLINE | ID: mdl-32102085

ABSTRACT

PURPOSE OF REVIEW: To review the shared pathology of atrial fibrillation and heart failure with preserved ejection fraction (HFpEF) and the prognostic, diagnostic, and treatment challenges incurred by the co-occurrence of these increasingly prevalent diseases. RECENT FINDINGS: Multiple risk factors and mechanisms have been proposed as potentially linking atrial fibrillation and HFpEF, with systemic inflammation more recently being invoked. Nonvitamin K oral anticoagulants, left atrial appendage occlusion devices, and catheter ablation have emerged as alternative treatment options. Other novel pharmacological agents, such as neprilysin inhibitors, need to be studied further in this patient population. SUMMARY: Atrial fibrillation and HFpEF commonly co-occur because of their shared risk factors and pathophysiology and incur increased morbidity and mortality relative to either condition alone. Although the presence of both diseases can often make each diagnosis difficult, it is important to do so early in the disease course as there are now a variety of treatment options aimed at improving symptoms and quality of life, slowing disease progression, and improving prognosis. However, more research needs to be performed on the role of catheter ablation in this population. Novel pharmacologic and procedural treatment options appear promising and may further improve the treatment options available to this growing population.


Subject(s)
Atrial Fibrillation/therapy , Catheter Ablation , Heart Failure/therapy , Humans , Quality of Life , Stroke Volume
10.
Am J Cardiol ; 123(1): 187-195, 2019 01 01.
Article in English | MEDLINE | ID: mdl-30352662

ABSTRACT

Atrial fibrillation (AF) and heart failure (HF) both have become major cardiovascular epidemics, adversely affecting quality of life, decreasing longevity, and imparting a large economic burden on the healthcare system. Both share similar risk factors and frequently coexist, leading to increased morbidity and mortality relative to patients with either condition alone. Although evidence-based treatment guidelines for both diseases exist, consensus treatment strategies are less clear when AF and HF co-occur. Given the risks of antiarrhythmic drugs and their incomplete success in maintaining sinus rhythm, catheter ablation has become an increasingly popular alternative to pharmacologic rhythm control in symptomatic patients with AF with normal cardiac function. Although multiple studies have demonstrated the efficacy of catheter ablation in AF, studies examining the use of catheter ablation specifically in patients with HF have recently begun to emerge and provide some guidance in this group of patients. In this review, we examine the effects of catheter ablation of AF in patients with HF on maintenance of sinus rhythm, left ventricular ejection fraction, exercise capacity, quality of life, hospitalization, and mortality rates. Data regarding both HF with reduced ejection fraction and preserved ejection fraction are discussed.


Subject(s)
Atrial Fibrillation/complications , Atrial Fibrillation/surgery , Heart Failure/complications , Radiofrequency Ablation , Humans
11.
Forensic Sci Int Genet ; 26: 77-84, 2017 01.
Article in English | MEDLINE | ID: mdl-27816849

ABSTRACT

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.


Subject(s)
DNA Fingerprinting/standards , DNA, Mitochondrial/genetics , Polymorphism, Single Nucleotide , Forensic Genetics/standards , Haplotypes , Humans , Laboratories/standards
12.
Forensic Sci Int Genet ; 19: 56-67, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26122263

ABSTRACT

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.


Subject(s)
Electrophoresis, Capillary/methods , Forensic Genetics , Genetic Markers , DNA/genetics , Genotype , Humans , Polymorphism, Single Nucleotide
13.
Brain Res ; 1112(1): 33-45, 2006 Sep 27.
Article in English | MEDLINE | ID: mdl-16904079

ABSTRACT

Previous studies showed poly C binding protein 1 (PCBP) participating in the mu-opioid receptor (MOR) gene regulation via binding to a single-stranded (ss) DNA element. In this report, we therefore investigate the molecular basis of PCBP regulating the MOR gene expression. Various truncated PCBPs, including one domain (KH1, KH2, variable or KH3), two- (K12, K2v or Kv3) or three-sequential domains (K12v or K2v3), were constructed. The MOR ssDNA binding abilities of these truncated PCBPs were examined using electrophoretic mobility shift assay (EMSA). KH1 domain possessed a strong MOR ssDNA binding activity. Variable domain displayed no binding, and KH2 or KH3 domain possessed a weak MOR ssDNA binding activity. Binding of two-domain PCBPs indicated an additive effect of two-domain combinations. Interestingly, K2v3, a three-domain PCBP, displayed as strong ssDNA binding as that of K12v, suggesting synergism of KH2, KH3 and variable domains for the binding activity. Functional analysis demonstrated one-domain PCBPs exhibiting no transactivation on the MOR proximal promoter. Two-domain PCBPs displayed approximately 20% activity, while three-domain PCBPs displayed 70%-85% of full-length PCBP activity. Taken together, these results suggested that no single domain possessed sufficient functional activity to serve as an independent transactivation domain, and the combination of three sequential domains was necessary for its optimal activity to activate the MOR proximal promoter. In summary, our data suggested that cooperativity of three sequential domains is essential for PCBP functioning as a MOR gene regulator. Various ways in which this cooperativity could occur are discussed.


Subject(s)
Carrier Proteins/physiology , Gene Expression Regulation/physiology , Promoter Regions, Genetic/physiology , Receptors, Opioid, mu/metabolism , Animals , Base Sequence , Cell Line, Tumor , DNA-Binding Proteins , Electrophoretic Mobility Shift Assay/methods , Methionine/metabolism , Mice , Neuroblastoma , Phosphorus Isotopes/metabolism , Protein Binding/drug effects , Protein Binding/physiology , Protein Structure, Tertiary/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins , Receptors, Opioid, mu/genetics , Regulatory Sequences, Nucleic Acid , Reverse Transcriptase Polymerase Chain Reaction/methods , Transcription, Genetic , Transfection/methods
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