Evaluating convolutional neural network-enhanced electrocardiography for hypertrophic cardiomyopathy detection in a specialized cardiovascular setting.

The efficacy of convolutional neural network (CNN)-enhanced electrocardiography (ECG) in detecting hypertrophic cardiomyopathy (HCM) and dilated HCM (dHCM) remains uncertain in real-world applications. This retrospective study analyzed data from 19,170 patients (including 140 HCM or dHCM) in the Shinken Database (2010-2017). We evaluated the sensitivity, positive predictive rate (PPR), and F1 score of CNN-enhanced ECG in a ''basic diagnosis'' model (total disease label) and a ''comprehensive diagnosis'' model (including disease subtypes). Using all-lead ECG in the "basic diagnosis" model, we observed a sensitivity of 76%, PPR of 2.9%, and F1 score of 0.056. These metrics improved in cases with a diagnostic probability of ≥ 0.9 and left ventricular hypertrophy (LVH) on ECG: 100% sensitivity, 8.6% PPR, and 0.158 F1 score. The ''comprehensive diagnosis'' model further enhanced these figures to 100%, 13.0%, and 0.230, respectively. Performance was broadly consistent across CNN models using different lead configurations, particularly when including leads viewing the lateral walls. While the precision of CNN models in detecting HCM or dHCM in real-world settings is initially low, it improves by targeting specific patient groups and integrating disease subtype models. The use of ECGs with fewer leads, especially those involving the lateral walls, appears comparably effective.

Identifying patients with acute aortic dissection using an electrocardiogram with convolutional neural network.

Background: The potential of utilizing artificial intelligence with electrocardiography (ECG) for initial screening of aortic dissection (AD) is promising. However, achieving a high positive predictive rate (PPR) remains challenging. Methods and results: This retrospective analysis of a single-center, prospective cohort study (Shinken Database 2010-2017, N = 19,170) used digital 12-lead ECGs from initial patient visits. We assessed a convolutional neural network (CNN) model's performance for AD detection with eight-lead (I, II, and V1-6), single-lead, and double-lead (I, II) ECGs via five-fold cross-validation. The mean age was 63.5 ± 12.5 years for the AD group (n = 147) and 58.1 ± 15.7 years for the non-AD group (n = 19,023). The CNN model achieved an area under the curve (AUC) of 0.936 (standard deviation [SD]: 0.023) for AD detection with eight-lead ECGs. In the entire cohort, the PPR was 7 %, with 126 out of 147 AD cases correctly diagnosed (sensitivity 86 %). When applied to patients with D-dimer levels ≥1 µg/dL and a history of hypertension, the PPR increased to 35 %, with 113 AD cases correctly identified (sensitivity 86 %). The single V1 lead displayed the highest diagnostic performance (AUC: 0.933, SD: 0.03), with PPR improvement from 8 % to 38 % within the same population. Conclusions: Our CNN model using ECG data for AD detection achieved an over 30% PPR when applied to patients with elevated D-dimer levels and hypertension history while maintaining sensitivity. A similar level of performance was observed with a single-lead V1 ECG in the CNN model.

Lead-Specific Performance for Atrial Fibrillation Detection in Convolutional Neural Network Models Using Sinus Rhythm Electrocardiography.

Background: We developed a convolutional neural network (CNN) model to detect atrial fibrillation (AF) using the sinus rhythm ECG (SR-ECG). However, the diagnostic performance of the CNN model based on different ECG leads remains unclear. Methods and Results: In this retrospective analysis of a single-center, prospective cohort study, we identified 616 AF cases and 3,412 SR cases for the modeling dataset among new patients (n=19,170). The modeling dataset included SR-ECGs obtained within 31 days from AF-ECGs in AF cases and SR cases with follow-up ≥1,095 days. We evaluated the CNN model's performance for AF detection using 8-lead (I, II, and V1-6), single-lead, and double-lead ECGs through 5-fold cross-validation. The CNN model achieved an area under the curve (AUC) of 0.872 (95% confidence interval (CI): 0.856-0.888) and an odds ratio of 15.24 (95% CI: 12.42-18.72) for AF detection using the eight-lead ECG. Among the single-lead and double-lead ECGs, the double-lead ECG using leads I and V1 yielded an AUC of 0.871 (95% CI: 0.856-0.886) with an odds ratio of 14.34 (95% CI: 11.64-17.67). Conclusions: We assessed the performance of a CNN model for detecting AF using eight-lead, single-lead, and double-lead SR-ECGs. The model's performance with a double-lead (I, V1) ECG was comparable to that of the 8-lead ECG, suggesting its potential as an alternative for AF screening using SR-ECG.

Identification of patients with dilated phase of hypertrophic cardiomyopathy using a convolutional neural network applied to multiple, dual, and single lead electrocardiograms.

Background: This study sought to develop an artificial intelligence-derived model to detect the dilated phase of hypertrophic cardiomyopathy (dHCM) on digital electrocardiography (ECG) and to evaluate the performance of the model applied to multiple-lead or single-lead ECG. Methods: This is a retrospective analysis using a single-center prospective cohort study (Shinken Database 2010-2017, n = 19,170). After excluding those without a normal P wave on index ECG (n = 1,831) and adding dHCM patients registered before 2009 (n = 39), 17,378 digital ECGs were used. Totally 54 dHCM patients were identified of which 11 diagnosed at baseline, 4 developed during the time course, and 39 registered before 2009. The performance of the convolutional neural network (CNN) model for detecting dHCM was evaluated using eight-lead (I, II, and V1-6), single-lead, and double-lead (I, II) ECGs with the five-fold cross validation method. Results: The area under the curve (AUC) of the CNN model to detect dHCM (n = 54) with eight-lead ECG was 0.929 (standard deviation [SD]: 0.025) and the odds ratio was 38.64 (SD 9.10). Among the single-lead and double-lead ECGs, the AUC was highest with the single lead of V5 (0.953 [SD: 0.038]), with an odds ratio of 58.89 (SD:68.56). Conclusion: Compared with the performance of eight-lead ECG, the most similar performance was achieved with the model with a single V5 lead, suggesting that this single-lead ECG can be an alternative to eight-lead ECG for the screening of dHCM.

Cardiovascular events and artificial intelligence-predicted age using 12-lead electrocardiograms.

Background: There is increasing evidence that 12-lead electrocardiograms (ECG) can be used to predict biological age, which is associated with cardiovascular events. However, the utility of artificial intelligence (AI)-predicted age using ECGs remains unclear. Methods: Using a single-center database, we developed an AI-enabled ECG using 17 042 sinus rhythm ECGs (SR-ECG) to predict chronological age (CA) with a convolutional neural network that yields AI-predicted age. Using the 5-fold cross validation method, AI-predicted age deriving from the test dataset was yielded for all ECGs. The incidence by AgeDiff and the areas under the curve by receiver operating characteristic curve with AI-predicted age for cardiovascular events were analyzed. Results: During the mean follow-up period of 460.1 days, there were 543 cardiovascular events. The annualized incidence of cardiovascular events was 2.24 %, 2.44 %, and 3.01 %/year for patients with AgeDiff < -6, -6 to ≤6, and >6 years, respectively. The areas under the curve for cardiovascular events with CA and AI-predicted age, respectively, were 0.673 and 0.679 (Delong's test, P = 0.388) for all patients; 0.642 and 0.700 (P = 0.003) for younger patients (CA < 60 years); and 0.584 and 0.570 (P = 0.268) for older patients (CA ≥ 60 years). Conclusions: AI-predicted age using 12-lead ECGs showed superiority in predicting cardiovascular events compared with CA in younger patients, but not in older patients.

Identifying patients with atrial fibrillation during sinus rhythm on ECG: Significance of the labeling in the artificial intelligence algorithm.

BACKGROUND: This study aimed to increase the knowledge on how to enhance the performance of artificial intelligence (AI)-enabled electrocardiography (ECG) to detect atrial fibrillation (AF) on sinus rhythm ECG (SR-ECG). METHODS: It is a retrospective analysis of a single-center, prospective cohort study (Shinken Database). We developed AI-enabled ECG using SR-ECG to predict AF with a convolutional neural network (CNN). Among new patients in our hospital (n = 19,170), 276 AF label (having ECG on AF [AF-ECG] in the ECG database) and 1896 SR label with following three conditions were identified in the derivation dataset: (1) without structural heart disease, (2) in AF label, SR-ECG was taken within 31 days from AF-ECG, and (3) in SR label, follow-up ≥ 1,095 days. Three patterns of AF label were analyzed by timing of SR-ECG to AF-ECG (before/after/before-or-after, CNN algorithm 1 to 3). The outcome measurement was area under the curve (AUC), sensitivity, specificity, accuracy, and F1 score. As an extra-testing dataset, the performance of AI-enabled ECG was tested in patients with structural heart disease. RESULTS: The AUC of AI-enabled ECG with CNN algorithm 1, 2, and 3 in the derivation dataset was 0.83, 0.88, and 0.86, respectively; when tested in patients with structural heart disease, 0.75, 0.81, and 0.78, respectively. CONCLUSION: We confirmed high performance of AI-enabled ECG to detect AF on SR-ECG in patients without structural heart disease. The performance enhanced especially when SR-ECG after index AF-ECG was included in the algorithm, which was consistent in patients with structural heart disease.

Correction to Discovery of the Orally Effective Thyrotropin-Releasing Hormone Mimetic: 1-{N-[(4S,5S)-(5-Methyl-2-oxooxazolidine-4-yl)carbonyl]-3-(thiazol-4-yl)-l-alanyl}-(2R)-2-methylpyrrolidine Trihydrate (Rovatirelin Hydrate).

[This corrects the article DOI: 10.1021/acsomega.8b01481.].

Discovery of the Orally Effective Thyrotropin-Releasing Hormone Mimetic: 1-{N-[(4S,5S)-(5-Methyl-2-oxooxazolidine-4-yl)carbonyl]-3-(thiazol-4-yl)-l-alanyl}-(2R)-2-methylpyrrolidine Trihydrate (Rovatirelin Hydrate).

We have explored orally effective thyrotropin-releasing hormone (TRH) mimetics, showing oral bioavailability and brain penetration by structure-activity relationship (SAR) study on the basis of in vivo antagonistic activity on reserpine-induced hypothermia in mice. By primary screening of the synthesized TRH mimetics, we found a novel TRH mimetic: l-pyroglutamyl-[3-(thiazol-4-yl)-l-alanyl]-l-prolinamide with a high central nervous system effect compared with TRH as a lead compound. Further SAR optimization studies of this lead compound led to discovery of a novel orally effective TRH mimetic: 1-{N-[(4S,5S)-(5-methyl-2-oxooxazolidine-4-yl)carbonyl]-3-(thiazol-4-yl)-l-alanyl}-(2R)-2-methylpyrrolidine trihydrate (rovatirelin hydrate), which was selected as a candidate for clinical trials.

How do user experiences with different transport modes affect the risk of traffic accidents? From the viewpoint of licence possession status.

Road accidents between different modes of transport-such as between automobiles and pedestrians, automobiles and bicycles, or automobiles and motorcycles-are frequent. In such cases, it is important to consider the other side's perspective. This involves the ability to correctly judge, for every given situation, how other people on the road perceive their surroundings and what they intend to do next. In this paper, we conduct two types of studies assuming that this kind of ability to consider perspectives is higher when the person is licenced to drive the mode of transport used by the other party. For Study 1, we analysed accidents involving senior citizens between the ages of 65 and 74 years, who collided with automobiles as pedestrians or cyclists (1656 and 3192 cases respectively), in terms of the accident category and type of road at the accident spot. The results indicate that possession or non-possession of a licence was irrelevant for accidents involving cyclists, but for accidents with pedestrians, senior citizens who did not possess a licence are likely to be involved in a greater number of accidents in places that require interaction with automobiles, such as while crossing at crosswalks or at intersections. For Study 2, we reviewed 875 ordinary first-class licence practical test examinees, categorised them according to their licence possession status (motorcycle licence, moped licence, or no licence), and made a category-wise comparison of the test instructor's assessment of their ability to make a left turn. The results showed that those who had a motorcycle or moped licence tended to make a left turn more safely. Thus, the results indicate that experience with different modes of transport is likely to reduce the risk of accidents. These findings may be used to popularise educational interventions encouraging users of various transport modes to consider the perspective of others (i.e. via perspective-taking).

[Stress reactions among bus drivers: towards the development of an educational resource for better management of emotions].

Interview and questionnaire surveys were conducted with bus drivers in Japan, with the goal of developing an educational program for better control of emotions among bus drivers. The interviews aimed at identifying stressors and ways in which stress negatively influenced bus services. The questionnaire survey. which was being developed as a self-diagnosis tool, further provided bus drivers with the opportunity to understand their own emotional tendencies. Factor analysis identified six factors underlying work-related stress: anger at unsafe behaviours of nearby road users, irritation caused by complaints from passengers, time pressures, anxiety about traffic accidents, impatience with slow passengers, and resentment of bad-mannered passengers. The influence of stress on the drivers comprised four factors: cognitive failure, sullen behaviour, abrupt acceleration/deceleration, and aggressive driving. Moreover, drivers with lower stress were relatively older and more experienced. Based on these results, educational materials were proposed with the aim of enhancing bus drivers' understanding of their emotional processes and coping skills.

Stringent response processes suppress DNA damage sensitivity caused by deficiency in full-length translation initiation factor 2 or PriA helicase.

When Escherichia coli grows in the presence of DNA-damaging agents such as methyl methanesulphonate (MMS), absence of the full-length form of Translation Initiation Factor 2 (IF2-1) or deficiency in helicase activity of replication restart protein PriA leads to a considerable loss of viability. MMS sensitivity of these mutants was contingent on the stringent response alarmone (p)ppGpp being at low levels. While zero levels (ppGpp°) greatly aggravated sensitivity, high levels promoted resistance. Moreover, M+ mutations, which suppress amino acid auxotrophy of ppGpp° strains and which have been found to map to RNA polymerase subunits, largely restored resistance to IF2-1- and PriA helicase-deficient mutants. The truncated forms IF2-2/3 played a key part in inducing especially severe negative effects in ppGpp° cells when restart function priB was knocked out, causing loss of viability and severe cell filamentation, indicative of SOS induction. Even a strain with the wild-type infB allele exhibited significant filamentation and MMS sensitivity in this background whereas mutations that prevent expression of IF2-2/3 essentially eliminated filamentation and largely restored MMS resistance. The results suggest different influences of IF2-1 and IF2-2/3 on the replication restart system depending on (p)ppGpp levels, each having the capacity to maximize survival under differing growth conditions.

A new role for translation initiation factor 2 in maintaining genome integrity.

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA-damaging agent is present.

Psychosocial and cognitive consequences of major neonatal surgery.

PURPOSE: To evaluate the long-term quality of life (QOL) of patients who had undergone major neonatal surgery, the psychosocial and cognitive consequences of neonatal surgical stress were assessed when the patients reached school age. MATERIALS AND METHODS: Seventy-two patients who had undergone major neonatal surgery were enrolled in this study. Their primary diseases were anorectal malformation (ARM) in 27 cases, esophageal atresia (EA) in 23, and congenital diaphragmatic hernia (CDH) in 22. Intelligence tests using Wechsler Intelligence Scale for Children III (WISC-III) or a developmental test and the Child Behavior Checklist were conducted through questionnaires and interviews with clinical psychologists. RESULTS: Mental retardation (MR) was apparent in 25% of EA, 20% of ARM, and 18% of CDH, significantly higher than the 2% to 3% commonly found in the general population. The clinical range (CR) of the Child Behavior Checklist was seen in 35% of EA, 59% of ARM, and 38% of CDH, which is also significantly higher than the 25% typically seen in the general population. No significant differences in MR and CR were seen among the primary diseases. The most important factors influencing MR and CR remain to be identified. CONCLUSIONS: To ensure true quality of life after neonatal surgical stress, pediatric surgeons must consider not only physical assessments but also cognitive, emotional, and psychosocial assessments.

Management of perianal abscess with hainosankyuto in neonates and young infants.

BACKGROUND: Perianal abscess (PA) is a common condition acquired in infancy, yet its treatment method remains controversial. We reviewed the outcome of neonates and young infants with PA who were treated with the traditional Japanese medicine, hainosankyuto (TJ-122). METHODS: Fifteen male infants with PA under the age of 3 months were reviewed. The median age of infants at disease onset was 33 days (range, 18-88 days) and the median bodyweight was 4.1 kg (range, 2.5-6.4 kg). TJ-122 was administered at a dose of 0.20 g/kg/day (n= 13) or 0.25 g/kg/day (n= 2) orally in two or three divided doses before meals. Antibiotics were not used in any of the patients. RESULTS: Of the 15 patients, 14 were cured and had no recurrence, with a median TJ-122 administration of 28 days (range, 14-117 days). Eight patients were cured within 28 days (53%) and 12 were cured within 60 days (80%). One patient, who was later diagnosed with growth hormone deficiency, showed incomplete healing of PA with intermittent pus discharge and recurrence. The patient was cured by 1 year of age following repeated administration of TJ-122 and juzentaihoto (TJ-48). CONCLUSION: Medical management with TJ-122 was effective in most neonates and young infants with PA. It appears prudent to manage these patients with hainosankyuto before resorting to surgical intervention.

Meconium-related ileus in extremely low-birthweight neonates: etiological considerations from histology and radiology.

BACKGROUND: A nationwide survey on neonatal surgery conducted by the Japanese Society of Pediatric Surgeons has demonstrated that the mortality of neonatal intestinal perforation has risen over the past 15 years. The incidence of intestinal perforation in extremely low-birthweight (ELBW) neonates has been increasing as more ELBW neonates survive and as the live-birth rate of ELBW has increased. In contrast to necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP), the pathogenesis of meconium-related ileus, defined as functional bowel obstruction characterized by delayed meconium excretion and microcolon, remains unclarified. METHODS: The histology of 13 ELBW neonates with intestinal perforation secondary to meconium-related ileus was reviewed, and the radiology of 33 cases of meconium-related ileus diagnosed on contrast enema was reviewed. Specimens obtained from 16 ELBW neonates without gastrointestinal disease served as age-matched controls for histological assessment. RESULTS: The size of the ganglion cell nucleus in meconium-related ileus and in control subjects was 47.3 ± 22.0 µm(2) and 37.8 ± 11.6 µm(2), respectively, which was not significantly different. In all cases of meconium-related ileus, contrast enema demonstrated a microcolon or small-sized colon, with a gradual caliber change in the ileum and filling defects due to meconium in the ileum or colon, showing not-identical locations of caliber changes and filling defects. CONCLUSION: Morphological immaturity of ganglia was not suggested to be the pathogenesis of meconium-related ileus. Impaction of inspissated meconium is not the cause of obstruction, but the result of excessive water absorption in the hypoperistaltic bowel before birth, although the underlying mechanism responsible for the fetal hypoperistalsis remains unclear.

Therapeutic strategy for persistent cloaca: the efficacy of antegrade continent enema as a salvage surgery.

PURPOSE: The aim of this study is to evaluate our therapeutic strategy for persistent cloaca from the viewpoint of long-term functional outcome. MATERIALS AND METHODS: This study covers 17 cases of persistent cloaca treated at our institution and followed for more than 3 years. As a definitive repair for anorectal and urogenital systems, simultaneous surgery with posterior sagittal approach or anorecto-urethrovagino-plasty (PSARUVP) was performed. The length of the common channel and the shape of the vagina determined the vaginoplasty methods. Fecal function was assessed with the scoring system of the Japan Study Group of Anorectal Anomalies. RESULTS: Anorectoplasty was performed with the posterior sagittal approach in 15 cases and with the perineal approach in two. Vaginoplasty was performed with total urogenital mobilization in nine cases, rectal interposition in four, vaginal flap in two and with other methods. Fecal function was classified as good in three cases, moderate in ten, and poor in four. In the poor cases, Malone's antegrade continence enema (MACE) was performed, which improved fecal function significantly. CONCLUSION: PSARUVP might be the optimal surgery for persistent cloaca at present; however, satisfactory fecal function could not be achieved in those cases with a longer common channel. MACE effectively compensated for the poor outcome and was especially successful at eliminating incontinence.

Physiological and clinical characteristics of gastroesophageal reflux after congenital diaphragmatic hernia repair.

BACKGROUND/PURPOSE: Gastroesophageal reflux (GER) is an important sequela of congenital diaphragmatic hernia (CDH) repair. This study investigated the physiological and clinical characteristics of GER in CDH survivors. METHODS: A total of 52 CDH survivors were investigated retrospectively. Esophageal acid exposure was evaluated with 24-h esophageal pH monitoring in all patients, and esophageal anatomical and motor functional abnormalities were examined with videomanometry in 16 patients. RESULTS: Fundoplication was necessary in 1 patient. Medical treatment with acid suppression or rikkunshito, a traditional Japanese medicine, was successful in nine patients, and the reflux symptoms were ameliorated at the age of 3 years. The percentage of total time the esophageal pH was below 4.0 (reflux index: RI) ranged from 0.1 to 44.3%. No patient with an RI < 10% had reflux symptoms requiring treatment. The basal lower esophageal sphincter (LES) tone ranged from 15 to 35 mmHg (median 25 mmHg). Esophageal peristalsis was preserved in all of the patients examined, except one who had failed peristalsis and poor clearance in the dilated esophagus. CONCLUSION: The motor function of the esophageal body and LES is usually preserved in CDH survivors despite the wide range of esophageal acid exposure in early infancy. Those with symptomatic GER outgrow it, unless associated with advanced respiratory distress or neurological impairment.

Should fundoplication be added at the time of gastrostomy placement in patients who are neurologically impaired?

BACKGROUND/PURPOSE: Patients who have advanced neurologic impairment (NI) and require gastrostomy placement (GP) frequently have symptomatic gastroesophageal reflux. We investigated the outcomes of GP without fundoplication in patients who had NI. METHODS: This was a retrospective review of 54 patients with NI (median, 7 years; range, 1-18 years) undergoing GP alone. The operative criteria included medically controllable or no reflux symptoms. The patients were divided into 2 groups based on the percentage of total esophageal time with a pH less than 4.0 (reflux index, or RI): group I (GI, n = 33), RI less than 5.0% (median age, 6 years; range, 2-15 years); group II (GII, n = 21), RI 5.0% or greater (median age, 10 years; range, 1-18 years). Data are expressed as medians and ranges. RESULTS: Nutritional management was successfully conducted after GP with or without the administration of lansoprazole, famotidine, or rikkunshito in all but 2 patients. One GI patient with alpha-thalassemia required fundoplication, and one GII patient with Cockayne syndrome required gastrojejunal tube feeding. The RI increased significantly in GI patients (2.1% [0%-4.8%] vs 4.5% [0.2%-11.4%], P = .004), whereas it decreased significantly in GII patients (11.2% [5.9%-41.6%] vs 9.8% [1.05-26.6%], P = .04). CONCLUSION: Gastroesophageal reflux and related symptoms rarely deteriorate to require additional treatment after GP in patients with NI. Gastrostomy placement is a less invasive and effective procedure for improving the quality of life in those patients.

Activation of a dormant ClpX recognition motif of bacteriophage Mu repressor by inducing high local flexibility.

The C-terminal domain (CTD) of bacteriophage Mu immunity repressor (Rep) regulates DNA binding by the N-terminal domain and degradation by ClpXP protease. Five residues at the Rep C terminus (CTD5) can serve as a ClpX recognition motif, but it is dormant unless activated, a state that can be induced by the presence of dominant-negative mutant repressors (Vir). Conversion of Rep to ClpXP-sensitive form was associated with not only increased exposure of CTD5 to solvent but also increased CTD motion or flexibility as measured by fluorescence anisotropy. CTD mutations (V183S, K193S, and V196S) promoting ClpXP resistance without destroying the recognition motif prevented increased CTD motion induced by Vir. Suppression of ClpXP protease resistance conferred by the V196S mutation also correlated with restoration of CTD motion. The temperature-sensitive R47Q mutation present in cis within the DNA-binding domain restored ClpXP protease sensitivity to the V196S mutant, and anisotropy analysis indicated that R47Q allows the V196S CTD to gain increased flexibility when Vir was present. The results indicate that the CTD functions to turn the recognition motif on and off, most likely by modulating flexibility of the domain that harbors the ClpX recognition motif, suggesting a general mechanism by which proteins can regulate their own degradation.

Trans-targeting of protease substrates by conformationally activating a regulable ClpX-recognition motif.

Conversion of bacteriophage Mu repressor to ClpXP-sensitive form correlates with induced local flexibility at the ClpX recognition motif located at the C-terminal end. Changing the C-terminal valine to an alanine (RepV196A) caused the degradation tag to be constitutively active like that of mutant repressors called Vir, which have a dominant ClpXP-sensitive conformation. However, unlike Vir, RepV196A was unable to convert wild-type repressor (Rep) to the ClpXP-sensitive form. In mixtures with Rep, only RepV196A was rapidly degraded by ClpXP. Unlike Rep, RepV196A was ClpXP sensitive without induced C-terminal flexibility. And unlike adaptor proteins that tether and deliver substrates to ClpX for trans-targeting, Vir promoted rapid degradation of Rep by ClpX deleted for the tethering site that binds adaptor proteins. Therefore, Rep's ClpX recognition motif has regulable properties, allowing an alternative trans-targeting mechanism in which an inactive degradation tag is turned on by induced conformational changes.