The hospital frailty risk score in patients with heart failure is strongly associated with outcomes but less so with pharmacotherapy.

BACKGROUND: Although frailty is known to be an important prognostic factor in heart failure (HF), HF risk-adjustment models do not incorporate frailty measures and the interplay between frailty, age and pharmacotherapy is unclear. OBJECTIVES: To explore the relationships between frailty, pharmacotherapy and outcomes in heart failure (HF). METHODS: Retrospective cohort study of all adults in Alberta, Canada hospitalized for the first time for HF between 2004 and 2016. Frailty was defined using the Hospital Frailty Risk Score (HFRS). RESULTS: In 26 626 patients (mean age 77.4 years), the 8887 (33.4%) defined as frail (HFRS ≥ 5) were older, had higher Charlson scores and more prior emergency department visits or hospitalizations. The HFRS and the Charlson Score were only weakly correlated (r = 0.35). Whilst more common in older patients (41.4% of patients 80 or older), frailty was present in 22.4% of patients younger than 65. Frail patients had longer lengths of stay and worse outcomes postdischarge, but adding the HFRS to age, sex and Charlson score did not improve prediction of events (c-statistics 0.69 for 30-day mortality after admission, and 0.54 for 30-day readmission/ED visit/or death after discharge). Frail patients younger than 65 were significantly more likely than nonfrail patients 80 or older to be prescribed high-dose evidence-based HF therapies (27.1% vs. 22.2%, P = 0.003). CONCLUSION: Although the HFRS reflects aspects of frailty that patient age and Charlson scores do not, the addition of the HFRS to standard risk prediction equations provides little additional information. Prescribing practices correlate more with patient age than frailty status.

Elevated fasting vs post-load glucose levels and pregnancy outcomes in gestational diabetes: a population-based study.

AIMS: To examine the relative association between fasting plasma glucose vs post-load (1-h and 2-h) glucose levels based on the oral glucose tolerance test in pregnancy and large-for-gestational-age and hypertensive disorders of pregnancy outcomes. METHODS: All live singleton births between October 2008 and December 2014 in Alberta, Canada were included. Gestational diabetes mellitus was diagnosed using Diabetes Canada criteria. Logistic regression models were used to examine the association between fasting plasma glucose vs post-load values and large-for-gestational-age infants and hypertensive disorders of pregnancy after adjusting for maternal characteristics and pharmaceutical intervention in gestational diabetes pregnancies. RESULTS: Among 257 547 pregnancies, 208 344 (80.9%) had negative 50-g glucose challenge tests, 36 261 (14.1%) had negative 75-g oral glucose tolerance tests, and 12 942 (5.0%) had gestational diabetes based on either elevated fasting plasma glucose (n=4130, 1.6%) or elevated 1-h and/or 2-h oral glucose tolerance test values (n=8812, 3.4%). Large-for-gestational-age and hypertensive disorders of pregnancy rates were 8.1% and 5.1% in negative glucose challenge test pregnancies, 11.0% and 7.0% in negative oral glucose tolerance test pregnancies, 22.4% and 11.9% in gestational diabetes pregnancies with elevated fasting plasma glucose, and 9.1% and 8% in gestational diabetes pregnancies with elevated post-load levels, respectively. Among gestational diabetes pregnancies, those with elevated fasting plasma glucose were at higher risk of large-for-gestational age (adjusted odds ratio 2.66, 95% CI 2.39-2.96) and hypertensive disorders of pregnancy (adjusted odds ratio 1.51, 95% CI 1.33-1.72) outcomes relative to pregnancies with post-load glucose elevations only. Fasting plasma glucose remained significantly associated with adverse outcomes in gestational diabetes pregnancies with and without pharmacological intervention. CONCLUSIONS: Elevated fasting plasma glucose in women with gestational diabetes is a stronger predictor of large-for-gestational-age and hypertensive disorders of pregnancy outcomes than elevated post-load glucose.

History of mood or anxiety disorders and risk of gestational diabetes mellitus in a population-based cohort.

AIM: To examine the association between mood and anxiety disorders and the development of gestational diabetes mellitus in a retrospective population-based cohort study. METHODS: Clinical data from a provincial perinatal health registry were linked to physician claims, hospitalization records and emergency visits to identify any diagnoses of mood or anxiety disorders in the 2 years prior to pregnancy and a subsequent diagnosis of gestational diabetes during pregnancy. The study population included all singleton pregnancies in the Canadian province of Alberta from 1 April 2000 to 31 March 2010. Generalized estimating equations were used to determine the adjusted odds ratio of gestational diabetes, comparing women with and without a history of mood or anxiety disorders. RESULTS: Among 373 674 pregnancies from 253 911 women, 25.7% had a history of mood or anxiety disorders, and 3.8% developed gestational diabetes. The multivariate-adjusted odds of developing gestational diabetes were higher among women with a history of mood or anxiety disorders (odds ratio 1.10, 95% CI 1.06-1.14). CONCLUSIONS: Women with a history of mood or anxiety disorders had a moderately increased risk of developing gestational diabetes.

Validation of administrative and clinical case definitions for gestational diabetes mellitus against laboratory results.

AIM: To examine the validity of International Classification of Disease, version 10 (ICD-10) codes for gestational diabetes mellitus in administrative databases (outpatient and inpatient), and in a clinical perinatal database (Alberta Perinatal Health Program), using laboratory data as the 'gold standard'. METHODS: Women aged 12-54 years with in-hospital, singleton deliveries between 1 October 2008 and 31 March 2010 in Alberta, Canada were included in the study. A gestational diabetes diagnosis was defined in the laboratory data as ≥2 abnormal values on a 75-g oral glucose tolerance test or a 50-g glucose screen ≥10.3 mmol/l. RESULTS: Of 58 338 pregnancies, 2085 (3.6%) met gestational diabetes criteria based on laboratory data. The gestational diabetes rates in outpatient only, inpatient only, outpatient or inpatient combined, and Alberta Perinatal Health Program databases were 5.2% (3051), 4.8% (2791), 5.8% (3367) and 4.8% (2825), respectively. Although the outpatient or inpatient combined data achieved the highest sensitivity (92%) and specificity (97%), it was associated with a positive predictive value of only 57%. The majority of the false-positives (78%), however, had one abnormal value on oral glucose tolerance test, corresponding to a diagnosis of impaired glucose tolerance in pregnancy. CONCLUSIONS: The ICD-10 codes for gestational diabetes in administrative databases, especially when outpatient and inpatient databases are combined, can be used to reliably estimate the burden of the disease at the population level. Because impaired glucose tolerance in pregnancy and gestational diabetes may be managed similarly in clinical practice, impaired glucose tolerance in pregnancy is often coded as gestational diabetes.

Validation of administrative data case definitions for gestational diabetes mellitus.

AIM: To examine, using administrative data, the validity of two algorithms for identifying gestational diabetes mellitus: 1) the current National Diabetes Surveillance System algorithm for excluding gestational diabetes cases and 2) gestational diabetes-specific ICD codes in the delivery-related hospitalization. METHODS: This was a retrospective study of all women, aged 18-54 years, residing in Alberta, Canada, with singleton deliveries between 1 April 1999 and 31 March 2010. We linked Alberta Perinatal Health Program data on all deliveries to administrative claims data from Alberta Health using the mother's personal health number. For both gestational diabetes algorithms, we calculated the sensitivity, specificity, positive predictive value, negative predictive value and agreement, using gestational diabetes identified in the Alberta Perinatal Health Program as the 'gold standard'. RESULTS: Our study sample consisted of 411 390 deliveries for 273 152 women. The mean (sd) age was 29.1 (5.6) years and 82.3% of the women were white. Crude rates of gestational diabetes were 3.9% (16 215 cases), 1.3% (5189 cases) and 4.0% (16 440 cases) according to the Alberta Perinatal Health Program, National Diabetes Surveillance System and ICD code-based algorithms, respectively. Compared with the Alberta Perinatal Health Program database, the National Diabetes Surveillance System algorithm had a sensitivity of 25% and specificity of 100%, whereas the gestational diabetes-specific ICD code-based algorithm had a sensitivity of 86% and specificity of 99%. CONCLUSIONS: The National Diabetes Surveillance System algorithm underestimates the number of gestational diabetes cases. A more valid mechanism to identify gestational diabetes prevalence using health administrative data is the use of gestational diabetes-specific ICD-9/10 codes in the delivery hospitalization.

Patterns of glucose-lowering therapies and neonatal outcomes in the treatment of gestational diabetes in Canada, 2009-2014.

AIM: To examine patterns of use of different glycaemic control agents for treating gestational diabetes mellitus. METHODS: This was a large, retrospective, population-based cohort study of pregnant women with gestational diabetes from Alberta, Canada. We linked data from the Alberta Vital Statistics - Birth database with administrative claims data. Alberta Vital Statistics - Birth data were used to identify births that occurred between 1 January 2009 and 31 December 2014. We used International Classification of Diseases version 9/10 codes to identify women with gestational diabetes, and we excluded women with pre-existing diabetes. RESULTS: Our cohort consisted of 16 857 women with gestational diabetes, with a total of 18 761 birth events between 2009 and 2014. Over the study period, the proportion of women with gestational diabetes who were treated with glycaemic control therapies increased from 25.0% to 31.4% (P<0.0001). The number of pregnancies treated with insulin only increased (from 23.6% to 28.3%; P<0.0001), as did the number treated with metformin, +/- insulin (from 1.4% to 3.2%; P<0.0001). Rates of large-for-gestational-age infants were significantly higher among pregnancies treated with insulin only (17%) or metformin (16.5%) than among pregnancies that did not receive any pharmacological treatment (12.8%). CONCLUSIONS: Our findings show increasing use of insulin and metformin in women with gestational diabetes. Rates of large-for-gestational-age infants were similar among pregnant women receiving either pharmacological treatment, and higher than among pregnant women who did not receive any pharmacological treatment. Future research should explore the long-term outcomes and safety of metformin as an alternative for treating gestational diabetes.

Impact of gestational diabetes mellitus and high maternal weight on the development of diabetes, hypertension and cardiovascular disease: a population-level analysis.

AIMS: To examine the association between gestational diabetes mellitus (GDM) and high maternal weight and the risk of development of chronic disease. METHODS: Women with singleton deliveries between April 1999 and March 2010 in Alberta, Canada, were categorized according to pre-pregnancy weight (overweight ≥ 91 kg) and GDM status. Obstetric and neonatal outcomes, as well as the long-term incidence of maternal diabetes, hypertension and cardiovascular disease were examined. RESULTS: Of 240 083 women, 213 765 (89%) had no GDM and were not overweight (reference group), 17 587 (7.3%) were overweight only, 7332 (3%) had GDM only and 1399 (0.6%) had GDM and were overweight. Significant differences in Caesarean section rates, induction rates and birthweight were observed across the four groups. During a median follow-up of 5.3 years, diabetes incidence was 36% in the GDM and overweight, 18.8% in the GDM only, 4.8% in the overweight only and 1.1% in the reference group. With respect to hypertension and cardiovascular disease, the GDM and overweight group had the highest rates (26.8% and 3.1%, respectively) and the reference group had the lowest rates (5.8% and 1.0%, respectively). However, rates were similar in the GDM only (14.9% and 1.9%, respectively) and overweight only groups (14.9% and 1.5%, respectively). CONCLUSIONS: Not surprisingly, the presence of both high maternal weight and GDM compounds the risk of developing diabetes. However, the association between overweight alone and GDM alone and hypertension and cardiovascular disease appears similar suggesting a need for effective interventions to manage both these conditions to improve the health of these patients.

Health Canada advisory impacts on the prevalence of oral codeine use in the Pediatric Canadian population: comparative study across provinces.

Health Canada (HC) has, since 2013, issued safety alerts restricting the use of codeine-containing drugs among breastfeeding women and children/adolescents under 18 years of age. These products are linked to breathing problems among ultra-rapid CYP2D6 metabolizers and early use of opioid can lead to future opioid misuse. Using a multi-province population-based cohort study, we estimate the impact of federal safety alerts on annual rates of codeine use in the Canadian pediatric population. We analyzed data from 8,156,948 children/adolescents in five Canadian provinces between 1996 and 2021, using a common protocol. Children/adolescents were categorized as: ≤ 12 years (children) or > 12 years (adolescents). We defined codeine exposure by ≥ 1 prescription filled for codeine alone or combined with other medications. For both age categories, we obtained province-specific codeine prescription filling rates per calendar year by dividing the number of children/adolescents with ≥ 1 codeine prescription filled by the number of person-time. Annual rates of codeine use per 1000 persons vary by province from 3.0 (Quebec) to 10.1 (Manitoba) in children, and from 5.5 to 51.3 in adolescents. After the 2013 HC advisory, exposure decreased in all provinces (adjusted level change from - 0.6 to - 18.4%) in children and from - 2.1 to - 17.9% in adolescents after the 2016 advisory. Annual rates declined over time in all provinces, following HC safety alerts specific to each of the two age categories.

Catheter-based renal denervation in the treatment of resistant hypertension.

Clinical trials have shown that catheter-based renal denervation (RD), i.e. interruption of afferent and efferent sympathetic nerves supplying the kidney, can reduce systolic blood pressure (BP) by approximately 30 mm Hg. This technology is currently being tested as a therapeutic option for patients with resistant hypertension, a condition in which BP remains elevated despite adherence to a rational medication regimen. This novel treatment approach was developed on the basis of a wealth of animal and human research demonstrating the importance of the sympathorenal axis in the pathogenesis of hypertension. Sympathetic efferent signals to the kidneys raise BP by stimulating sodium retention and renin release, and the kidneys influence central sympathetic drive via afferent nerves. But as is true with many therapeutic advances, RD has shown benefit in clinical studies long before the mechanisms are fully understood. Additional research is needed to understand the contribution of afferent sympathetic nerve interruption to BP reductions observed with RD; to examine the degree and significance of re-innervation following RD; to elucidate factors that may lead to a lack of response to RD in some patients; to determine whether the modulation of the sympathetic nervous system via RD can have beneficial effects independent of BP reduction; and to develop methods to measure the effectiveness of RD in real time.

Prognostic significance of soft tissue deposits in head and neck squamous cell carcinoma: a systematic review and meta-analysis.

Soft tissue deposits, also known as tumour deposits (TDs), have not been studied well in head and neck squamous cell carcinoma (HNSCC) and are not included in any of the staging systems or treatment guidelines. The aim of this systematic review was to determine the prevalence and prognostic implications of TDs in patients with HNSCC. This systematic review of the literature was conducted following the PRISMA guidelines. The PubMed, Embase, and Scopus electronic databases were searched for relevant studies, from inception to August 2022. Although 14 studies qualified for inclusion, only eight were finally included in the review due to the considerable overlap of patients in several studies. Data from 7127 patients were analysed. The pooled prevalence of TDs was 21% (95% confidence interval (CI) 9-33%). The presence of TDs was adversely associated with overall survival and disease-free survival, with hazard ratios of 2.08 (95% CI 1.60-2.70) and 2.56 (95% CI 1.97-3.32), respectively. TDs are detected in a significant number of patients with HNSCC and adversely affect survival. Longitudinal prospective studies are needed to evaluate the prognostic implications of TDs in HNSCC for their potential role in cancer staging and adjuvant treatment planning.

Innominate truncal dissection and rupture into right pleural cavity following acute type A dissection of the aorta with right coronary ostial avulsion and inferior STEMI.

An innominate truncal dissection and rupture into the right pleural cavity with massive hemothorax is the initial presentation in this 66-year-old lady with type A dissection of the aorta complicated by right coronary ostial avulsion and inferior STEMI. She underwent supracoronary interposition graft replacement of the ascending aorta and hemiarch, interposition graft replacement of the innominate trunk and saphenous vein bypass grafting of the right coronary artery successfully. Innominate truncal rupture following aortic dissection is practically unknown and has not been described before in the absence of aortic rupture. Innominate truncal rupture secondary to other pathologies presents with supraaortic and mediastinal hematomas, but almost never with right hemothorax. On the backdrop of this unusual presentation with no neurological injury, we review the literature for innominate truncal dissection and rupture, other etiologies for innominate truncal rupture, the complex interplay of factors determining neurological injury and discuss the changes in the strategies and conduct of arterial return during cardiopulmonary bypass and selective antegrade perfusion imposed by this previously undescribed instance of innominate truncal rupture due to dissection.

CD46 polymorphism: a probable risk factor for recurrent spontaneous abortion in a northern Indian population.

BACKGROUND: CD46 is a complement regulatory glycoprotein. Certain polymorphic forms of the CD46 gene have been associated with recurrent pregnancy loss in the Caucasian population. We assessed the role of CD46 polymorphism in recurrent spontaneous abortion in our setting, as this has not been done on Indian subjects till date. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out on 44 samples each from women with recurrent spontaneous abortion and normal pregnancy. Genotyping of the CD46 gene was done using 2.5% agarose gel. Statistical analysis was done using the TFPGA software. RESULTS: The absence of CD46H*1 homozygosity was more pronounced in women with recurrent spontaneous abortion in the Indian population. Of recurrent aborters, 9% had the H*1/*l genotype as compared to 30% of normal pregnant women. CONCLUSION: Although our data did not fit the Hardy-Weinberg equilibrium, this pilot study indicates that further increasing the sample size might clarify whether polymorphism in the first intron of the CD46 gene can be regarded as a risk factor for recurrent spontaneous abortion.

Single photon emission computed tomography myocardial imaging: clinical applications and future directions.

Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) has proven to be an invaluable tool in the non-invasive assessment of patients with cardiovascular diseases. Exercise and pharmacologic stress SPECT MPI has been extensively studied and validated in the diagnosis and prognosis of patients with known and suspected coronary artery disease. In addition to the assessment of perfusion, electrocardiography-gated SPECT offers additional functional and volumetric information that can have significant implications on patient care. SPECT also has well-developed applications in systolic heart failure for identifying viable myocardium and predicting response to revascularization. Emerging applications of SPECT imaging include: l) phase analysis for quantitative assessment of LV dyssynchrony that may serve to improve patient selection for CRT; 2) application of heart-to-mediastinal ratio via 123I meta-iodobenzylguanidine (I-MIBG) imaging for the prediction of sudden death in patients with reduced left ventricular function, that may serve to improve patient selection for ICD therapy, and 3) use of metabolic tracers to identify the changes of ''ischemic memory'', which may help with rapid and appropriate triage of patients in the emergency room setting. SPECT MPI remains a cornerstone of clinical care that is evolving with emerging and novel applications that will continue to improve the care of patients with cardiovascular disease in the future.

Population-Level Outcomes with a 2-Step Approach for Gestational Diabetes Screening and Diagnosis.

OBJECTIVES: To examine outcomes associated with alternative glucose thresholds in a 2-step approach for screening and diagnosing gestational diabetes mellitus (GDM). METHODS: We studied 178,527 pregnancies between 2008 and 2012 in Alberta, Canada. They were categorized retrospectively as normal 50 g screen (n=144,191); normal 75 g oral glucose tolerance test (OGTT) (n=21,248); abnormal at glucose thresholds suggested by the International Association of Diabetes and Pregnancy Group (IADPSG) (HAPO 1.75, n=4308); abnormal at glucose thresholds associated with an odds ratio of 2.0 for adverse events in the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study. This latter group, which would have been treated for GDM based on customary care, was further divided into those with 1 (HAPO 2-1 n=5528) or 2 or more abnormal glucose values (HAPO 2-2 n=3252). Main outcomes were large for gestational age (LGA), induced labour and Cesarean-section rates. RESULTS: LGA rates were 8.2%, 10.5%, 14.2%, 11.8% and 16.5% among normal 50 g, normal 75 g OGTT, HAPO 1.75, HAPO 2-1, and HAPO 2-2 groups, respectively. Labour induction and caesarean-section rates were 29.6% and 36.2% in the IADPSG, 38.2% and 36.8% in the HAPO 2-1 group, and 42.3% and 41.1% in the HAPO 2-2 groups, respectively. Excessive maternal weight (≥91 kg) was associated with a higher risk for all adverse outcomes. CONCLUSIONS: The 2-step approach effectively identifies pregnancies at low risk for adverse outcomes. Labelling influences induction practice. Any glucose intolerance increases risk for adverse outcomes, and pregnancies with highest (2 or higher) abnormal glucose values remain at greatest risk. Further research is needed to determine whether glycemic thresholds for GDM diagnosis should incorporate information about maternal weight.

Does the North American Society for Pediatric and Adolescent Gynecology Short Curriculum Increase Resident Knowledge in Pediatric and Adolescent Gynecology?

STUDY OBJECTIVE: To determine if the North American Society for Pediatric and Adolescent Gynecology (NASPAG) Short Curriculum improves self-reported knowledge in pediatric and adolescent gynecology (PAG) among obstetrics and gynecology (Ob/Gyn) residents, at programs without PAG-trained faculty. DESIGN: Prospective, cross-sectional exposure to the NASPAG short curriculum with a follow-up questionnaire. SETTING: Ob/Gyn residency training programs without PAG faculty. PARTICIPANTS: Ob/Gyn residents in training from February 2015 to June 2015. INTERVENTIONS: Exposure to the NASPAG Short Curriculum. MAIN OUTCOME MEASURES: Improvement in self-perceived knowledge after completion of curriculum. RESULTS: Two hundred twenty-seven residents met inclusion criteria; 34 completed the study (15% response). Less than 50% of residents reported adequate knowledge in the areas of prepubertal vaginal bleeding, vulvovaginitis, precocious and delayed puberty, Home environment, Education and Employment, Eating, peer-related Activities, Drugs, Sexuality, Suicide/depression, Safety from injury and violence (HEEADSSS) interview, pelvic pain, and bleeding management in teens with developmental delay. After completion of the curriculum, self-reported knowledge improved in 8 of 10 learning objectives, with no significant improvement in bleeding disorders or Müllerian anomalies. There was no association between pretest knowledge and level of residency training, type of residency program, previous exposure to PAG lectures, and previous exposure to patients with PAG complaints. CONCLUSION: Significant deficiencies exist regarding self-reported knowledge of core PAG topics among Ob/Gyn residents at programs without PAG-trained faculty. Use of the NASPAG Short Curriculum by residents without access to PAG-trained faculty resulted in improved self-reported knowledge in PAG.

Prognostic value of ST segment depression in acute coronary syndromes: insights from PARAGON-A applied to GUSTO-IIb. PARAGON-A and GUSTO IIb Investigators. Platelet IIb/IIIa Antagonism for the Reduction of Acute Global Organization Network.

OBJECTIVES: Our objectives were to develop a risk-stratification model addressing the importance of the magnitude and distribution of ST segment depression in predicting long-term outcomes and to validate the model in an analogous patient population. BACKGROUND: Although patients without ST segment elevation presenting with acute coronary syndromes represent an increasingly frequent population admitted to coronary care units, little attention has been paid to quantifying their ST segment abnormalities. METHODS: ST segment depression was categorized into three groups: 1) no ST segment depression; 2) 1-mm ST segment depression in two contiguous leads; and 3) ST segment depression > or =2 mm in two contiguous leads. A logistic regression model was developed using Platelet IIb/IIIa Antagonism for the Reduction of Acute coronary syndrome events in a Global Organization Network (PARAGON-A) data to assess the prognostic value of the extent and distribution of ST segment depression in predicting one-year mortality. The model was validated using the non-ST segment elevation population in Global Use of Strategies To Open occluded arteries in acute coronary syndromes (GUSTO-IIb). RESULTS: ST segment depression was the strongest predictor of one-year mortality, accounting for 35% of the model's predictive power. Patients with ST segment depression > or =2 mm were approximately 6 times (odds ratio [OR] 5.73, 95% confidence interval [CI] 2.8 to 11.6) more likely to die within one year than patients with no ST segment depression. On validation, the model showed good discriminatory power (c-index = 0.75). Patients with ST segment depression > or =2 mm in more than one region were almost 10 times more likely to die within one year than patients with no ST segment depression. CONCLUSIONS: These data provide new evidence supporting the powerful prognostic value of the baseline electrocardiogram and, in particular, the magnitude and distribution of ST segment depression in predicting unfavorable events.

Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin.

Acidic conditions within the endosomal lumen induce the T domain of receptor-bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A + D352A and fourfold for E349Q + D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.