Thermal fatalities in Pretoria: A 5-year retrospective review.

In South Africa, research on burn mortality has emanated primarily from specialised burn centres and has focused on specific age groups and hospital-based fatalities. This study describes the demographic profile and the pathology of trauma related to burn fatalities as seen at the Pretoria Medico-Legal Laboratory (PTA MLL), a large urban medico-legal mortuary over a 5-year period from January 2011 to December 2015. Mortuary admission records and autopsy reports were used to gather information on demographics, circumstances of injury, apparent manner and cause of death, pathology of burns, toxicology and histology reports and identification of the decedents. RESULTS: Of the 9558 unnatural deaths admitted to the PTA MLL during this time period, 291 (3.0%) of the fatalities met the inclusion criteria. The male:female ratio was 2.9:1. Most fatalities occurred between the ages of 0-4 years. One hundred and forty-two (142) decedents were charred beyond recognition. Identification was confirmed in 134 (94.4%) of the charred remains. In 208 (69.8%) of the cases the manner of death was deemed to be accidental, 23 (7.9%) were homicidal and 11 (3.8%) were suicides. Two hundred and fifty-five (87.4%) of the fatalities were as a result of open flames/fires. Shack fires were responsible for 105 (36%) of all fatalities. In 32 (11.0%) cases of open flame/fire fatalities where death occurred at the scene of injury, more than one fatality was reported per incident. In 122 (79.2%) of scene fatalities, soot deposition was noted in the upper and lower airways. Forty-five (32.8%) of hospital fatalities occurred within 24 h of admission. The most common complications in hospital fatalities were from the respiratory system. The mean blood alcohol concentrations (BAC) was 0.09 g/100 ml. The mean carboxyhaemoglobin concentrations (COHb) was 19.9%. All available cyanide results were negative. CONCLUSION: The study is the first of its kind in South Africa to generate bimodal descriptive statistics for burn fatalities. Approximately 3% of unnatural deaths at the PTA-MLL were due to burns, occurring at a rate of ±1 death per week. The data provides a platform for funding, collaborative research, planning and development of public health programs.

Association between non-alcoholic hepatic steatosis and hyper reactive blood pressure response on the exercise treadmill test.

AIMS: Non-alcoholic hepatic steatosis (HS) is associated with hypertension and increased cardiovascular risk. While Blood pressure hyper-reactive response (HRR) during peak exercise indicates an increased risk of incident hypertension and increased cardiovascular risk, no data on the association of non-alcoholic HS and HRR exists. In this study, we have evaluated the association of HS with HRR. METHODS: We included 13 410 consecutive individuals with a mean age: 42.4 ± 8.9 years, 3561 (26.6%) female with normal resting blood pressure and without a previous diagnosis of hypertension, who underwent symptom limited exercise treadmill test, abdominal ultrasonography and clinical and laboratory evaluation. HS was detected by abdominal ultrasonography. HRR was defined by a peak exercise systolic blood pressure >220 mmHg and/or elevation of 15 mmHg or more in diastolic blood pressure from rest to peak exercise. RESULTS: The prevalence of HS was 29.5% (n = 3956). Overall, 4.6% (n = 619) of the study population presented a HRR. Subjects with HS had a higher prevalence of HRR (8.1 vs. 3.1%, odds ratio 2.8, 95% CI 2.4-3.3, P < 0.001). After adjustment for body mass index, waist circumference, fasting plasma glucose and low density lipoprotein cholesterol, HS (odds ratio 1.4, 95% CI 1.1-1.6, P = 0.002) remained independently associated with HRR. HS was additive to obesity markers in predicting exercise HRR. CONCLUSIONS: Non-alcoholic HS is independently associated with hyper-reactive exercise blood pressure response.

Associations of pentraxin 3 with cardiovascular disease: the Multi-Ethnic Study of Atherosclerosis.

OBJECTIVE: Pentraxin 3 (PTX3) is probably a specific marker of vascular inflammation. However, associations of PTX3 with cardiovascular disease (CVD) risk have not been well studied in healthy adults or multi-ethnic populations. We examined associations of PTX3 with CVD risk factors, measures of subclinical CVD, coronary artery calcification (CAC) and CVD events in the Multi-Ethnic Study of Atherosclerosis. APPROACH AND RESULTS: Two thousand eight hundred and thirty-eight participants free of prevalent CVD with measurements of PTX3 were included in the present study. After adjustment for age, sex, and ethnicity, PTX3 was positively associated with age, obesity, insulin, systolic blood pressure, C-reactive protein (CRP), and carotid intima-media thickness (all P < 0.045). A one standard deviation increase in PTX3 level (1.62 ng mL(-1) ) was associated with the presence of CAC in fully adjusted models including multiple CVD risk factors (relative risk of 1.05; 95% confidence interval [CI] 1.01-1.08). In fully adjusted models, a standard deviation higher level of PTX3 was associated with an increased risk of myocardial infarction (hazard ratio [HR] 1.51; 95% [CI] 1.16-1.97), combined CVD events (HR 1.23; 95% [CI] 1.05-1.45), and combined CHD events (HR 1.33; 95% [CI] 1.10-1.60), but not stroke, CVD-related mortality, or all-cause death. CONCLUSIONS: In these apparently healthy adults, PTX3 was associated with CVD risk factors, subclinical CVD, CAC and incident coronary heart disease events independently of CRP and CVD risk factors. These results support the hypothesis that PTX3 reflects different aspects of inflammation than CRP, and may provide additional insights into the development and progression of atherosclerosis.

Cardiovascular disease prevention: matching evidence-based algorithms with individualized care.

The appropriate use of statins in primary prevention remains a matter of debate. Although statins reduce cardiovascular events at all levels of baseline risk, they are associated with rare but important side effects including incident diabetes. Herein, we review strategies for statin allocation ranging from strict "evidence-based" adherence to randomized controlled clinical trial (RCT) entry criteria to more "personalized" risk assessment using high-sensitivity C-reactive protein (hsCRP), coronary artery calcification (CAC), or genetic testing. Current guidelines advocate an unusual middle ground between an evidence-based approach and a personalized approach.

The relationship between inflammation, obesity and risk for hypertension in the Multi-Ethnic Study of Atherosclerosis (MESA).

It has been suggested that inflammation is important in the aetiology of hypertension and that this may be most relevant among obese persons. To study this, we examined the independent relationships between obesity, inflammation-related proteins (interleukin-6 (IL-6), C-reactive protein (CRP) and fibrinogen) and risk for hypertension in the Multi-Ethnic Study of Atherosclerosis (MESA). Hypertension status, defined as a blood pressure ≥140/90 mm Hg or a history of hypertension and use of blood pressure medications, was determined at baseline and two subsequent exams over 5 years. Among 3543 non-hypertensives at baseline, 714 individuals developed incident hypertension by Exam 3. Cox proportional hazard models were used to determine the relationship between baseline levels of IL-6, CRP and fibrinogen and future risk of hypertension. One s.d. difference in baseline concentration of IL-6, CRP or fibrinogen was associated with 20-40% greater risk of incident hypertension. This risk was attenuated after accounting for other hypertension risk factors (hazard ratio (HR) IL-6: 1.13 (95% CI: 1.04-1.23); CRP: 1.11 (95% CI: 1.02-1.21); fibrinogen 1.0 (95% CI: 0.92-1.08)). Conversely, obesity was an independent risk factor for hypertension risk, minimally impacted by other covariates, including IL-6 and CRP (HR 1.72 (95% CI: 1.36-2.16)). IL-6 and CRP did not modify the relationship between obesity and hypertension, though an adjusted twofold greater risk was observed for obese individuals with a CRP >3 mg l⁻¹ compared with CRP <1 mg l⁻¹. The relationship between inflammation-related proteins and hypertension risk was predominantly explained by other hypertension risk factors. Obesity, independent of inflammation, remained a potent risk factor for future hypertension.

Improved nonviral cancer suicide gene therapy using survivin promoter-driven mutant Bax.

Suicide gene vectors are being developed in many laboratories as an attractive approach to cancer therapy. However, the development of these therapies is hampered by safety concerns and limitations of efficacy. The use of tumor-specific promoters, such as survivin promoter, can provide much needed specificity to target tumor cells. However, the expression levels from these promoters is often suboptimal and hence it is imperative to enhance the activity of the cytotoxic gene of interest. We tested apoptotic activity of several mutants of proapoptotic gene bax that constitutively translocate to the mitochondria and induce apoptosis. One of these mutants with deletion of serine at position S184 (S184del) was found to be most active and showed significant antitumor activity when expressed by the survivin promoter. In vitro testing shows that this vector (Sur-BaxS184del) induces cell killing in a variety of tumor cell lines of different origin with significantly higher efficacy than wild-type bax (Sur-BaxWT). The increase in cytotoxicity was a result of enhanced induction of apoptosis in tumor cells. In contrast to cytomegalovirus (CMV) promoter-driven bax (CMV-Bax), Sur-BaxS184del caused minimum toxicity in normal human dermal fibroblasts validating its specificity and safety. In a mouse tumor model (DA-3, murine breast cancer cells), we show that intratumoral injection of Sur-BaxS184del resulted in tumor growth retardation to the same level as CMV-Bax. This study highlights the effectiveness of using bax mutants in combination with survivin promoter for tumor-targeted suicide gene therapy in a nonviral vector.

Role of HIV Gp41 mediated fusion/hemifusion in bystander apoptosis.

Mechanisms of HIV-mediated CD4+ T cell loss leading to immunodeficiency are amongst the most extensively studied yet unanswered questions in HIV biology. The level of CD4+ T cell depletion in HIV infected patients far exceeds the number of infected T cells, suggesting an indirect mechanism of HIV pathogenesis termed bystander cell death. Evidence is accumulating that the HIV envelope glycoprotein (Env) is a major determinant of HIV pathogenesis and plays a critical role in bystander cell death. The complex structure and function of HIV Env makes the determination of the mechanism of Env mediated apoptosis more complex than previously thought. This review will examine the complex relationship between HIV Env phenotype, coreceptor expression and immune activation in determining HIV pathogenesis. We review data here corresponding to the role of HIV Env hemifusion activity in HIV pathogenesis and how it interplays with other AIDS associated factors such as chemokine receptor expression and immune activation.

Dissecting HIV fusion: identifying novel targets for entry inhibitors.

Significant momentum has been recently generated in understanding the HIV fusion process. This has led to the development of a host of HIV entry inhibitors which are currently in preclinical and/or clinical development or have been approved for clinical use. In this review we update our understanding of HIV fusion, specifically highlighting novel mechanisms and agents that inhibit this process. Major focus will be placed on three key areas. Initially viral attachment will be reviewed as recent developments in this field emphasize the importance of understanding cell type specific interactions with HIV. This has aided in identifying promising targets for the development of attachment inhibitors. Secondly, we will review the role of cellular lipids in HIV entry. Glycosphingolipids have been shown to interact with different components of the HIV fusion machinery and agents that perturb glycosphingolipid biosynthesis have inhibitory effects on HIV fusion. Likewise, manipulating ceramide biosynthesis also inhibits HIV fusion. Here, we describe how manipulating cellular lipids inhibits HIV fusion and how lipid biosynthesis can be modulated to potentially prevent HIV infection. We end this review by discussing the notion of targeting select host cell proteins for HIV therapy. We will review the role of the cellular proteins PDI, defensins and cytoskeletal proteins in facilitating the fusion reaction. As our understanding of the HIV fusion process increases, the identification of targets for developing entry inhibitors becomes more diverse. Given the rapid resistance of HIV to any selective pressure this is an important avenue in the advancement of drug therapy.

Association of white blood cell count with systolic blood pressure within the normotensive range.

Hypertension and inflammation promote cardiovascular disease (CVD). Even high normal systolic blood pressure (SBP) is associated with increased CVD risk. We assessed the relationship of elevated SBP within the normotensive range and white blood cell (WBC) count. This is a cross-sectional study of 3484 white asymptomatic individuals (mean age: 43+/-8 years, 79% males) without hypertension with SBP<140 mm Hg. White blood cell count >or=75th percentile (8.35 x 10(9) cells/l) was considered cutoff for elevated WBC. Subjects were classified into three levels of SBP (first: <120 mm Hg, n=1,176, 34%; second: 120-129 mm Hg, n=1,654, 47%; third: 130-139 mm Hg, n=654, 19%). Mean WBC count increased linearly across SBP categories (first: 6.14+/-1.54, second: 6.20+/-1.52, third: 6.41+/-1.62, P=0.02 for trend). There was a linear increase in prevalence of elevated WBC across higher SBP categories (22, 24 and 28%, P=0.02). As compared to those with SBP<120 mm Hg, in multivariate linear regression analyses (adjusting for age, gender, smoking status, diabetes, body mass index, physical activity, cholesterol/high-density lipoprotein cholesterol ratio) WBC count was significantly higher among participants with SBP 130-139 mm Hg (regression coefficient: 2.64, 95% confidence interval: 1.04-4.24, P=0.001). Odds ratio for prevalence of elevated WBC with SBP<120 mm Hg as reference group was 1.14 (0.92-1.41) for SBP 120-129 mm Hg and 1.50 (1.15-1.92) for SBP 130-139 mm Hg. In conclusion, Higher SBP within the normotensive range is also associated with elevated WBC count. Further studies are needed to clarify the role of inflammation in high normal SBP and associated CVD risk.

The clinical potential of sphingolipid-based therapeutics.

The era of sphingolipid-based therapeutics is upon us. A large body of work has been accumulating that demonstrates the distinct biological roles of sphingolipids in maintaining a homeostatic environment and in responding to environmental stimuli to regulate cellular processes. It is thus necessary to further investigate alterations in sphingolipid-metabolism in pathological conditions and, in turn, try to exploit altered sphingolipid-metabolizing enzymes and their metabolites as therapeutic targets. This review will examine how advances in the fields of drug delivery, drug discovery, synthetic chemistry, enzyme replacement therapy, immunobiology, infectious disease and nanotechnology have delivered the potential and promise of utilizing and/or targeting sphingolipid metabolites as therapies for diverse diseases.

Genome-wide linkage scan in a large bipolar disorder sample from the National Institute of Mental Health genetics initiative suggests putative loci for bipolar disorder, psychosis, suicide, and panic disorder.

We conducted a 9-cM genome scan in a large bipolar pedigree sample from the National Institute of Mental Health genetics initiative (1060 individuals from 154 multiplex families). We performed parametric and nonparametric analyses using both standard diagnostic models and comorbid conditions thought to identify phenotypic subtypes: psychosis, suicidal behavior, and panic disorder. Our strongest linkage signals (genome-wide significance) were observed on chromosomes 10q25, 10p12, 16q24, 16p13, and 16p12 using standard diagnostic models, and on 6q25 (suicidal behavior), 7q21 (panic disorder) and 16p12 (psychosis) using phenotypic subtypes. Several other regions were suggestive of linkage, including 1p13 (psychosis), 1p21 (psychosis), 1q44, 2q24 (suicidal behavior), 2p25 (psychosis), 4p16 (psychosis, suicidal behavior), 5p15, 6p25 (psychosis), 8p22 (psychosis), 8q24, 10q21, 10q25 (suicidal behavior), 10p11 (psychosis), 13q32 and 19p13 (psychosis). Over half the implicated regions were identified using phenotypic subtypes. Several regions - 1p, 1q, 6q, 8p, 13q and 16p - have been previously reported to be linked to bipolar disorder. Our results suggest that dissection of the disease phenotype can enrich the harvest of linkage signals and expedite the search for susceptibility genes. This is the first large-scale linkage scan of bipolar disorder to analyze simultaneously bipolar disorder, psychosis, suicidal behavior, and panic disorder.

Treatment of obstetric hemorrhage with recombinant activated factor VII (rFVIIa).

Recombinant activated factor VII (rFVIIa, NovoSeven) was used in three patients with massive obstetric hemorrhage due to placenta previa accreta, rupture of the uterus and pre-eclampsia with HELLP. Administration of the drug markedly decreased the bleeding and enabled control of the hemorrhage. rFVIIa seems to be an adjunctive hemostatic measure for the treatment of severe obstetric hemorrhage.

Colonic tumor CEA, CSAp and MUC-1 expression following radioimmunotherapy or chemotherapy.

Understanding the changes in tumor biology following cytotoxic therapy may lead to a better understanding of the properties of surviving tumor cell populations and to an improved ability to target and treat these cells. This report addressed the time-dependent dynamic alterations in the expression of three tumor-associated antigens: carcinoembryonic antigen (CEA), colon-specific antigen (CSAp) and mucin-1 (MUC-1) following chemotherapy with 5-fluorouracil (5-FU) or radioimmunotherapy (RAIT; (131)I-labeled anti-CEA IgG) in human colonic tumor xenografts. Immunoassay results show that CEA and MUC-1 expression all increase rapidly after either 5-FU or RAIT. GW-39 tumors show a 2.7-fold increase in CEA expression after a maximum tolerated dose of RAIT, being highest after 21 days, while LS174T and HT-29 tumors maximally increase expression 8.3- and 2.6-fold on day 7 after RAIT, respectively. The change in LS174T is short-term, whereas the change in HT-29 is maintained for at least 4 weeks. Serum CEA levels in these tumor- bearing mice also increase in parallel to the changes observed in tumor. MUC-1 increases 2.5-fold by day 5-7 following RAIT in LS174T tumors and 6-fold by day 14 following RAIT in GW-39 tumors, with a corresponding increase in serum MUC-1. Dramatic increases in CSAp after RAIT were also demonstrated in GW-39 tissue by immunohistochemistry. Thus, these data indicate that the response of tumor cells to low-dose-rate radiation from RAIT or to chemotherapy is associated with an increase of CEA, MUC-1 and CSAp.

Tumor-specific dose scheduling of bimodal radioimmunotherapy and chemotherapy.

INTRODUCTION: Radioimmunotherapy (RAIT) has been shown to enhance the efficacy of chemotherapy in a variety of tumor models. When multiple drugs are available for a given tumor type, several questions arise, such as how one selects the appropriate drug to be used with RAIT, in what sequence to administer the two therapeutic agents and how to space the two modalities. Unique molecular characteristics affecting chemo- and radiosensitivity are found in every tumor. We postulated that the agents and dose scheduling of bimodal RAIT plus chemotherapy might have to be tailored to each tumor based on the expression of specific relevant genes. MATERIALS AND METHODS: To determine whether a single choice of agents and sequence and spacing of agents would be effective in 4 established human ovarian cancer cell lines, the cytotoxic effect of four standard first- and second-line chemotherapeutic agents used to treat ovarian cancer (doxorubicin, carboplatin, paclitaxel, or topotecan) or radioimmunotherapy (90Y-RS-7 IgG anti-EGP1) were evaluated as single agents or as a bimodal therapy. The four human ovarian cancer lines differed with respect to expression of p53 and drug resistance proteins: SKOV3 (p53null, mdr-, mrp+), A2780 (p53wt, mdr+, mrp), IGROV-1 (p53mut, mdr, mrp+), and OVCAR3 (p53mut, mdr-, mrp-). RESULTS: The profile of chemo- and radiosensitivity for the 4 drugs and for RAIT was unique for each of the four tumor lines. As a result, combinations of radio-antibody and chemotherapy that resulted in good growth inhibition in one tumor line were ineffective in another. Several specific synergistic and antagonistic combinations were identified for each of the ovarian cancer cell lines studied. CONCLUSION: These studies provide evidence that individualized bimodal RAIT and chemotherapy has to be used for each tumor. Therefore, the influence of individual molecular traits on growth inhibition effects from different combinations of agents needs to be studied. This work should then permit rational choices of which drug to add to RAIT, which dose-schedule to use (sequential with RAIT first or sequential with drug first) and how to space the two modalities, based on certain phenotypic markers of the tumors.

Morbidity and outcome after sentinel lymph node dissection in patients with early-stage malignant cutaneous melanoma.

OBJECTIVE: Prospective analysis of the morbidity and outcome of the sentinel lymph node (SLN) technique in a consecutive series of patients with early-stage melanoma. METHODS: Between 1997 and 1998, 60 patients with stage IB-II malignant melanoma underwent SLN dissection. Preoperative dynamic lymphoscintigraphy with mapping of the lymph vessels and lymph nodes and location of the sentinel node was performed the day before SLN dissection. SLN was identified by use of the blue dye technique. SLN was assessed for histopathological and immunohistochemical examination. Postoperative morbidity and mortality were recorded. Follow-up consisted of repetitive clinical examination with lymph nodes status, laboratory and radiologic findings. RESULTS: Tumor-positive SLN was observed in 18% of the patients and stage II disease was found in 91% of the patients with positive SLN. Breslow thickness was the only significant factor predicting involvement of a SLN (p = 0.02). In 36% of the positive SLN, metastases could be assessed only by immunohistochemical examination. Postoperative complications after SLN dissection were observed in 5% in comparison with 36% after elective lymph node dissection. After a mean follow-up of 32 months, recurrence was observed in 3% with a mean disease-free survival of 8 months. Overall survival was 82% and 90% in patients with positive and negative SLN, respectively. Overall mortality was 15%, due to distant metastases in 78% of the cases. CONCLUSIONS: Staging of early-stage melanoma with the SLN dissection by use of the blue dye technique combined to lymphoscintigraphy and immunohistochemistry is reliable and safe, with less morbidity than elective lymphadenectomy. Long-term follow-up is mandatory to establish the exact reliability of SLN dissection.

Optimizing the use of combined radioimmunotherapy and hypoxic cytotoxin therapy as a function of tumor hypoxia.

Combined radioimmunotherapy (RAIT) and hypoxic cytotoxin therapy (SR4233 or NLCQ-1) have been evaluated with both modalities administered on the same day with only moderate improvement compared with the effects of RAIT alone. In a series of studies using oxygen electrodes, immunohistochemistry and radiotracers, we have demonstrated that RAIT induces a prolonged state of hypoxia in most tumors, without affecting the pO(2) levels in normal tissues. Using serial microelectrode measurements through subcutaneous (s.c.) GW-39 human colonic xenografts, we established that the median pO(2) was unrelated to the initial size of the tumor, over a range of sizes from 1.0 to 4.0 cm. Fourteen days after mice were given a 240-microCi dose of (131)I-MN-14 anti-carcinoembryonic antigen immunoglobulin G, their median pO(2) declined from 26.1 +/- 9.6 mmHg to 9.8 +/- 3.9 mmHg (p < 0.001). Using the radiotracer (3)H-MISO that accumulates in hypoxic regions, uptake in GW-39, LoVo and LS174T s.c. human colonic tumors increased 3.0- to 4.2-fold from day 14 through day 28 post-RAIT, but uptake of (3)H-MISO in CALU-3 tumors remained unchanged after RAIT. Normal tissue (liver, kidney, lung) uptake of (3)H-MISO did not exhibit significant changes. The increase in tumor hypoxia was also demonstrated visually using anti-PIMO staining of tumor sections. We postulated that sequential delivery of the 2 therapeutic agents, with the hypoxic cytotoxin given 2 weeks after RAIT when tumor pO(2) levels were at their nadir, would improve the therapeutic response above either modality alone or above the 2 agents delivered on the same day. Tumor growth was compared in mice given either RAIT or cytotoxin alone, the combined treatment on the same day or with the cytotoxin delivered 14 days after RAIT. Tumor size on day 35 for RAIT-treated and SR4233-treated GW-39 were 3.56 +/- 0.40 and 7.98 +/- 2.50 cm(3). When RAIT + SR4233 were delivered on the same day, tumor size dropped to 2.78 +/- 0.80 cm(3). If RAIT was given on day 0 and SR4233 on day 14, size further declined further to 1.74 +/- 0.32 cm(3) (p < 0.05 compared with same day delivery). For LS174T, tumor size on day 28 for RAIT-treated and SR4233-treated tumors were 1.14 +/- 0.36 cm(3) and 3.65 +/- 0.78 cm(3), respectively. When RAIT + SR4233 were delivered on the same day, size was 0.51 +/- 0.174 cm(3). If RAIT was dosed on day 0 and SR4233 was given on day 14, tumor size was 0.13 +/- 0.07 cm(3) (p < 0.05). Similar results were obtained for LoVo, but not for CALU-3 tumors. Another hypoxic cytotoxin, NLCQ-1, was also more efficacious 2 weeks after RAIT, compared with same-day dosing. Thus, information on tumor hypoxia after radioantibody therapy could be important for ascertaining a window of opportunity when the surviving tumor regions are most responsive to hypoxic cytotoxins.

Role of the Glu residues of the influenza hemagglutinin fusion peptide in the pH dependence of fusion activity.

To elucidate the role of the fusion peptide in influenza hemagglutinin (HA)-mediated fusion, we compared pH-dependent conformational changes and fusion mediated by wild-type and a mutant HA in which Glu residues at positions 11 and 15 of the fusion peptide are substituted for valine. The pH dependence of conformational changes and kinetics of fusion with erythrocytes was the same for both forms of HA. The time for commitment and the temperature dependence of HA-mediated fusion were also the same. However, striking differences were observed between wild-type and mutant fusion peptides in their interactions with lipid membranes at neutral and acidic pH. Since elimination of the negatively charged residues allows the exposed fusion peptide to penetrate the bilayer at pH values closer to neutral, but does not affect conformational changes and fusion activity in intact HA, we conclude that conformational changes are tightly coupled to fusion peptide insertion in the overall HA-mediated fusion cascade.