Strikingly different roles of SARS-CoV-2 fusion peptides uncovered by neutron scattering

Coronavirus disease-2019 (COVID-19), a lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of the SARS-CoV-2 Spike protein, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium however re-orients the protein to the lipid leaflet in contact with the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed. SIGNIFICANCEWe have recreated here important elements of the critical membrane fusion mechanism of the SARS-CoV-2 coronavirus by simplifying the system down to its core elements, amenable to experimental analysis by neutron scattering. Neutrons are well suited for the study of protein - membrane interactions under physiological conditions, since they allow structural and dynamics characterization at room temperature. Our results revealed strikingly different functions encoded in the viral Spike fusion domain and thereby provide a potential calcium-dependent cell entry mechanism for SARS-CoV-2. In particular, calcium drives the proteins N-terminal to harpoon through the host membrane, while removing calcium re-orients the protein so that it is able to bridge and dehydrate lipid membranes, facilitating their fusion.

Exploratory Evaluation of Bezlotoxumab on Outcomes Associated With Clostridioides difficile Infection in MODIFY I/II Participants With Cancer.

BACKGROUND: The incidence of Clostridioides difficile infection (CDI) is reportedly higher and the cure rate lower in individuals with cancer vs those without cancer. An exploratory post hoc analysis of the MODIFY I/II trials (NCT01241552/NCT01513239) investigated how bezlotoxumab affected the rate of CDI-related outcomes in participants with cancer. METHODS: Participants received a single infusion of bezlotoxumab (10 mg/kg) or placebo during anti-CDI antibacterial treatment. A post hoc analysis of CDI-related outcomes was conducted in subgroups of MODIFY I/II participants with and without cancer. RESULTS: Of 1554 participants in the modified intent-to-treat (mITT) population, 382 (24.6%) were diagnosed with cancer (bezlotoxumab 190, placebo 192). Of participants without cancer, 591 and 581 received bezlotoxumab and placebo, respectively. In the placebo group, initial clinical cure (ICC) was achieved by fewer cancer participants vs participants without cancer (71.9% vs 83.1%; absolute difference, -11.3%; 95% CI, -18.6% to -4.5%); however, CDI recurrence (rCDI) rates were similar in cancer (30.4%) and noncancer (34.0%) participants. In participants with cancer, bezlotoxumab treatment had no effect on ICC rate compared with placebo (76.8% vs 71.9%), but resulted in a statistically significant reduction in rCDI vs placebo (17.8% vs 30.4%; absolute difference, -12.6%; 95% CI, -22.5% to -2.7%). CONCLUSIONS: In this post hoc analysis of participants with cancer enrolled in MODIFY I/II, the rate of rCDI in bezlotoxumab-treated participants was lower than in placebo-treated participants. Additional studies are needed to confirm these results. CLINICAL TRIAL REGISTRATION: MODIFY I (NCT01241552), MODIFY II (NCT01513239).

Efficacy and safety of ridinilazole compared with vancomycin for the treatment of Clostridium difficile infection: a phase 2, randomised, double-blind, active-controlled, non-inferiority study.

BACKGROUND: Clostridium difficile infection is the most common health-care-associated infection in the USA. We assessed the safety and efficacy of ridinilazole versus vancomycin for treatment of C difficile infection. METHODS: We did a phase 2, randomised, double-blind, active-controlled, non-inferiority study. Participants with signs and symptoms of C difficile infection and a positive diagnostic test result were recruited from 33 centres in the USA and Canada and randomly assigned (1:1) to receive oral ridinilazole (200 mg every 12 h) or oral vancomycin (125 mg every 6 h) for 10 days. The primary endpoint was achievement of a sustained clinical response, defined as clinical cure at the end of treatment and no recurrence within 30 days, which was used to establish non-inferiority (15% margin) of ridinilazole versus vancomycin. The primary efficacy analysis was done on a modified intention-to-treat population comprising all individuals with C difficile infection confirmed by the presence of free toxin in stool who were randomly assigned to receive one or more doses of the study drug. The study is registered with ClinicalTrials.gov, number NCT02092935. FINDINGS: Between June 26, 2014, and August 31, 2015, 100 patients were recruited; 50 were randomly assigned to receive ridinilazole and 50 to vancomycin. 16 patients did not complete the study, and 11 discontinued treatment early. The primary efficacy analysis included 69 patients (n=36 in the ridinilazole group; n=33 in the vancomycin group). 24 of 36 (66·7%) patients in the ridinilazole group versus 14 of 33 (42·4%) of those in the vancomycin group had a sustained clinical response (treatment difference 21·1%, 90% CI 3·1-39·1, p=0·0004), establishing the non-inferiority of ridinilazole and also showing statistical superiority at the 10% level. Ridinilazole was well tolerated, with an adverse event profile similar to that of vancomycin: 82% (41 of 50) of participants reported adverse events in the ridinilazole group and 80% (40 of 50) in the vancomycin group. There were no adverse events related to ridinilazole that led to discontinuation. INTERPRETATION: Ridinilazole is a targeted-spectrum antimicrobial that shows potential in treatment of initial C difficile infection and in providing sustained benefit through reduction in disease recurrence. Further clinical development is warranted. FUNDING: Wellcome Trust and Summit Therapeutics.

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection.

BACKGROUND: Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. METHODS: We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. RESULTS: In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, -10.1 percentage points; 95% confidence interval [CI], -15.9 to -4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, -9.9 percentage points; 95% CI, -15.5 to -4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, -11.6 percentage points; 95% CI, -17.4 to -5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, -10.7 percentage points; 95% CI, -16.4 to -5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. CONCLUSIONS: Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239 .).

Defining the optimal formulation and schedule of a candidate toxoid vaccine against Clostridium difficile infection: A randomized Phase 2 clinical trial.

BACKGROUND: Clostridium difficile, a major cause of nosocomial and antibiotic-associated diarrhea, carries a significant disease and cost burden. This study aimed to select an optimal formulation and schedule for a candidate toxoid vaccine against C. difficile toxins A and B. METHODS: Randomized, placebo-controlled, two-stage, Phase 2 study in a total of 661 adults aged 40-75 years. Stage I: low (50 µg antigen) or high (100 µg antigen) dose with or without aluminum hydroxide (AlOH) adjuvant, or placebo, administered on Days 0-7-30. Stage II: Days 0-7-30, 0-7-180, and 0-30-180, using the formulation selected in Stage I through a decision tree defined a priori and based principally on a bootstrap ranking approach. Administration was intramuscular. Blood samples were obtained on Days 0, 7, 14, 30, 60 (Stage I and II), 180, and 210 (Stage II); IgG to toxins A and B was measured by ELISA and in vitro functional activity was measured by toxin neutralizing assay (TNA). Safety data were collected using diary cards. RESULTS: In Stage I the composite immune response against toxins A and B (percentage of participants who seroconverted for both toxins) was highest in the high dose+adjuvant group (97% and 92% for Toxins A and B, respectively) and was chosen for Stage II. In Stage II the immune response profile for this formulation through Day 180 given on Days 0-7-30 ranked above the other two administration schedules. There were no safety issues. CONCLUSIONS: The high dose+adjuvant (100 µg antigen+AlOH) formulation administered at 0-7-30 days elicited the best immune response profile, including functional antibody responses, through Day 180 and was selected for use in subsequent clinical trials.

Efficacy of high-dose versus standard-dose influenza vaccine in older adults.

BACKGROUND: As compared with a standard-dose vaccine, a high-dose, trivalent, inactivated influenza vaccine (IIV3-HD) improves antibody responses to influenza among adults 65 years of age or older. This study evaluated whether IIV3-HD also improves protection against laboratory-confirmed influenza illness. METHODS: We conducted a phase IIIb-IV, multicenter, randomized, double-blind, active-controlled trial to compare IIV3-HD (60 µg of hemagglutinin per strain) with standard-dose trivalent, inactivated influenza vaccine (IIV3-SD [15 µg of hemagglutinin per strain]) in adults 65 years of age or older. Assessments of relative efficacy, effectiveness, safety (serious adverse events), and immunogenicity (hemagglutination-inhibition [HAI] titers) were performed during the 2011-2012 (year 1) and the 2012-2013 (year 2) northern-hemisphere influenza seasons. RESULTS: A total of 31,989 participants were enrolled from 126 research centers in the United States and Canada (15,991 were randomly assigned to receive IIV3-HD, and 15,998 to receive IIV3-SD). In the intention-to-treat analysis, 228 participants in the IIV3-HD group (1.4%) and 301 participants in the IIV3-SD group (1.9%) had laboratory-confirmed influenza caused by any viral type or subtype associated with a protocol-defined influenza-like illness (relative efficacy, 24.2%; 95% confidence interval [CI], 9.7 to 36.5). At least one serious adverse event during the safety surveillance period was reported by 1323 (8.3%) of the participants in the IIV3-HD group, as compared with 1442 (9.0%) of the participants in the IIV3-SD group (relative risk, 0.92; 95% CI, 0.85 to 0.99). After vaccination, HAI titers and seroprotection rates (the percentage of participants with HAI titers ≥ 1:40) were significantly higher in the IIV3-HD group. Conclusions: Among persons 65 years of age or older, IIV3-HD induced significantly higher antibody responses and provided better protection against laboratory-confirmed influenza illness than did IIV3-SD. (Funded by Sanofi Pasteur; ClinicalTrials.gov number, NCT01427309.).

Extremely low excretion of daptomycin into breast milk of a nursing mother with methicillin-resistant Staphylococcus aureus pelvic inflammatory disease.

Antibiotic treatment for pelvic inflammatory disease (PID) is often broad spectrum and targets a diverse range of vaginal flora. Treatment of PID in nursing mothers presents a particular clinical challenge because use of antimicrobials during breastfeeding poses several potential risks to infants. Excretion of drugs into breast milk can occur through different mechanisms and depends on the characteristics of both the drug and the mother. Whether daptomycin is excreted into breast milk is unknown, as is its subsequent exposure to breastfeeding infants and the associated risks. We describe a case of PID caused by methicillin-resistant Staphylococcus aureus, an uncommon pathogen in PID, in a breastfeeding mother who was successfully treated with daptomycin. Daptomycin concentrations in her breast milk were measured to determine potential exposure to her infant. These concentrations were extremely low, with an estimated milk:plasma ratio of 0.0012. Although additional confirmatory studies are needed, daptomycin may be a reasonable option in the treatment of PID caused by gram-positive organisms that are resistant to other antibiotics.

Simulated ischemia enhances L-type calcium current in pacemaker cells isolated from the rabbit sinoatrial node.

Ischemic-like conditions (a glucose-free, pH 6.6 Tyrode solution bubbled with 100% N(2)) enhance L-type Ca current (I(Ca,L)) in single pacemaker cells (PCs) isolated from the rabbit sinoatrial node (SAN). In contrast, studies of ventricular myocytes have shown that acidic extracellular pH, as employed in our "ischemic" Tyrode, reduces I(Ca,L). Therefore, our goal was to explain why I(Ca,L) is increased by "ischemia" in SAN PCs. The major findings were the following: 1) blockade of Ca-induced Ca release with ryanodine, exposure of PCs to BAPTA-AM, or replacement of extracellular Ca(2+) with Ba(2+) failed to prevent the ischemia-induced enhancement of I(Ca,L); 2) inhibition of protein kinase A with H-89, or calcium/calmodulin-dependent protein kinase II with KN-93, reduced I(Ca,L) but did not prevent its augmentation by ischemia; 3) ischemic Tyrode or pH 6.6 Tyrode shifted the steady-state inactivation curve in the positive direction, thereby reducing inactivation; 4) ischemic Tyrode increased the maximum conductance but did not affect the activation curve; 5) in rabbit atrial myocytes isolated and studied with exactly the same techniques used for SAN PCs, ischemic Tyrode reduced the maximum conductance and shifted the activation curve in the positive direction; pH 6.6 Tyrode also shifted the steady-state inactivation curve in the positive direction. We conclude that the acidic pH of ischemic Tyrode enhances I(Ca,L) in SAN PCs, because it increases the maximum conductance and reduces inactivation. Furthermore, the opposite results obtained with rabbit atrial myocytes cannot be explained by differences in cell isolation or patch-clamp techniques.

Ionic basis of ischemia-induced bradycardia in the rabbit sinoatrial node.

To investigate the basis of ischemia-induced bradycardia (<60 beats/min), we isolated pacemaker cells from the rabbit sinoatrial node and exposed them to ischemic-like conditions, including omission of glucose, pH 6.6, and either 5.4 or 10 mM KCl to evaluate the role of increased serum [K]. A perforated-patch technique was employed to test the hypothesis that the arrhythmia is caused by attenuation of inward currents that contribute to the diastolic depolarization. After exposure to "ischemic" Tyrode containing 5.4 mM KCl, the pacemaker cells exhibited 13% slower beat rates and action potentials with 6-mV greater overshoots and 44% longer durations. In contrast, after exposure to "ischemic" Tyrode containing 10 mM KCl, the pacemaker cells exhibited a 7-mV depolarization of the maximum diastolic potential but no significant change in the overshoot. Beat rates were slowed by 43%, and the action potentials were prolonged by 46%. "Ischemic" Tyrode containing 5.4 mM KCl increased L-type Ca current, decreased T-type Ca current and reduced Ni-sensitive inward current tails (presumably Na-Ca exchange current), even after treatment with 40 muM ryanodine to block Ca release from the sarcoplasmic reticulum. "Ischemic" Tyrode containing 10 mM KCl increased hyperpolarization-activated inward current at diastolic potentials and reduced the slowly activating component, but not the rapidly activating component, of delayed rectifier K current. Our results suggest that reductions of inward Na-Ca exchange current and T-type Ca current contribute to "ischemia"-induced "bradycardia" in sinoatrial node pacemaker cells.

Nocifensive reflex-related on- and off-cells in the pedunculopontine tegmental nucleus, cuneiform nucleus, and lateral dorsal tegmental nucleus.

Cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to the rostral ventromedial medulla (RVM) have been implicated in nociceptive modulation. The goal of this study was to identify neurons with nocifensive reflex-related activity in the mesopontine tegmentum including the PPTg. This study used the same behavioral neurophysiological classification system to identify neurons as has been extensively described in the RVM. Extracellular microelectrode recording was conducted in lightly anesthetized rats. Changes in firing associated with the noxious heat-evoked tail flick reflex were used to classify neurons as "on-cells" (displayed a burst in neuronal activity associated with the reflex), "off-cells" (displayed a pause in activity), and neutral cells (showed no response). Of 188 neurons studied in 23 rats, 77 were classified as on-cells, 14 as off-cells, the remainder as neutral cells. Recordings during periods without noxious stimulation found that some of the on- and off-cells displayed spontaneous transitions between active and silent periods termed cell cycling. The distribution of on- and off-cells in the mesopontine tegmentum overlapped and included the cholinergic PPTg and lateral dorsal tegmental nucleus identified by NADPH diaphorase staining, as well as the cuneiform nucleus and periaqueductal gray. The mesopontine tegmentum thus contains nocifensive reflex-related neurons with neurophysiological characteristics similar to those reported in the RVM. Neurons showing reflex-related activity were frequently encountered in the cholinergic PPTg and LDTg. Further studies will be required to determine whether these neurons modulate nociception through a link to the RVM.

Federalism and health policy.

This paper presents a cyclical theory of U.S. federalism and social policy: Many social policy initiatives are tested and refined at the state level, especially during conservative periods, and later morph into national policies. The paper describes such federalism cycles and offers an interpretation of why and how they occur, focusing on Medicaid. State activism has preserved and expanded Medicaid through policy innovation and resistance to retrenchment, especially in conservative periods, by taking advantage of the flexibility the program provides. I conclude that Medicaid's incremental/partnership approach is appropriate and feasible to build on for a future expansion of health care coverage.

Recovery of stroke hemiplegia through neurosurgical intervention in the chronic stage.

Objective: We evaluated quantitatively the further recovery from impairment and disability in the hemiplegic stroke survivors who required neurosurgical intervention, i.e. cranioplasty or ventriculoperitoneal (V-P) shunt, in chronic stage. Setting: Rehabilitation (RH) ward affiliated with university hospitals. Patients: Eleven first-ever stroke patients with hemiplegia (mean age, 56.3+/-2.5 years) out of 498 survivors required delayed (between 4 and 10 months after the onset) neurosurgical intervention during continuous RH therapy. Six patients received cranioplasty for preexisting hemicraniectomy, and five required V-P shunt for normal pressure hydrocephalus with later complications. Main outcome measures: Recovery grade (1--12) of hemiplegia and Barthel index were assessed monthly before (the 1st RH) and after the intervention (the 2nd RH). Results: The recovery grade of upper and lower extremity movements significantly increased both in the 1st and 2nd RH. Changes in the upper and lower extremity grades were significantly larger in the 2nd RH (0.5+/-0.3 in the 1st vs. 2.5+/-0.6 in the 2nd RH for upper extremity, p<0.005; 0.9+/-0.3 in the 1st vs. 3.4+/-0.5 in the 2nd RH for lower extremity, p<0.001). Barthel index increased significantly only in the 2nd RH (from 48+/-7 to 90+/-3, p<0.001); all patients regained the ability to walk independently. Conclusions: Significant recovery of functional grade and recovery from disability occurred after the neurosurgical intervention in the chronic stage (geq 4 months) of stroke.