Cell culture–based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act “universally” against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing. Key points • An automated perfusion process for production of IAV DIPs was established. • DIP titers of 7.40E + 9 plaque forming units per mL were reached. • A novel tubular cell retention device enabled continuous virus harvesting. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-021-11561-y

On the optimality of gluing over scales

We show that for every $\alpha > 0$, there exist $n$-point metric spaces (X,d) where every "scale" admits a Euclidean embedding with distortion at most $\alpha$, but the whole space requires distortion at least $\Omega(\sqrt{\alpha \log n})$. This shows that the scale-gluing lemma [Lee, SODA 2005] is tight, and disproves a conjecture stated there. This matching upper bound was known to be tight at both endpoints, i.e. when $\alpha = \Theta(1)$ and $\alpha = \Theta(\log n)$, but nowhere in between. More specifically, we exhibit $n$-point spaces with doubling constant $\lambda$ requiring Euclidean distortion $\Omega(\sqrt{\log \lambda \log n})$, which also shows that the technique of "measured descent" [Krauthgamer, et. al., Geometric and Functional Analysis] is optimal. We extend this to obtain a similar tight result for $L_p$ spaces with $p > 1$.Comment: minor revision

Identification of Map4k4 as a novel suppressor of skeletal muscle differentiation

Myoblast differentiation into mature myotubes is a critical step in the development and repair of human skeletal muscle. Here we show that small interfering RNA (siRNA)-based silencing of the Ste20-like mitogen-activated protein 4 kinase 4 (Map4k4) in C2C12 myoblasts markedly enhances expression of myogenic differentiation genes, myoblast fusion, and myotube diameter. In contrast, adenovirus-mediated expression of native Map4k4 in C2C12 cells attenuates each of these processes, indicating that Map4k4 is a negative regulator of myogenic differentiation and hypertrophy. Expression of a Map4k4 kinase-inactive mutant enhances myotube formation, suggesting that the kinase activity of Map4k4 is essential for its inhibition of muscle differentiation. Map4k4 regulation of myogenesis is unlikely to be mediated by classic mitogen-activated protein kinase (MAPK) signaling pathways, because no significant difference in phosphorylation of extracellular signal-regulated kinase (ERK), p38, or c-Jun N-terminal kinase (JNK) is observed in Map4k4-silenced cells. Furthermore, silencing of these other MAPKs does not result in a hypertrophic myotube phenotype like that seen with Map4k4 depletion. Uniquely, Map4k4 silencing upregulates the expression of the myogenic regulatory factor Myf5, whose depletion inhibits myogenesis. Furthermore, Myf5 is required for enhancement of myotube formation in Map4k4-silenced cells, while Myf5 overexpression rescues Map4k4-mediated inhibition of myogenic differentiation. These results demonstrate that Map4k4 is a novel suppressor of skeletal muscle differentiation, acting through a Myf5-dependent mechanism

Patient engagement with antibiotic messaging in secondary care: a qualitative feasibility study of the ‘review & revise’ experience

Background: We aimed to investigate and optimise the acceptability and usefulness of a patient leaflet about antibiotic prescribing decisions made during hospitalisation, and to explore individual patient experiences and preferences regarding the process of antibiotic prescription ‘review & revise’ which is a key strategy to minimise antibiotic overuse in hospitals. Methods: In this qualitative study, run within the feasibility study of a large, cluster-randomised stepped wedge trial of 36 hospital organisations, a series of semi-structured, think-aloud telephone interviews were conducted and data were analysed using thematic analysis. Fifteen adult patients who had experienced a recent acute medical hospital admission during which they had been prescribed antimicrobials and offered a patient leaflet about antibiotic prescribing were recruited to the study. Results: Participants reacted positively to the leaflet, reporting that it was both an accessible and important source of information which struck the appropriate balance between informing and reassuring. Participants all valued open communication with clinicians, and were keen to be involved in antibiotic prescribing decisions, with individuals reporting positive experiences regarding antibiotic prescription changes or stopping. Many participants had prior experience or knowledge of antibiotics and resistance, and generally welcomed efforts to reduce antibiotic usage. Overall, there was a feeling that healthcare professionals (HCPs) are trusted experts providing the most appropriate treatment for individual patient conditions. Conclusions: This study offers novel insights into how patients within secondary care are likely to respond to messages advocating a reduction in the use of antibiotics through the ‘review & revise’ approach. Due to the level of trust that patients place in their care provider, encouraging HCPs within secondary care to engage patients with greater communication and information provision could provide great advantages in the drive to reduce antibiotic use. It may also be beneficial for HCPs to view patient experiences as cumulative events that have the potential to impact future behaviour around antibiotic use. Finally, pre-testing messages about antibiotic prescribing and resistance is vital to dispelling any misconceptions either around effectiveness of treatment for patients, or perceptions of how messages may be received

Software defect prediction: do different classifiers find the same defects?

Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.During the last 10 years, hundreds of different defect prediction models have been published. The performance of the classifiers used in these models is reported to be similar with models rarely performing above the predictive performance ceiling of about 80% recall. We investigate the individual defects that four classifiers predict and analyse the level of prediction uncertainty produced by these classifiers. We perform a sensitivity analysis to compare the performance of Random Forest, Naïve Bayes, RPart and SVM classifiers when predicting defects in NASA, open source and commercial datasets. The defect predictions that each classifier makes is captured in a confusion matrix and the prediction uncertainty of each classifier is compared. Despite similar predictive performance values for these four classifiers, each detects different sets of defects. Some classifiers are more consistent in predicting defects than others. Our results confirm that a unique subset of defects can be detected by specific classifiers. However, while some classifiers are consistent in the predictions they make, other classifiers vary in their predictions. Given our results, we conclude that classifier ensembles with decision-making strategies not based on majority voting are likely to perform best in defect prediction.Peer reviewedFinal Published versio

Prophet Inequalities for IID Random Variables from an Unknown Distribution

A central object in optimal stopping theory is the single-choice prophet inequality for independent, identically distributed random variables: given a sequence of random variables X1, . . . , Xn drawn independently from a distribution F , the goal is to choose a stopping time τ so as to maximize α such that for all distributions F we have E[Xτ ] ≥ α · E[maxt Xt ]. What makes this problem challenging is that the decision whether τ = t may only depend on the values of the random variables X1, . . . , Xt and on the distribution F . For a long time the best known bound for the problem had been α ≥ 1 − 1/e ≈ 0.632, but quite recently a tight bound of α ≈ 0.745 was obtained. The case where F is unknown, such that the decision whether τ = t may depend only on the values of the random variables X1, . . . , Xt , is equally well motivated but has received much less attention. A straightforward guarantee for this case of α ≥ 1/e ≈ 0.368 can be derived from the solution to the secretary problem, where an arbitrary set of values arrive in random order and the goal is to maximize the probability of selecting the largest value. We show that this bound is in fact tight. We then investigate the case where the stopping time may additionally depend on a limited number of samples from F , and show that even with o(n) samples α ≤ 1/e. On the other hand, n samples allow for a significant improvement, while O(n2) samples are equivalent to knowledge of the distribution: specifically, with n samples α ≥ 1 − 1/e ≈ 0.632 and α ≤ ln(2) ≈ 0.693, and with O(n2) samples α ≥ 0.745 − ε for any ε > 0

Barriers to enrollment in a randomized controlled trial of hydrocortisone for cardiovascular insufficiency in term and late preterm newborn infants.

ObjectiveTo analyze reasons for low enrollment in a randomized controlled trial (RCT) of the effect of hydrocortisone for cardiovascular insufficiency on survival without neurodevelopmental impairment (NDI) in term/late preterm newborns.Study designThe original study was a multicenter RCT. Eligibility: ⩾34 weeks' gestation, <72 h old, mechanically ventilated, receiving inotrope. Primary outcome was NDI at 2 years; infants with diagnoses at high risk for NDI were excluded. This paper presents an analysis of reasons for low patient enrollment.ResultsTwo hundred and fifty-seven of the 932 otherwise eligible infants received inotropes; however, 207 (81%) had exclusionary diagnoses. Only 12 infants were randomized over 10 months; therefore, the study was terminated. Contributing factors included few eligible infants after exclusions, open-label steroid therapy and a narrow enrollment window.ConclusionDespite an observational study to estimate the population, very few infants were enrolled. Successful RCTs of emergent therapy may require fewer exclusions, a short-term primary outcome, waiver of consent and/or other alternatives

Deformation-induced microstructural banding in TRIP steels

Microstructure inhomogeneities can strongly influence the mechanical properties of advanced high-strength steels in a detrimental manner. This study of a transformation-induced plasticity (TRIP) steel investigates the effect of pre-existing contiguous grain boundary networks (CGBNs) of hard second-phases and shows how these develop into bands during tensile testing using in situ observations in conjunction with digital image correlation (DIC). The bands form by the lateral contraction of the soft ferrite matrix, which rotates and displaces the CGBNs of second-phases and the individual features within them to become aligned with the loading direction. The more extensive pre-existing CGBNs that were before the deformation already aligned with the loading direction are the most critical microstructural feature for damage initiation and propagation. They induce micro-void formation between the hard second-phases along them, which coalesce and develop into long macroscopic fissures. The hard phases, retained austenite and martensite, were not differentiated as it was found that the individual phases do not play a role in the formation of these bands. It is suggested that minimizing the presence of CGBNs of hard second-phases in the initial microstructure will increase the formability

Varespladib and cardiovascular events in patients with an acute coronary syndrome: the VISTA-16 randomized clinical trial

IMPORTANCE: Secretory phospholipase A2(sPLA2) generates bioactive phospholipid products implicated in atherosclerosis. The sPLA2inhibitor varespladib has favorable effects on lipid and inflammatory markers; however, its effect on cardiovascular outcomes is unknown. OBJECTIVE: To determine the effects of sPLA2inhibition with varespladib on cardiovascular outcomes. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized, multicenter trial at 362 academic and community hospitals in Europe, Australia, New Zealand, India, and North America of 5145 patients randomized within 96 hours of presentation of an acute coronary syndrome (ACS) to either varespladib (n = 2572) or placebo (n = 2573) with enrollment between June 1, 2010, and March 7, 2012 (study termination on March 9, 2012). INTERVENTIONS: Participants were randomized to receive varespladib (500 mg) or placebo daily for 16 weeks, in addition to atorvastatin and other established therapies. MAIN OUTCOMES AND MEASURES: The primary efficacy measurewas a composite of cardiovascular mortality, nonfatal myocardial infarction (MI), nonfatal stroke, or unstable angina with evidence of ischemia requiring hospitalization at 16 weeks. Six-month survival status was also evaluated. RESULTS: At a prespecified interim analysis, including 212 primary end point events, the independent data and safety monitoring board recommended termination of the trial for futility and possible harm. The primary end point occurred in 136 patients (6.1%) treated with varespladib compared with 109 patients (5.1%) treated with placebo (hazard ratio [HR], 1.25; 95%CI, 0.97-1.61; log-rank P = .08). Varespladib was associated with a greater risk of MI (78 [3.4%] vs 47 [2.2%]; HR, 1.66; 95%CI, 1.16-2.39; log-rank P = .005). The composite secondary end point of cardiovascular mortality, MI, and stroke was observed in 107 patients (4.6%) in the varespladib group and 79 patients (3.8%) in the placebo group (HR, 1.36; 95% CI, 1.02-1.82; P = .04). CONCLUSIONS AND RELEVANCE: In patients with recent ACS, varespladib did not reduce the risk of recurrent cardiovascular events and significantly increased the risk of MI. The sPLA2inhibition with varespladib may be harmful and is not a useful strategy to reduce adverse cardiovascular outcomes after ACS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01130246. Copyright 2014 American Medical Association. All rights reserved

Charge separation relative to the reaction plane in Pb-Pb collisions at sNN=2.76\sqrt{s_{\rm NN}}= 2.76 TeV

Measurements of charge dependent azimuthal correlations with the ALICE detector at the LHC are reported for Pb-Pb collisions at $\sqrt{s_{\rm NN}} = 2.76$ TeV. Two- and three-particle charge-dependent azimuthal correlations in the pseudo-rapidity range $|\eta| < 0.8$ are presented as a function of the collision centrality, particle separation in pseudo-rapidity, and transverse momentum. A clear signal compatible with a charge-dependent separation relative to the reaction plane is observed, which shows little or no collision energy dependence when compared to measurements at RHIC energies. This provides a new insight for understanding the nature of the charge dependent azimuthal correlations observed at RHIC and LHC energies.Comment: 12 pages, 3 captioned figures, authors from page 2 to 6, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/286