The production, design and application of antimicrobial peptides

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.Includes bibliographical references (p. 248-268).With the spread of drug-resistant bacteria, existing antibiotics are losing their potency. Antimicrobial peptides (AmPs) represent an exciting class of drug candidates, particularly because their mechanism of action is unlikely to induce drug resistance. If resistance to AmPs were also slower to emerge in the clinic, they would have longer useful lifetimes than existing antibiotics. Nevertheless, a number of limitations exist for AmPs in the clinic. The high cost of peptide manufacture requires that highly potent sequences are created. Additionally, AmP selectivity must be improved if effective systemic doses are to be given without hemolytic activity or other toxicity. Improved high-throughput methods for AmP design or discovery could enable the achievement of both of these goals. To this end, we developed an approach based on the discovery of semi-conserved motifs across natural AmPs, which we demonstrated are associated with antimicrobial activity. Additionally, we created novel AmP formulations that may bypass some of these clinical limitations. In order to evaluate AmP design approaches, a high-throughput production and assay platform was created using in vitro translation. This technology may produce peptides that would be toxic to recombinant hosts and synthesize peptides of arbitrary length.(cont.) The cost per peptide was minimized through a series of process improvements. First, we created methods to construct oligonucleotides that mimicked our motif-based design of AmPs. This approach allowed the reuse of primers for many peptides, reducing cost and enabling the study of pattern synergy. Additionally, we found peptide translation was enhanced by co-translating a fusion partner in frame with the AmP. The AmP could be freed from the fusion partner after translation using enterokinase digestion. Further, we increased yield 3-fold by optimizing the length of fusion partner. The partner was made as short as possible to limit the translational resources required to synthesize the fusion partner, while being long enough to ensure stability from proteases. The solubility of the fusion partner-AmP construct was also improved through the selection of a highly soluble partner of the optimal length. Finally, we developed a purification scheme to ensure that the in vitro translation extract would not impact measurement of antimicrobial activity. We also developed and evaluated the design of AmPs using semi-conserved motifs. We used a database of over 500 natural AmPs as a training set for pattern discovery.(cont.) The resulting motifs were exhaustively recombined to create all 20 amino acid sequences that were entirely covered by these patterns. These sequences were clustered, and 42 diverse members selected for characterization using representative Gram negative and Gram positive bacteria. Approximately 50% of the designed AmPs were active against at least one of the bacteria at 256 ug/ml. Control peptides were created in which the amino acids in the designed peptides were rearranged such that they were not homologous to any antimicrobial motifs. Thus, these controls had the same bulk physiochemical properties frequently associated with antimicrobial activity as the designed sequences, but we hypothesized they would not be active because they did not match the antimicrobial motifs. In fact, only 5% of the control sequences had activity at 256 ug/ml, indicating that the antimicrobial motifs give a 10-fold enrichment in activity. Further, two highly active designed peptides had MICs of 16 ug/ml against Bacillus cereus and 64 ug/ml against Escherichia coli. Additionally, AmPs active against B. cereus were all active against the hospital pathogen Staphylococcus aureus, and the bioterror agent, Bacillus anthracis.(cont.) Our motif-based design may be most effective as the first stage of a two-stage design tool. In the first stage, highly diverse leads with novel profiles are created and evaluated. Promising leads could then be optimized using a variety of techniques. By creating just 44 variants of one lead, we designed an AmP with broad spectrum activity that had MICs of 16 ug/ml against E. coli and 8 ug/ml against B. cereus and 4 ug/ml against S. aureus. Another approach to build on our design tool would be to incorporate activity and toxicity characteristics of members of the training set into the design or scoring of new sequences. In order to begin assembling this data using a standardized method, a representative set of 100 natural, linear AmPs was chosen through clustering. Their antimicrobial activity against E. coli, S. epidermidis, and S. aureus were evaluated, along with hemolytic activity. When further supplemented, this information may enable an improved scoring metric to be created. Additionally, we systematically demonstrated that amidating the c-terminus of natural AmPs improves both antimicrobial activity and therapeutic index. Finally, we recognized that AmP's mechanism of action would allow activity to be retained when they are permanently tethered to medical device surfaces.(cont.) Unlike existing coatings which rely on the slow release of silver or other antibiotics, a permanently tethered approach could have a longer lifetime and reduced systemic toxicity concerns. A versatile chemistry was developed to create immobilized AmP coatings. These formulations had broad spectrum antimicrobial activity without significant hemolytic activity. Further, the coatings were effective through multiple bacterial challenges. The combination of the AmP design tool along with localized formulations represent a significant advance in the process of moving AmPs to the clinic to combat drug-resistant infections.by Christopher Loose.Ph.D

The Impact of Real-Time Whole-Genome Sequencing in Controlling Healthcare-Associated SARS-CoV-2 Outbreaks.

Nosocomial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have severely affected bed capacity and patient flow. We utilized whole-genome sequencing (WGS) to identify outbreaks and focus infection control resources and intervention during the United Kingdom's second pandemic wave in late 2020. Phylogenetic analysis of WGS and epidemiological data pinpointed an initial transmission event to an admission ward, with immediate prior community infection linkage documented. High incidence of asymptomatic staff infection with genetically identical viral sequences was also observed, which may have contributed to the propagation of the outbreak. WGS allowed timely nosocomial transmission intervention measures, including admissions ward point-of-care testing and introduction of portable HEPA14 filters. Conversely, WGS excluded nosocomial transmission in 2 instances with temporospatial linkage, conserving time and resources. In summary, WGS significantly enhanced understanding of SARS-CoV-2 clusters in a hospital setting, both identifying high-risk areas and conversely validating existing control measures in other units, maintaining clinical service overall

Developing decision support tools incorporating personalised predictions of likely visual benefit versus harm for cataract surgery:research programme

Background Surgery for established cataract is highly cost-effective and uncontroversial, yet uncertainty remains for individuals about when to proceed and when to delay surgery during the earlier stages of cataract. Objective We aimed to improve decision-making for cataract surgery through the development of evidence-based clinical tools that provide general information and personalised risk/benefit information. Design We used a mixed methodology consisting of four work packages. Work package 1 involved the development and psychometric validation of a brief, patient self-reported measure of visual difficulty from cataract and its relief from surgery, named Cataract Patient-Reported Outcome Measure, five items (Cat-PROM5). Work package 2 involved the review and refinement of risk models for adverse surgical events (posterior capsule rupture and visual acuity loss related to cataract surgery). Work package 3 involved the development of prediction models for the Cat-PROM5-based self-reported outcomes from a cohort study of 1500 patients; assessment of the validity of preference-based health economic indices for cataract surgery and the calibration of these to Cat-PROM5; assessment of patients’ and health-care professionals’ views on risk–benefit presentation formats, the perceived usefulness of Cat-PROM5, the value of personalised risk–benefit information, high-value information items and shared decision-making; development of cataract decision aid frequently asked questions, incorporation of personalised estimates of risks and benefits; and development of a cataract decision quality measure to assess the quality of decision-making. Work package 4 involved a mixed-methods feasibility study for a fully powered randomised controlled trial of the use of the cataract decision aid and a qualitative study of discordant or mismatching perceptions of outcome between patients and health-care professionals. Setting Four English NHS recruitment centres were involved: Bristol (lead centre), Brighton, Gloucestershire and Torbay. Multicentre NHS cataract surgery data were obtained from the National Ophthalmology Database. Participants Work package 1 – participants (n = 822) were from all four centres. Work package 2 – electronic medical record data were taken from the National Ophthalmology Database (final set > 1M operations). Work package 3 – cohort study participants were from Bristol (n = 1200) and Gloucestershire (n = 300); qualitative and development work was undertaken with patients and health-care professionals from all four centres. Work package 4 – Bristol, Brighton and Torbay participated in the recruitment of patients (n = 42) for the feasibility trial and recruitment of health-care professionals for the qualitative elements. Interventions For the feasibility trial, the intervention was the use of the cataract decision aid, incorporating frequently asked questions and personalised estimations of both adverse outcomes and self-reported benefit. Main outcome measures There was a range of quantitative and qualitative outcome measures: questionnaire psychometric performance metrics, risk indicators of adverse surgical events and visual outcome, predictors of self-reported outcome following cataract surgery, patient and health-care practitioner views, health economic calibration measures and randomised controlled trial feasibility measures. Data sources The data sources were patient self-reported questionnaire responses, study clinical data collection forms, recorded interviews with patients and health-care professionals, and anonymised National Ophthalmology Database data. Results Work package 1 – Cat-PROM5 was developed and validated with excellent to good psychometric properties (Rasch reliability 0.9, intraclass correlation repeatability 0.9, unidimensionality with residual eigenvalues ≤ 1.5) and excellent responsiveness to surgical intervention (Cohen delta –1.45). Work package 2 – earlier risk models for posterior capsule rupture and visual acuity loss were broadly affirmed (C-statistic for posterior capsule rupture 0.64; visual acuity loss 0.71). Work package 3 – the Cat-PROM5-based self-reported outcome regression models were derived based on 1181 participants with complete data (R2 ≈ 30% for each). Of the four preference-based health economic indices assessed, two demonstrated reasonable performance. Cat-PROM5 was successfully calibrated to health economic indices; adjusted limited dependent variable mixture models offered good to excellent fit (root-mean-square error 0.10–0.16). The personalised quantitative risk information was generally perceived as beneficial. A cataract decision aid and cataract decision quality measure were successfully developed based on the views of patients and health-care professionals. Work package 4 – data completeness was good for the feasibility study primary and secondary variables both before and after intervention/surgery (data completeness range 100–88%). Considering ability to recruit, the sample size required, instrumentation and availability of necessary health economic data, a fully powered randomised controlled trial (patients, n = 800, effect size 0.2 standard deviations, power 80%; p = 0.05) of the cataract decision aid would be feasible following psychometric refinement of the primary outcome (the cataract decision quality measure). The cataract decision aid was generally well-received by patients and health-care professionals, with cautions raised regarding perceived time and workload barriers. Discordant outcomes mostly related to patient dissatisfaction, with no clinical problem found. Limitations The National Ophthalmology Database data are expected to include some errors (mitigated by large multicentre data aggregations). The feasibility randomised controlled trial primary outcome (the cataract decision quality measure) displayed psychometric imperfections requiring refinement. The clinical occurrence of discordant outcomes is uncommon and the study team experienced difficulty identifying patients in this situation. Future work Future work could include regular review of the risk models for adverse outcomes to ensure currency, and the technical precision of complex-numbers analysis of refractive outcome to invite opportunities to improve post-operative spectacle-free vision. In addition, a fully powered randomised controlled trial of the cataract decision aid would be feasible, following psychometric refinement of the primary outcome (the cataract decision quality measure); this would clarify its potential role in routine service delivery. Conclusions In this research programme, evidence-based clinical tools have been successfully developed to improve pre-operative decision-making in cataract surgery. These include a psychometrically robust, patient-reported outcome measure (Cat-PROM5); prediction models for patient self-reported outcomes using Cat-PROM5; prediction models for clinically adverse surgical events and adverse visual acuity outcomes; and a cataract decision aid with relevant general information and personalised risk/benefit predictions. In addition, the successful mapping of Cat-PROM5 to existing health economic indices was achieved and the performances of indices were assessed in patients undergoing cataract surgery. A future full-powered randomised controlled trial of the cataract decision aid would be feasible (patients, n = 800, effect size 0.2 standard deviations, power 80%; p = 0.05). Trial registration This trial is registered as ISRCTN11309852. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Programme Grants for Applied Research programme and will be published in full in Programme Grants for Applied Research; Vol. 10, No. 9. See the NIHR Journals Library website for further project information

Retrospective screening of routine respiratory samples revealed undetected community transmission and missed intervention opportunities for SARS-CoV-2 in the United Kingdom.

In the early phases of the SARS coronavirus type 2 (SARS-CoV-2) pandemic, testing focused on individuals fitting a strict case definition involving a limited set of symptoms together with an identified epidemiological risk, such as contact with an infected individual or travel to a high-risk area. To assess whether this impaired our ability to detect and control early introductions of the virus into the UK, we PCR-tested archival specimens collected on admission to a large UK teaching hospital who retrospectively were identified as having a clinical presentation compatible with COVID-19. In addition, we screened available archival specimens submitted for respiratory virus diagnosis, and dating back to early January 2020, for the presence of SARS-CoV-2 RNA. Our data provides evidence for widespread community circulation of SARS-CoV-2 in early February 2020 and into March that was undetected at the time due to restrictive case definitions informing testing policy. Genome sequence data showed that many of these early cases were infected with a distinct lineage of the virus. Sequences obtained from the first officially recorded case in Nottinghamshire - a traveller returning from Daegu, South Korea - also clustered with these early UK sequences suggesting acquisition of the virus occurred in the UK and not Daegu. Analysis of a larger sample of sequences obtained in the Nottinghamshire area revealed multiple viral introductions, mainly in late February and through March. These data highlight the importance of timely and extensive community testing to prevent future widespread transmission of the virus.Whole genome sequencing of SARS-CoV-2 was funded by COG-UK; COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research and Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute

The Impact of Real-Time Whole-Genome Sequencing in Controlling Healthcare-Associated SARS-CoV-2 Outbreaks

Nosocomial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have severely affected bed capacity and patient flow. We utilized whole-genome sequencing (WGS) to identify outbreaks and focus infection control resources and intervention during the United Kingdom’s second pandemic wave in late 2020. Phylogenetic analysis of WGS and epidemiological data pinpointed an initial transmission event to an admission ward, with immediate prior community infection linkage documented. High incidence of asymptomatic staff infection with genetically identical viral sequences was also observed, which may have contributed to the propagation of the outbreak. WGS allowed timely nosocomial transmission intervention measures, including admissions ward point-of-care testing and introduction of portable HEPA14 filters. Conversely, WGS excluded nosocomial transmission in 2 instances with temporospatial linkage, conserving time and resources. In summary, WGS significantly enhanced understanding of SARS-CoV-2 clusters in a hospital setting, both identifying high-risk areas and conversely validating existing control measures in other units, maintaining clinical service overall

Electrophysiological measurements of peripheral vestibular function—A review of electrovestibulography

Electrocochleography (EcochG), incorporating the Cochlear Microphonic (CM), the Summating Potential (SP), and the cochlear Compound Action Potential (CAP), has been used to study cochlear function in humans and experimental animals since the 1930s, providing a simple objective tool to assess both hair cell (HC) and nerve sensitivity. The vestibular equivalent of ECochG, termed here Electrovestibulography (EVestG), incorporates responses of the vestibular HCs and nerve. Few research groups have utilized EVestG to study vestibular function. Arguably, this is because stimulating the cochlea in isolation with sound is a trivial matter, whereas stimulating the vestibular system in isolation requires significantly more technical effort. That is, the vestibular system is sensitive to both high-level sound and bone-conducted vibrations, but so is the cochlea, and gross electrical responses of the inner ear to such stimuli can be difficult to interpret. Fortunately, several simple techniques can be employed to isolate vestibular electrical responses. Here, we review the literature underpinning gross vestibular nerve and HC responses, and we discuss the nomenclature used in this field. We also discuss techniques for recording EVestG in experimental animals and humans and highlight how EVestG is furthering our understanding of the vestibular system

ClimSim: A large multi-scale dataset for hybrid physics-ML climate emulation

Modern climate projections lack adequate spatial and temporal resolution due to computational constraints. A consequence is inaccurate and imprecise predictions of critical processes such as storms. Hybrid methods that combine physics with machine learning (ML) have introduced a new generation of higher fidelity climate simulators that can sidestep Moore's Law by outsourcing compute-hungry, short, high-resolution simulations to ML emulators. However, this hybrid ML-physics simulation approach requires domain-specific treatment and has been inaccessible to ML experts because of lack of training data and relevant, easy-to-use workflows. We present ClimSim, the largest-ever dataset designed for hybrid ML-physics research. It comprises multi-scale climate simulations, developed by a consortium of climate scientists and ML researchers. It consists of 5.7 billion pairs of multivariate input and output vectors that isolate the influence of locally-nested, high-resolution, high-fidelity physics on a host climate simulator's macro-scale physical state.The dataset is global in coverage, spans multiple years at high sampling frequency, and is designed such that resulting emulators are compatible with downstream coupling into operational climate simulators. We implement a range of deterministic and stochastic regression baselines to highlight the ML challenges and their scoring. The data (https://huggingface.co/datasets/LEAP/ClimSim_high-res) and code (https://leap-stc.github.io/ClimSim) are released openly to support the development of hybrid ML-physics and high-fidelity climate simulations for the benefit of science and society

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Overview of the MOSAiC expedition - Atmosphere

With the Arctic rapidly changing, the needs to observe, understand, and model the changes are essential. To support these needs, an annual cycle of observations of atmospheric properties, processes, and interactions were made while drifting with the sea ice across the central Arctic during the Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) expedition from October 2019 to September 2020. An international team designed and implemented the comprehensive program to document and characterize all aspects of the Arctic atmospheric system in unprecedented detail, using a variety of approaches, and across multiple scales. These measurements were coordinated with other observational teams to explore cross-cutting and coupled interactions with the Arctic Ocean, sea ice, and ecosystem through a variety of physical and biogeochemical processes. This overview outlines the breadth and complexity of the atmospheric research program, which was organized into 4 subgroups: atmospheric state, clouds and precipitation, gases and aerosols, and energy budgets. Atmospheric variability over the annual cycle revealed important influences from a persistent large-scale winter circulation pattern, leading to some storms with pressure and winds that were outside the interquartile range of past conditions suggested by long-term reanalysis. Similarly, the MOSAiC location was warmer and wetter in summer than the reanalysis climatology, in part due to its close proximity to the sea ice edge. The comprehensiveness of the observational program for characterizing and analyzing atmospheric phenomena is demonstrated via a winter case study examining air mass transitions and a summer case study examining vertical atmospheric evolution. Overall, the MOSAiC atmospheric program successfully met its objectives and was the most comprehensive atmospheric measurement program to date conducted over the Arctic sea ice. The obtained data will support a broad range of coupled-system scientific research and provide an important foundation for advancing multiscale modeling capabilities in the Arctic

Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant

SARS-CoV-2 infections were rising during early summer 2021 in many countries associated with the Delta variant. We assessed RT-PCR swab-positivity in the REal-time Assessment of Community Transmission-1 (REACT-1) study in England. We observed sustained exponential growth with average doubling time (June-July 2021) of 25 days driven by complete replacement of Alpha variant by Delta, and by high prevalence at younger less-vaccinated ages. Unvaccinated people were three times more likely than double-vaccinated people to test positive. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination