Plug and play virus-like particles for the generation of anti-toxin antibodies.

Snakebite is a major global health concern, for which antivenom remains the only approved treatment to neutralise the harmful effects of the toxins. However, some medically important toxins are poorly immunogenic, resulting in reduced efficacy of the final product. Boosting the immunogenicity of these toxins in the commercial antivenom immunising mixtures could be an effective strategy to improve the final dose efficacy, and displaying snake antigens on Virus-like particles (VLPs) is one method for this. However, despite some applications in the field of snakebite, VLPs have yet to be explored in methods that could be practical at an antivenom manufacturing scale. Here we describe the utilisation of a "plug and play" VLP system to display immunogenic linear peptide epitopes from three finger toxins (3FTxs) and generate anti-toxin antibodies. Rabbits were immunised with VLPs displaying individual consensus linear epitopes and their antibody responses were characterised by immunoassay. Of the three experimental consensus sequences, two produced antibodies capable of recognising the consensus peptides, whilst only one of these could also recognise native whole toxins. Further characterisation of antibodies raised against this peptide demonstrated a sub-class specific response, and that these were able to elicit partially neutralising antibody responses, resulting in increased survival times in a murine snakebite envenoming model.

Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5.

There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.

Ketamine and major ketamine metabolites function as allosteric modulators of opioid receptors.

Ketamine is a glutamate receptor antagonist that was developed over 50 years ago as an anesthetic agent. At subanesthetic doses, ketamine and some metabolites are analgesics and fast-acting antidepressants, presumably through targets other than glutamate receptors. We tested ketamine and its metabolites for activity as allosteric modulators of opioid receptors expressed in recombinant receptors in heterologous systems and native receptors in rodent brain; signaling was examined by measuring GTP binding, b-arrestin recruitment, MAPK activation and neurotransmitter release. While micromolar concentrations of ketamine alone had weak agonist activity at mu opioid receptors, the combination of submicromolar concentrations of ketamine with endogenous opioid peptides produced robust synergistic responses with statistically significant increases in efficacies. All three opioid receptors (mu, delta, and kappa) showed synergism with submicromolar concentrations of ketamine and either Met-enkephalin, Leu-enkephalin, and/or dynorphin A17, albeit the extent of synergy was variable between receptors and peptides. S-ketamine exhibited higher modulatory effect compared to R-ketamine or racemic ketamine with nearly ~100% increase in efficacy. Importantly, the ketamine metabolite 6-hydroxynorketamine showed robust allosteric modulatory activity at mu opioid receptors; this metabolite is known to have analgesic and antidepressant activity but does not bind to glutamate receptors. Ketamine enhanced potency and efficacy of Met-enkephalin signaling both in mouse midbrain membranes and in rat ventral tegmental area neurons, as determined by electrophysiology recordings in brain slices. Taken together, these findings support the hypothesis that some of the therapeutic effects of ketamine and its metabolites are mediated by directly engaging the endogenous opioid system. Significance Statement We found that ketamine and its major biologically-active metabolites function as potent allosteric modulators of mu, delta, and kappa opioid receptors, with submicromolar concentrations of these compounds synergizing with endogenous opioid peptides such as enkephalin and dynorphin. This allosteric activity may contribute to ketamine's therapeutic effectiveness for treating acute and chronic pain and as a fast-acting antidepressant drug.

Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype.

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Deeper and stronger North Atlantic Gyre during the Last Glacial Maximum.

Subtropical gyre (STG) depth and strength are controlled by wind stress curl and surface buoyancy forcing1,2. Modern hydrographic data reveal that the STG extends to a depth of about 1 km in the Northwest Atlantic, with its maximum depth defined by the base of the subtropical thermocline. Despite the likelihood of greater wind stress curl and surface buoyancy loss during the Last Glacial Maximum (LGM)3, previous work suggests minimal change in the depth of the glacial STG4. Here we show a sharp glacial water mass boundary between 33° N and 36° N extending down to between 2.0 and 2.5 km-approximately 1 km deeper than today. Our findings arise from benthic foraminiferal δ18O profiles from sediment cores in two depth transects at Cape Hatteras (36-39° N) and Blake Outer Ridge (29-34° N) in the Northwest Atlantic. This result suggests that the STG, including the Gulf Stream, was deeper and stronger during the LGM than at present, which we attribute to increased glacial wind stress curl, as supported by climate model simulations, as well as greater glacial production of denser subtropical mode waters (STMWs). Our data suggest (1) that subtropical waters probably contributed to the geochemical signature of what is conventionally identified as Glacial North Atlantic Intermediate Water (GNAIW)5-7 and (2) the STG helped sustain continued buoyancy loss, water mass conversion and northwards meridional heat transport (MHT) in the glacial North Atlantic.

Preclinical development of a stabilized RH5 virus-like particle vaccine that induces improved antimalarial antibodies.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage malaria vaccine antigen target, currently in a phase 2b clinical trial as a full-length soluble protein/adjuvant vaccine candidate called RH5.1/Matrix-M. We identify that disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees and that a re-engineered and stabilized immunogen (including just the alpha-helical core of RH5) induces a qualitatively superior growth inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this immunogen, termed "RH5.2," to hepatitis B surface antigen virus-like particles (VLPs) using the "plug-and-display" SpyTag-SpyCatcher platform technology also enables superior quantitative antibody immunogenicity over soluble protein/adjuvant in vaccinated mice and rats. These studies identify a blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in phase 1a/b clinical trials.

Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells.

Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

Blood-stage malaria vaccine candidate RH5.1/Matrix-M in healthy Tanzanian adults and children; an open-label, non-randomised, first-in-human, single-centre, phase 1b trial.

BACKGROUND: A blood-stage Plasmodium falciparum malaria vaccine would provide a second line of defence to complement partially effective or waning immunity conferred by the approved pre-erythrocytic vaccines. RH5.1 is a soluble protein vaccine candidate for blood-stage P falciparum, formulated with Matrix-M adjuvant to assess safety and immunogenicity in a malaria-endemic adult and paediatric population for the first time. METHODS: We did a non-randomised, phase 1b, single-centre, dose-escalation, age de-escalation, first-in-human trial of RH5.1/Matrix-M in Bagamoyo, Tanzania. We recruited healthy adults (aged 18-45 years) and children (aged 5-17 months) to receive the RH5.1/Matrix-M vaccine candidate in the following three-dose regimens: 10 µg RH5.1 at 0, 1, and 2 months (Adults 10M), and the higher dose of 50 µg RH5.1 at 0 and 1 month and 10 µg RH5.1 at 6 months (delayed-fractional third dose regimen; Adults DFx). Children received either 10 µg RH5.1 at 0, 1, and 2 months (Children 10M) or 10 µg RH5.1 at 0, 1, and 6 months (delayed third dose regimen; Children 10D), and were recruited in parallel, followed by children who received the dose-escalation regimen (Children DFx) and children with higher malaria pre-exposure who also received the dose-escalation regimen (High Children DFx). All RH5.1 doses were formulated with 50 µg Matrix-M adjuvant. Primary outcomes for vaccine safety were solicited and unsolicited adverse events after each vaccination, along with any serious adverse events during the study period. The secondary outcome measures for immunogenicity were the concentration and avidity of anti-RH5.1 serum IgG antibodies and their percentage growth inhibition activity (GIA) in vitro, as well as cellular immunogenicity to RH5.1. All participants receiving at least one dose of vaccine were included in the primary analyses. This trial is registered at ClinicalTrials.gov, NCT04318002, and is now complete. FINDINGS: Between Jan 25, 2021, and April 15, 2021, we recruited 12 adults (six [50%] in the Adults 10M group and six [50%] in the Adults DFx group) and 48 children (12 each in the Children 10M, Children 10D, Children DFx, and High Children DFx groups). 57 (95%) of 60 participants completed the vaccination series and 55 (92%) completed 22 months of follow-up following the third vaccination. Vaccinations were well-tolerated across both age groups. There were five serious adverse events involving four child participants during the trial, none of which were deemed related to vaccination. RH5-specific T cell and serum IgG antibody responses were induced by vaccination and purified total IgG showed in vitro GIA against P falciparum. We found similar functional quality (ie, GIA per µg RH5-specific IgG) across all age groups and dosing regimens at 14 days after the final vaccination; the concentration of RH5.1-specific polyclonal IgG required to give 50% GIA was 14·3 µg/mL (95% CI 13·4-15·2). 11 children were vaccinated with the delayed third dose regimen and showed the highest median anti-RH5 serum IgG concentration 14 days following the third vaccination (723 µg/mL [IQR 511-1000]), resulting in all 11 who received the full series showing greater than 60% GIA following dilution of total IgG to 2·5 mg/mL (median 88% [IQR 81-94]). INTERPRETATION: The RH5.1/Matrix-M vaccine candidate shows an acceptable safety and reactogenicity profile in both adults and 5-17-month-old children residing in a malaria-endemic area, with all children in the delayed third dose regimen reaching a level of GIA previously associated with protective outcome against blood-stage P falciparum challenge in non-human primates. These data support onward efficacy assessment of this vaccine candidate against clinical malaria in young African children. FUNDING: The European and Developing Countries Clinical Trials Partnership; the UK Medical Research Council; the UK Department for International Development; the National Institute for Health and Care Research Oxford Biomedical Research Centre; the Division of Intramural Research, National Institute of Allergy and Infectious Diseases; the US Agency for International Development; and the Wellcome Trust.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex.

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Towards co-design of rehabilitation technologies: a collaborative approach to prioritize usability issues.

Introduction: Early stakeholder engagement is critical to the successful development and translation of rehabilitation technologies, a pivotal step of which is usability testing with intended end-users. To this end, several methods employ end-user feedback to identify usability and implementation issues. However, the process of prioritizing identified issues seldom leverages the knowledge and expertise of the range of stakeholders who will ultimately affect the demand and supply of a device. This paper describes a novel method to prioritize end-user feedback using transdisciplinary stakeholder consultation and address it in subsequent product development. The proposed approach was demonstrated using a case study relating to the development of a novel technology for neural recovery after spinal cord injury. Method: Feedback from five individuals with chronic spinal cord injury was collected during two-hour usability evaluation sessions with a fully functional high-fidelity system prototype. A think-aloud and semi-structured interview protocol was used with each participant to identify usability and acceptability issues relating to the system in a 3-phase approach. Phase 1 involved extracting usability issues from think-aloud and semi-structured interview data. Phase 2 involved rating the usability issues based on their significance, technical feasibility, and implementation priority by relevant internal and external stakeholders. Finally, Phase 3 involved aggregating the usability issues according to design and implementation elements to facilitate solution generation, and these solutions were then raised as action tasks for future design iterations. Results: Sixty usability issues representing nine facets of usability were rated. Eighty percent of issues were rated to be of moderate to high significance, 83% were rated as being feasible to address, and 75% were rated as addressable using existing project resources. Fifty percent of the issues were rated to be a high priority for implementation. Evaluation of the grouped issues identified 21 tasks which were mapped to the product roadmap for integration into future design iterations. Discussion: This paper presents a method for meaningful transdisciplinary stakeholder engagement in rehabilitation technology development that can extended to other projects. Alongside a worked example, we offer practical considerations for others seeking to co-develop rehabilitation technologies.

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension.

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease.

Two Different Strategies for Stabilization of Brain Tissue and Extraction of Neuropeptides.

Neuropeptides are bioactive peptides that are synthesized and secreted by neurons in signaling pathways in the brain. Peptides and proteins are extremely vulnerable to proteolytic cleavage when their biological surrounding changes. This makes neuropeptidomics challenging due to the rapid alterations that occur to the peptidome after harvesting of brain tissue samples. For a successful neuropeptidomic study, the biological tissue sample analyzed should resemble the living state as much as possible. Heat stabilization has been proven to inhibit postmortem degradation by denaturing proteolytic enzymes, hence increasing identification rates of neuropeptides. Here, we describe two different stabilization protocols for rodent brain samples that increase the number of intact mature neuropeptides and minimize interference from degradation products of abundant proteins. Additionally, we present an extraction protocol that aims to extract a wide range of hydrophilic and hydrophobic neuropeptides by sequentially using an aqueous and an organic extraction medium.

Quantitative Peptidomics Using Reductive Methylation of Amines.

A number of different approaches have been used for quantitative peptidomics. In this protocol, we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (2H), and reagents containing deuterium and 13C, it is possible to produce five isotopically distinct forms of the methylated peptides, which can be quantified by mass spectrometry. Peptides with free N-termini that are primary amines incorporate two methyl groups using this procedure, which differ by 2 Da for each of the five isotopic combinations. Peptides that contain unmodified lysine residues incorporate additional pairs of methyl groups, leading to larger mass differences between isotopic forms. The reagents are commercially available, relatively inexpensive, and chemically stable.

Quantitative Peptidomics: General Considerations.

Peptidomics is the detection and identification of the peptides present in a sample, and quantitative peptidomics provides additional information about the amounts of these peptides. It is possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards of each peptide. More commonly, relative quantitation is performed to compare peptide levels between two or more samples. Relative quantitation can measure differences between all peptides that are detectable, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available.

Neuropeptidomics of Genetically Defined Cell Types in Mouse Brain.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. In the case of brain, which contains a complex mixture of cell types, standard peptidomics procedures reveal the major peptides in a dissected brain region. It is difficult to obtain information on peptides within a specific cell type using standard approaches, unless that cell type can be isolated. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. When this method is used with mice lacking CPE activity in genetically defined cell types, it allows the detection of peptides specifically produced in that cell type.

Current Challenges and Future Directions in Peptidomics.

The field of peptidomics has been under development since its start more than 20 years ago. In this chapter we provide a personal outlook for future directions in this field. The applications of peptidomics technologies are spreading more and more from classical research of peptide hormones and neuropeptides towards commercial applications in plant and food-science. Many clinical applications have been developed to analyze the complexity of biofluids, which are being addressed with new instrumentation, automization, and data processing. Additionally, the newly developed field of immunopeptidomics is showing promise for cancer therapies. In conclusion, peptidomics will continue delivering important information in classical fields like neuropeptides and peptide hormones, benefiting from improvements in state-of-the-art technologies. Moreover, new directions of research such as immunopeptidomics will further complement classical omics technologies and may become routine clinical procedures. Taken together, discoveries of new substances, networks, and applications of peptides can be expected in different disciplines.

Pulmonary primary oxysterol and bile acid synthesis as a predictor of outcomes in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare and fatal vascular disease with heterogeneous clinical manifestations. To date, molecular determinants underlying the development of PAH and related outcomes remain poorly understood. Herein, we identify pulmonary primary oxysterol and bile acid synthesis (PPOBAS) as a previously unrecognized pathway central to PAH pathophysiology. Mass spectrometry analysis of 2,756 individuals across five independent studies revealed 51 distinct circulating metabolites that predicted PAH-related mortality and were enriched within the PPOBAS pathway. Across independent single-center PAH studies, PPOBAS pathway metabolites were also associated with multiple cardiopulmonary measures of PAH-specific pathophysiology. Furthermore, PPOBAS metabolites were found to be increased in human and rodent PAH lung tissue and specifically produced by pulmonary endothelial cells, consistent with pulmonary origin. Finally, a poly-metabolite risk score comprising 13 PPOBAS molecules was found to not only predict PAH-related mortality but also outperform current clinical risk scores. This work identifies PPOBAS as specifically altered within PAH and establishes needed prognostic biomarkers for guiding therapy in PAH.

Recombinant inbred lines derived from wide crosses in Pisum.

Genomic resources are becoming available for Pisum but to link these to phenotypic diversity requires well marked populations segregating for relevant traits. Here we describe two such resources. Two recombinant inbred populations, derived from wide crosses in Pisum are described. One high resolution mapping population involves cv Caméor, for which the first pea whole genome assembly was obtained, crossed to JI0281, a basally divergent P. sativum sativum landrace from Ethiopia. The other is an inter sub-specific cross between P. s. sativum and the independently domesticated P. s. abyssinicum. The corresponding genetic maps provide information on chromosome level sequence assemblies and identify structural differences between the genomes of these two Pisum subspecies. In order to visualise chromosomal translocations that distinguish the mapping parents, we created a simplified version of Threadmapper to optimise it for interactive 3-dimensional display of multiple linkage groups. The genetic mapping of traits affecting seed coat roughness and colour, plant height, axil ring pigmentation, leaflet number and leaflet indentation enabled the definition of their corresponding genomic regions. The consequence of structural rearrangement for trait analysis is illustrated by leaf serration. These analyses pave the way for identification of the underlying genes and illustrate the utility of these publicly available resources. Segregating inbred populations derived from wide crosses in Pisum, together with the associated marker data, are made publicly available for trait dissection. Genetic analysis of these populations is informative about chromosome scale assemblies, structural diversity in the pea genome and has been useful for the fine mapping of several discrete and quantitative traits.

Evaluation of a Deep Learning Reconstruction for High-Quality T2-Weighted Breast Magnetic Resonance Imaging.

Deep learning (DL) reconstruction techniques to improve MR image quality are becoming commercially available with the hope that they will be applicable to multiple imaging application sites and acquisition protocols. However, before clinical implementation, these methods must be validated for specific use cases. In this work, the quality of standard-of-care (SOC) T2w and a high-spatial-resolution (HR) imaging of the breast were assessed both with and without prototype DL reconstruction. Studies were performed using data collected from phantoms, 20 retrospectively collected SOC patient exams, and 56 prospectively acquired SOC and HR patient exams. Image quality was quantitatively assessed via signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and edge sharpness. Qualitatively, all in vivo images were scored by either two or four radiologist readers using 5-point Likert scales in the following categories: artifacts, perceived sharpness, perceived SNR, and overall quality. Differences in reader scores were tested for significance. Reader preference and perception of signal intensity changes were also assessed. Application of the DL resulted in higher average SNR (1.2-2.8 times), CNR (1.0-1.8 times), and image sharpness (1.2-1.7 times). Qualitatively, the SOC acquisition with DL resulted in significantly improved image quality scores in all categories compared to non-DL images. HR acquisition with DL significantly increased SNR, sharpness, and overall quality compared to both the non-DL SOC and the non-DL HR images. The acquisition time for the HR data only required a 20% increase compared to the SOC acquisition and readers typically preferred DL images over non-DL counterparts. Overall, the DL reconstruction demonstrated improved T2w image quality in clinical breast MRI.