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Objective. Propose a highly automated treatment plan re-optimization strategy suitable for online adaptive proton therapy. The strategy includes a rapid re-optimization method that generates quality replans and a novel solution that efficiently addresses the planning constraint infeasibility issue that can significantly prolong the re-optimization process.Approach. We propose a systematic reference point method (RPM) model that minimizes the l-infinity norm from the initial treatment plan in the daily objective space for online re-optimization. This model minimizes the largest objective value deviation among the objectives of the daily replan from their reference values, leading to a daily replan similar to the initial plan. Whether a set of planning constraints is feasible with respect to the daily anatomy cannot be known before solving the corresponding optimization problem. The conventional trial-and-error-based relaxation process can cost a significant amount of time. To that end, we propose an optimization problem that first estimates the magnitude of daily violation of each planning constraint. Guided by the violation magnitude and clinical importance of the constraints, the constraints are then iteratively converted into objectives based on their priority until the infeasibility issue is solved.Main results.The proposed RPM-based strategy generated replans similar to the offline manual replans within the online time requirement for six head and neck and four breast patients. The average targetD95and relevant organ at risk sparing parameter differences between the RPM replans and clinical offline replans were -0.23, -1.62 Gy for head and neck cases and 0.29, -0.39 Gy for breast cases. The proposed constraint relaxation solution made the RPM problem feasible after one round of relaxation for all four patients who encountered the infeasibility issue.Significance. We proposed a novel RPM-based re-optimization strategy and demonstrated its effectiveness on complex cases, regardless of whether constraint infeasibility is encountered.

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Objective. Clinical outcomes after proton therapy have shown some variability that is not fully understood. Different approaches have been suggested to explain the biological outcome, but none has yet provided a comprehensive and satisfactory rationale for observed toxicities. The relatively recent transition from passive scattering (PS) to pencil beam scanning (PBS) treatments has significantly increased the voxel-wise dose rate in proton therapy. In addition, the dose rate distribution is no longer uniform along the cross section of the target but rather highly heterogeneous, following the spot placement. We suggest investigating dose rate as potential contributor to a more complex proton RBE model.Approach. Due to the time structure of the PBS beam delivery the instantaneous dose rate is highly variable voxel by voxel. Several possible parameters to represent voxel-wise dose rate for a given clinical PBS treatment plan are detailed. These quantities were implemented in the scripting environment of our treatment planning system, and computations experimentally verified. Sample applications to treated patient plans are shown.Main results. Computed dose rates we experimentally confirmed. Dose rate maps vary depending on which method is used to represent them. Mainly, the underlying time and dose intervals chosen determine the topography of the resultant distributions. The maximum dose rates experienced by any target voxel in a given PBS treatment plan in our system range from ∼100 to ∼450 Gy(RBE)/min, a factor of 10-100 increase compared to PS. These dose rate distributions are very heterogeneous, with distinct hot spots.Significance. Voxel-wise dose rates for current clinical PBS treatment plans vary greatly from clinically established practice with PS. The exploration of different dose rate measures to evaluate potential correlations with observed clinical outcomes is suggested, potentially adding a missing component in the understanding of proton relative biological effectiveness (RBE).

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PURPOSE: The primary goal of this study was to examine changes in peak insole force and cumulative weighted peak force (CWPF)/km with increased step rate in collegiate runners. The secondary goal was to determine whether sacral acceleration correlates with insole force when increasing step rate. METHODS: Twelve collegiate distance runners ran 1000 m outdoors at 3.83 m·s -1 at preferred and 10% increased step rates while insole force and sacral acceleration were recorded. Cumulative weighted peak force/km was calculated from insole force based on cumulative damage models. The effects of step rate on peak insole force and CWPF·km -1 were tested using paired t tests or Wilcoxon tests. Correlation coefficients between peak axial (approximately vertical) sacral acceleration times body mass and peak insole force were calculated on cohort and individual levels. RESULTS: Peak insole force and CWPF·km -1 decreased ( P < 0.001) with increased step rate. Peak axial sacral acceleration did not correlate with peak insole force on the cohort level ( r = 0.35, P = 0.109) but did within individuals (mean, r = 0.69-0.78; P < 0.05). CONCLUSIONS: Increasing step rate may reduce peak vGRF and CWPF·km -1 in collegiate runners. Therefore, clinicians should consider step rate interventions to reduce peak and cumulative vGRF in this population. Individual-specific calibrations may be required to assess changes in peak vGRF in response to increasing step rate using wearable accelerometers.

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BACKGROUND: Biomedical researchers use alignments produced by BLAST (Basic Local Alignment Search Tool) to categorize their query sequences. Producing such alignments is an essential bioinformatics task that is well suited for the cloud. The cloud can perform many calculations quickly as well as store and access large volumes of data. Bioinformaticians can also use it to collaborate with other researchers, sharing their results, datasets and even their pipelines on a common platform. RESULTS: We present ElasticBLAST, a cloud native application to perform BLAST alignments in the cloud. ElasticBLAST can handle anywhere from a few to many thousands of queries and run the searches on thousands of virtual CPUs (if desired), deleting resources when it is done. It uses cloud native tools for orchestration and can request discounted instances, lowering cloud costs for users. It is supported on Amazon Web Services and Google Cloud Platform. It can search BLAST databases that are user provided or from the National Center for Biotechnology Information. CONCLUSION: We show that ElasticBLAST is a useful application that can efficiently perform BLAST searches for the user in the cloud, demonstrating that with two examples. At the same time, it hides much of the complexity of working in the cloud, lowering the threshold to move work to the cloud.

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Background: Biomedical researchers use alignments produced by BLAST (Basic Local Alignment Search Tool) to categorize their query sequences. Producing such alignments is an essential bioinformatics task that is well suited for the cloud. The cloud can perform many calculations quickly as well as store and access large volumes of data. Bioinformaticians can also use it to collaborate with other researchers, sharing their results, datasets and even their pipelines on a common platform. Results: We present ElasticBLAST, a cloud native application to perform BLAST alignments in the cloud. ElasticBLAST can handle anywhere from a few to many thousands of queries and run the searches on thousands of virtual CPUs (if desired), deleting resources when it is done. It uses cloud native tools for orchestration and can request discounted instances, lowering cloud costs for users. It is supported on Amazon Web Services and Google Cloud Platform. It can search BLAST databases that are user provided or from the National Center for Biotechnology Information. Conclusion: We show that ElasticBLAST is a useful application that can efficiently perform BLAST searches for the user in the cloud, demonstrating that with two examples. At the same time, it hides much of the complexity of working in the cloud, lowering the threshold to move work to the cloud.

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The National Center for Biotechnology Information (NCBI) provides online information resources for biology, including the GenBank® nucleic acid sequence database and the PubMed® database of citations and abstracts published in life science journals. NCBI provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for most of these databases. New resources include the Comparative Genome Resource (CGR) and the BLAST ClusteredNR database. Resources receiving significant updates in the past year include PubMed, PMC, Bookshelf, IgBLAST, GDV, RefSeq, NCBI Virus, GenBank type assemblies, iCn3D, ClinVar, GTR, dbGaP, ALFA, ClinicalTrials.gov, Pathogen Detection, antimicrobial resistance resources, and PubChem. These resources can be accessed through the NCBI home page at https://www.ncbi.nlm.nih.gov.

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PURPOSE: In proton therapy, dose distributions are currently often conformed to organs at risk (OARs) using the less sharp dose fall-off at the lateral beam edge to reduce the effects of uncertainties in the in vivo proton range. However, range uncertainty reductions may make greater use of the sharper dose fall-off at the distal beam edge feasible, potentially improving OAR sparing. We quantified the benefits of such novel beam arrangements. METHODS: For each of 10 brain or skull base cases, five treatment plans robust to 2 mm setup and 0%-4% range uncertainty were created for the traditional clinical beam arrangement and a novel beam arrangement making greater use of the distal beam edge to conform the dose distribution to the brainstem. Metrics including the brainstem normal tissue complication probability (NTCP) with the endpoint of necrosis were determined for all plans and all setup and range uncertainty scenarios. RESULTS: For the traditional beam arrangement, reducing the range uncertainty from the current level of approximately 4% to a potentially achievable level of 1% reduced the brainstem NTCP by up to 0.9 percentage points in the nominal and up to 1.5 percentage points in the worst-case scenario. Switching to the novel beam arrangement at 1% range uncertainty improved these values by a factor of 2, that is, to 1.8 percentage points and 3.2 percentage points, respectively. The novel beam arrangement achieved a lower brainstem NTCP in all cases starting at a range uncertainty of 2%. CONCLUSION: The benefits of novel beam arrangements may be of the same magnitude or even exceed the direct benefits of range uncertainty reductions. Indirect effects may therefore contribute markedly to the benefits of reducing proton range uncertainties.

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PURPOSE: Proton therapy allows for more conformal dose distributions and lower organ at risk and healthy tissue doses than conventional photon-based radiotherapy, but uncertainties in the proton range currently prevent proton therapy from making full use of these advantages. Numerous developments therefore aim to reduce such range uncertainties. In this work, we quantify the benefits of reductions in range uncertainty for treatments of skull base tumors. METHODS: The study encompassed 10 skull base patients with clival tumors. For every patient, six treatment plans robust to setup errors of 2 mm and range errors from 0% to 5% were created. The determined metrics included the brainstem and optic chiasm normal tissue complication probability (NTCP) with the endpoints of necrosis and blindness, respectively, as well as the healthy tissue volume receiving at least 70% of the prescription dose. RESULTS: A range uncertainty reduction from the current level of 4% to a potentially achievable level of 1% reduced the probability of brainstem necrosis by up to 1.3 percentage points in the nominal scenario in which neither setup nor range errors occur and by up to 2.9 percentage points in the worst-case scenario. Such a range uncertainty reduction also reduced the optic chiasm NTCP with the endpoint of blindness by up to 0.9 percentage points in the nominal scenario and by up to 2.2 percentage points in the worst-case scenario. The decrease in the healthy tissue volume receiving at least 70% of the prescription dose ranged from -7.8 to 24.1 cc in the nominal scenario and from -3.4 to 38.4 cc in the worst-case scenario. CONCLUSION: The benefits quantified as part of this study serve as a guideline of the OAR and healthy tissue dose benefits that range monitoring techniques may be able to achieve. Benefits were observed between all levels of range uncertainty. Even smaller range uncertainty reductions may therefore be beneficial.

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Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.

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The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed® database of citations and abstracts published in life science journals. The Entrez system provides search and retrieval operations for most of these data from 34 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Custom implementations of the BLAST program provide sequence-based searching of many specialized datasets. New resources released in the past year include a new PubMed interface and NCBI datasets. Additional resources that were updated in the past year include PMC, Bookshelf, Genome Data Viewer, SRA, ClinVar, dbSNP, dbVar, Pathogen Detection, BLAST, Primer-BLAST, IgBLAST, iCn3D and PubChem. All of these resources can be accessed through the NCBI home page at https://www.ncbi.nlm.nih.gov.

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Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50 ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates.

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The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed database of citations and abstracts published in life science journals. The Entrez system provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Custom implementations of the BLAST program provide sequence-based searching of many specialized datasets. New resources released in the past year include a new PubMed interface, a sequence database search and a gene orthologs page. Additional resources that were updated in the past year include PMC, Bookshelf, My Bibliography, Assembly, RefSeq, viral genomes, the prokaryotic genome annotation pipeline, Genome Workbench, dbSNP, BLAST, Primer-BLAST, IgBLAST and PubChem. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

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BACKGROUND: Next-generation sequencing technologies can produce tens of millions of reads, often paired-end, from transcripts or genomes. But few programs can align RNA on the genome and accurately discover introns, especially with long reads. We introduce Magic-BLAST, a new aligner based on ideas from the Magic pipeline. RESULTS: Magic-BLAST uses innovative techniques that include the optimization of a spliced alignment score and selective masking during seed selection. We evaluate the performance of Magic-BLAST to accurately map short or long sequences and its ability to discover introns on real RNA-seq data sets from PacBio, Roche and Illumina runs, and on six benchmarks, and compare it to other popular aligners. Additionally, we look at alignments of human idealized RefSeq mRNA sequences perfectly matching the genome. CONCLUSIONS: We show that Magic-BLAST is the best at intron discovery over a wide range of conditions and the best at mapping reads longer than 250 bases, from any platform. It is versatile and robust to high levels of mismatches or extreme base composition, and reasonably fast. It can align reads to a BLAST database or a FASTA file. It can accept a FASTQ file as input or automatically retrieve an accession from the SRA repository at the NCBI.

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Despite the enormous effort in the development of effective vaccines against HIV-1, no vaccine candidate has elicited broadly neutralizing antibodies in humans. Thus, generation of more effective anti-HIV vaccines is critically needed. Here we characterize the immune responses induced by nucleoside-modified and purified mRNA-lipid nanoparticle (mRNA-LNP) vaccines encoding the clade C transmitted/founder HIV-1 envelope (Env) 1086C. Intradermal vaccination with nucleoside-modified 1086C Env mRNA-LNPs elicited high levels of gp120-specific antibodies in rabbits and rhesus macaques. Antibodies generated in rabbits neutralized a tier 1 virus, but no tier 2 neutralization activity could be measured. Importantly, three of six non-human primates developed antibodies that neutralized the autologous tier 2 strain. Despite stable anti-gp120 immunoglobulin G (IgG) levels, tier 2 neutralization titers started to drop 4 weeks after booster immunizations. Serum from both immunized rabbits and non-human primates demonstrated antibody-dependent cellular cytotoxicity activity. Collectively, these results are supportive of continued development of nucleoside-modified and purified mRNA-LNP vaccines for HIV. Optimization of Env immunogens and vaccination protocols are needed to increase antibody neutralization breadth and durability.

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The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed database of citations and abstracts published in life science journals. The Entrez system provides search and retrieval operations for most of these data from 38 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. New resources released in the past year include PubMed Labs and a new sequence database search. Resources that were updated in the past year include PubMed, PMC, Bookshelf, genome data viewer, Assembly, prokaryotic genomes, Genome, BioProject, dbSNP, dbVar, BLAST databases, igBLAST, iCn3D and PubChem. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

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Systemic administration of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) leads predominantly to hepatic uptake and expression. Here, we conjugated nucleoside-modified mRNA-LNPs with antibodies (Abs) specific to vascular cell adhesion molecule, PECAM-1. Systemic (intravenous) administration of Ab/LNP-mRNAs resulted in profound inhibition of hepatic uptake concomitantly with ~200-fold and 25-fold elevation of mRNA delivery and protein expression in the lungs compared to non-targeted counterparts. Unlike hepatic delivery of LNP-mRNA, Ab/LNP-mRNA is independent of apolipoprotein E. Vascular re-targeting of mRNA represents a promising, powerful, and unique approach for novel experimental and clinical interventions in organs of interest other than liver.

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Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.

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T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

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