Publications by authors named "David Baker"

1,452 Publications

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Highly effective disease-modifying treatment as initial MS therapy.

Curr Opin Neurol 2021 Mar 31. Epub 2021 Mar 31.

The Blizard Institute, Centre for Neuroscience, Surgery & Trauma, Barts and The London School of Medicine and Dentistry, Queen Mary University of London Clinical Board Medicine (Neuroscience), The Royal London Hospital, Barts Health NHS Trust, London, UK Danish Multiple Sclerosis Center, Department of Neurology, Copenhagen University Hospital, Rigshospitalet Department of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark.

Purpose Of Review: Using highly effective (HE) compounds right from the beginning of disease-modifying immunotherapy (DMT) in people with multiple sclerosis (pwMS) has gained popularity among clinicians and pwMS alike. We discuss the most recent evidence supporting this approach, and whether any of the associated risks should stop us adopting it as a default strategy.

Recent Findings: With the addition of injectable ofatumumab, and the two oral sphingosines one phosphate modulators siponimod and ozanimod, ten HE DMTs are now available for pwMS, though variation in licensing status and cost may limit their use in some healthcare environments. Real World evidence based on large MS registry data suggests the superiority of early HE DMT over a slow treatment escalation approach; delaying HE DMT leads to more rapid and often irreversible disability accrual. Mechanistically, B-cell depletion, particularly memory B-cell suppression, is a common denominator closely associated with DMT efficacy.

Summary: The concept that HE DMTs are necessarily associated with a high risk of adverse effects, is no longer supported by the evidence. The rather predictable and manageable risk profile of most HE DMTs should lower the threshold for clinicians to discuss such treatment with pwMS as a first line approach.
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http://dx.doi.org/10.1097/WCO.0000000000000937DOI Listing
March 2021

In Vitro Permeability, Irritability, and Release Evaluation of Commonly Used Topical Diclofenac Gel Preparations (1%, 5%, and 10%).

Int J Pharm Compd 2021 Mar-Apr;25(2):146-155

Western New England University College of Pharmacy & Health Sciences, Springfield, Massachusetts.

Recently, there has been an increase in the use of compounded topical pain preparations, raising concerns that clinicians and patients may not be aware of the potential safety risks. Topical diclofenac is one of the most widely used pain medications, often used for joint ailments such as osteoarthritis and other musculoskeletal pain. Systemic exposure to diclofenac has a dose-related risk for gastrointestinal, cardiovascular, and renal adverse events, particularly in elderly patients. Topical diclofenac preparations are frequently compounded in pharmacies at the concentrations of 1% to 10% (or higher) with or without other active ingredients such as camphor. Considering the significantly higher strengths of the compounded preparations as compared to the commercially available products (1% to 3%) and the frequency of application (sometimes up to six times a day), concerns arise as to the levels of absorption with these formulations and their potential toxicity. The objective of this initial study was formulated in an attempt to shed light on safety concerns of topical diclofenac preparations. A study was designed to evaluate the in vitro release, irritability, and permeability of three different concentrations of compounded diclofenac gels (1%, 5%, and 10%) in PLO GEL MEDIFLO and VersaPro Gel bases. Using MatTek's EpiDerm system, skin irritability and the in vitro permeation of compounded diclofenac gels were evaluated. Additionally, the in vitro release profile, drug content, content uniformity, and physical properties of the compounded gels (pH, homogeneity) were assessed. In all cases, the drug content, content uniformity, physical properties, and preparation stability during the recommended beyond-use dating (90 days) were acceptable. The release profiles of all tested preparations followed the Higuchi model. The in vitro skin irritation evaluation of the tested formulations indicated no irritant preparation. The permeability assessment of the formulated gels revealed that there is a correlation between drug release and percutaneous absorption. VersaPro Gelbased preparations, which showed a lower percentage of drug release over the experiment time, showed a significantly lower average flux at steady-state and the average percentage of absorbed dose after 24 hours. The percentage absorption (%abs) from different formulations ranged from 11.18% to 19.6% depending on the gel base. The permeability coefficient, kp, (cm/hr) ranged from 0.019 to 0.037, and the average flux (µg/cm2/hr) ranged from 8.7 to 103 depending on the gel base and the diclofenac concentration. Based on our findings and previously reported data, the possibility exists that higher diclofenac concentrations in compounded topical preparations may lead to significantly higher blood concentrations as compared to commercially available products, which in turn may also lead to serious side effects. Accordingly, there is a need for clinical studies to evaluate the safety of compounded diclofenac preparations with higher diclofenac contents than United States Food and Drug Administrationapproved formulations.
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April 2021

Designed proteins assemble antibodies into modular nanocages.

Science 2021 04;372(6537)

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)-mediated apoptosis, angiopoietin-1 receptor (Tie2)-mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-angiotensin-converting enzyme 2 (ACE2) fusion proteins.
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http://dx.doi.org/10.1126/science.abd9994DOI Listing
April 2021

Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson's disease individuals.

Microbiome 2021 03 26;9(1):75. Epub 2021 Mar 26.

Gut Microbes & Health, Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UA, UK.

Background: Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson's disease (PD) and Alzheimer's disease (AD) or as a "brain microbiome" in the context of healthy patients' brain samples.

Methods: Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR.

Results: Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue's sterility and could also be found in independent brain 16S rRNA gene sequences.

Conclusions: Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains. Video abstract.
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http://dx.doi.org/10.1186/s40168-021-01012-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004470PMC
March 2021

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Nature 2021 Mar 24. Epub 2021 Mar 24.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization. Here we show that computationally designed, two-component nanoparticle immunogens induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines.
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http://dx.doi.org/10.1038/s41586-021-03365-xDOI Listing
March 2021

Ca signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Sci Adv 2021 Mar 24;7(13). Epub 2021 Mar 24.

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.
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http://dx.doi.org/10.1126/sciadv.abe5396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990342PMC
March 2021

Peptidomics of enteroendocrine cells and characterisation of potential effects of a novel preprogastrin derived-peptide on glucose tolerance in lean mice.

Peptides 2021 Mar 17:170532. Epub 2021 Mar 17.

University of Cambridge Metabolic Research Laboratories, WT-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK. Electronic address:

Objectives: To analyse the peptidomics of mouse enteroendocrine cells (EECs) and human gastrointestinal (GI) tissue and identify novel gut derived peptides.

Methods: High resolution nano-flow liquid chromatography mass spectrometry (LC-MS/MS) was performed on (i) flow-cytometry purified NeuroD1 positive cells from mouse and homogenised human intestinal biopsies, (ii) supernatants from primary murine intestinal cultures, (iii) intestinal homogenates from mice fed high fat diet. Candidate bioactive peptides were selected on the basis of species conservation, high expression/biosynthesis in EECs and evidence of regulated secretionin vitro. Candidate novel gut-derived peptides were chronically administered to mice to assess effects on food intake and glucose tolerance.

Results: A large number of peptide fragments were identified from human and mouse, including known full-length gut hormones and enzymatic degradation products. EEC-specific peptides were largely from vesicular proteins, particularly prohormones, granins and processing enzymes, of which several exhibited regulated secretionin vitro. No regulated peptides were identified from previously unknown genes. High fat feeding particularly affected the distal colon, resulting in reduced peptide levels from GCG, PYY and INSL5. Of the two candidate novel peptides tested in vivo, a peptide from Chromogranin A (ChgA 435-462a) had no measurable effect, but a progastrin-derived peptide (Gast p59-79), modestly improved glucose tolerance in lean mice.

Conclusion: LC-MS/MS peptidomic analysis of murine EECs and human GI tissue identified the spectrum of peptides produced by EECs, including a potential novel gut hormone, Gast p59-79, with minor effects on glucose tolerance.
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http://dx.doi.org/10.1016/j.peptides.2021.170532DOI Listing
March 2021

Modification of fatty acid profile and biosynthetic pathway in symbiotic corals under eutrophication.

Sci Total Environ 2021 Jun 22;771:145336. Epub 2021 Jan 22.

School of Biological Sciences, The University of Hong Kong, Kadoorie Biological Sciences Building, Pokfulam Road, Hong Kong Special Administrative Region; Swire Institute of Marine Science, The University of Hong Kong, Cape d'Aguilar Road, Shek O, Hong Kong Special Administrative Region. Electronic address:

Symbiotic corals receive energy not only by ingesting food (e.g. plankton, inorganic/organic matter, i.e. heterotrophy), but also by endosymbiosis, which supplies photosynthates (dissolved inorganic carbon, i.e. autotrophy). These two sources of energy have distinct fatty acid (FA) profiles, which can be used to differentiate corals by their primary feeding mode. FA profiles have been applied as biomarkers to evaluate the quality of nutrition in the midst of environmental change. However, species-specific responses of coral FA profiles and biosynthetic pathway under cultural eutrophication are still unknown. We collected two coral species (Acropora samoensis, Platygyra carnosa) from sites with different levels of eutrophication to test for variations in FA profiles. Gas Chromatography-Mass Spectrometry (GC-MS) was performed to identify FA profiles and quantify their concentration. Our main findings are threefold: 1) chronic eutrophication inhibits corals' ability to synthesize essential FA; 2) PUFA:SFA ratio and certain FA biomarkers or their pathway can be successfully utilized to determine the relative degree of autotrophy and heterotrophy in corals; 3) under eutrophication, different FA profiles of coral host tissue are attributed to different feeding strategies. Thus, our research provides significant new insights into the roles of FA as a risk assessment tool in coral reef ecosystems under the pressure of eutrophication.
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http://dx.doi.org/10.1016/j.scitotenv.2021.145336DOI Listing
June 2021

O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity.

Nat Chem 2021 Mar 15. Epub 2021 Mar 15.

Departments of Chemistry, University of Southern California, Los Angeles, CA, USA.

A major role for the intracellular post-translational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area focused on O-GlcNAc modification of the amyloid-forming proteins themselves. Here we used synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we found that O-GlcNAc increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aβ(1-42). Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Finally, we found that, although O-GlcNAc levels are globally reduced in Alzheimer's disease brains, the modification of relevant sHSPs is either maintained or increased, which suggests a mechanism to maintain these potentially protective O-GlcNAc modifications. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.
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http://dx.doi.org/10.1038/s41557-021-00648-8DOI Listing
March 2021

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Institute for Protein Design, Department of Biochemistry, Molecular Engineering and Sciences, University of Washington, Seattle, WA 98195.

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.
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http://dx.doi.org/10.1073/pnas.2012800118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000195PMC
March 2021

Protein sequence design by conformational landscape optimization.

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Faculty of Arts and Sciences, Division of Science, Harvard University, Cambridge, MA 02138;

The protein design problem is to identify an amino acid sequence that folds to a desired structure. Given Anfinsen's thermodynamic hypothesis of folding, this can be recast as finding an amino acid sequence for which the desired structure is the lowest energy state. As this calculation involves not only all possible amino acid sequences but also, all possible structures, most current approaches focus instead on the more tractable problem of finding the lowest-energy amino acid sequence for the desired structure, often checking by protein structure prediction in a second step that the desired structure is indeed the lowest-energy conformation for the designed sequence, and typically discarding a large fraction of designed sequences for which this is not the case. Here, we show that by backpropagating gradients through the transform-restrained Rosetta (trRosetta) structure prediction network from the desired structure to the input amino acid sequence, we can directly optimize over all possible amino acid sequences and all possible structures in a single calculation. We find that trRosetta calculations, which consider the full conformational landscape, can be more effective than Rosetta single-point energy estimations in predicting folding and stability of de novo designed proteins. We compare sequence design by conformational landscape optimization with the standard energy-based sequence design methodology in Rosetta and show that the former can result in energy landscapes with fewer alternative energy minima. We show further that more funneled energy landscapes can be designed by combining the strengths of the two approaches: the low-resolution trRosetta model serves to disfavor alternative states, and the high-resolution Rosetta model serves to create a deep energy minimum at the design target structure.
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http://dx.doi.org/10.1073/pnas.2017228118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980421PMC
March 2021

Consistent ultra-long DNA sequencing with automated slow pipetting.

BMC Genomics 2021 Mar 12;22(1):182. Epub 2021 Mar 12.

Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, USA.

Background: Oxford Nanopore Technologies' instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing.

Results: We have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries' average read length over manual slow pipetting.

Conclusions: SNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.
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http://dx.doi.org/10.1186/s12864-021-07500-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953553PMC
March 2021

Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice.

bioRxiv 2021 Mar 1. Epub 2021 Mar 1.

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2021.03.01.433110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941621PMC
March 2021

Malaria Parasite Schizont Egress Antigen-1 Plays an Essential Role in Nuclear Segregation during Schizogony.

mBio 2021 03 9;12(2). Epub 2021 Mar 9.

Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom

Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle. In human malarial infections, an antibody response specific for the protein PF3D7_1021800 was previously associated with protection against malaria, leading to an interest in PF3D7_1021800 as a candidate vaccine antigen. Antibodies to the protein were reported to inhibit egress, resulting in it being named schizont egress antigen-1 (SEA1). A separate study found that SEA1 undergoes phosphorylation in a manner dependent upon the parasite cGMP-dependent protein kinase PKG, which triggers egress. While these findings imply a role for SEA1 in merozoite egress, this protein has also been implicated in kinetochore function during schizont development. Therefore, the function of SEA1 remains unclear. Here, we show that SEA1 localizes in proximity to centromeres within dividing nuclei and that conditional disruption of SEA1 expression severely impacts the distribution of DNA and formation of merozoites during schizont development, with a proportion of SEA1-null merozoites completely lacking nuclei. SEA1-null schizonts rupture, albeit with low efficiency, suggesting that neither SEA1 function nor normal segmentation is a prerequisite for egress. We conclude that SEA1 does not play a direct mechanistic role in egress but instead acts upstream of egress as an essential regulator required to ensure the correct packaging of nuclei within merozoites. Malaria is a deadly infectious disease. Rationally designed novel therapeutics will be essential for its control and eradication. The protein PF3D7_1021800, annotated as SEA1, is under investigation as a prospective component of a malaria vaccine, based on previous indications that antibodies to SEA1 interfere with parasite egress from infected erythrocytes. However, a consensus on the function of SEA1 is lacking. Here, we demonstrate that SEA1 localizes to dividing parasite nuclei and is necessary for the correct segregation of replicated DNA into individual daughter merozoites. In the absence of SEA1, merozoites develop defectively, often completely lacking a nucleus, and, consequently, egress is impaired and/or aberrant. Our findings provide insights into the divergent mechanisms by which intraerythrocytic malaria parasites develop and divide. Our conclusions regarding the localization and function of SEA1 are not consistent with the hypothesis that antibodies against it confer protective immunity to malaria by blocking merozoite egress.
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http://dx.doi.org/10.1128/mBio.03377-20DOI Listing
March 2021

Protein sequence optimization with a pairwise decomposable penalty for buried unsatisfied hydrogen bonds.

PLoS Comput Biol 2021 Mar 8;17(3):e1008061. Epub 2021 Mar 8.

Institute for Protein Design, University of Washington, Seattle, Washington, United States of America.

In aqueous solution, polar groups make hydrogen bonds with water, and hence burial of such groups in the interior of a protein is unfavorable unless the loss of hydrogen bonds with water is compensated by formation of new ones with other protein groups. For this reason, buried "unsatisfied" polar groups making no hydrogen bonds are very rare in proteins. Efficiently representing the energetic cost of unsatisfied hydrogen bonds with a pairwise-decomposable energy term during protein design is challenging since whether or not a group is satisfied depends on all of its neighbors. Here we describe a method for assigning a pairwise-decomposable energy to sidechain rotamers such that following combinatorial sidechain packing, buried unsaturated polar atoms are penalized. The penalty can be any quadratic function of the number of unsatisfied polar groups, and can be computed very rapidly. We show that inclusion of this term in Rosetta sidechain packing calculations substantially reduces the number of buried unsatisfied polar groups.
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http://dx.doi.org/10.1371/journal.pcbi.1008061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971855PMC
March 2021

A Symbiosis of Access: Proliferating STEM PhD Training in the U.S. from 1920-2010.

Minerva 2021 Mar 23;59(1):79-98. Epub 2020 Oct 23.

The Pennsylvania State University.

Over the course of the 20 century, unprecedented growth in scientific discovery was fueled by broad growth in the number of university-based scientists. During this period the American undergraduate enrollment rate and number of universities with STEM graduate programs each doubled three times and the annual volume of new PhDs doubled six times. This generated the research capacity that allowed the United States to surpass early European-dominated science production and lead for the rest of the century. Here, we focus on origins in the organizational environment and institutional dynamics instead of conventional economic factors. We argue that three trends of such dynamics in the development of American higher education not often considered together-mass undergraduate education, decentralized founding of universities, and flexible mission charters for PhD training-form a process characterized by a term coined here: . Then using a 90-year data series on STEM PhD production and institutional development, we demonstrate the historical progression of these mutually beneficial trends. This access symbiosis in the U.S., and perhaps versions of it in other nations, is likely one critical component of the integration of higher education development with the growing global capacity for scientific discovery. These results are discussed in terms of the contributions of American universities to the Century of Science, recent international trends, and its future viability.
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http://dx.doi.org/10.1007/s11024-020-09422-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923690PMC
March 2021

CRISPR-Cas9 Genome Editing of .

Bio Protoc 2020 Feb 20;10(4):e3522. Epub 2020 Feb 20.

Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, United Kingdom.

is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After is the only other malaria parasite capable of being maintained in long term culture with human red blood cells (RBCs). Its closer ancestry to other non-falciparum human malaria parasites, more balanced AT-content, larger merozoites and higher transfection efficiencies, gives some key advantages over for the study of malaria parasite cell/molecular biology. Here, we describe the generation of marker-free CRISPR gene-edited parasites, the fast and scalable production of transfection constructs and analysis of transfection efficiencies. Our protocol allows rapid, reliable and unlimited rounds of genome editing in requiring only a single recyclable selection marker.
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http://dx.doi.org/10.21769/BioProtoc.3522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842605PMC
February 2020

Improved protein structure refinement guided by deep learning based accuracy estimation.

Nat Commun 2021 02 26;12(1):1340. Epub 2021 Feb 26.

Department of Biochemistry and Institute for Protein Design, University of Washington, Washington, WA, USA.

We develop a deep learning framework (DeepAccNet) that estimates per-residue accuracy and residue-residue distance signed error in protein models and uses these predictions to guide Rosetta protein structure refinement. The network uses 3D convolutions to evaluate local atomic environments followed by 2D convolutions to provide their global contexts and outperforms other methods that similarly predict the accuracy of protein structure models. Overall accuracy predictions for X-ray and cryoEM structures in the PDB correlate with their resolution, and the network should be broadly useful for assessing the accuracy of both predicted structure models and experimentally determined structures and identifying specific regions likely to be in error. Incorporation of the accuracy predictions at multiple stages in the Rosetta refinement protocol considerably increased the accuracy of the resulting protein structure models, illustrating how deep learning can improve search for global energy minima of biomolecules.
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http://dx.doi.org/10.1038/s41467-021-21511-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910447PMC
February 2021

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

PLoS Pathog 2021 02 26;17(2):e1009373. Epub 2021 Feb 26.

Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.
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http://dx.doi.org/10.1371/journal.ppat.1009373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946358PMC
February 2021

De novo design of transmembrane β barrels.

Science 2021 02;371(6531)

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a "hypothesis, design, and test" approach to determine TMB design principles, notably, the importance of negative design to slow β-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.
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http://dx.doi.org/10.1126/science.abc8182DOI Listing
February 2021

Investigating the link between Pearl River-induced eutrophication and hypoxia in Hong Kong shallow coastal waters.

Sci Total Environ 2021 Jun 2;772:145007. Epub 2021 Feb 2.

Department of Earth Science, Swire Institute of Marine Science, The University of Hong Kong, Hong Kong, China. Electronic address:

We present geochemical analysis of 75 surface water samples collected in 2016 in Hong Kong coastal waters. We found that nitrogen distribution around Hong Kong can be characterized by two regimes driven by the influence of the Pearl River: 1) a regime where nitrate is the dominant species of nitrogen, associated with lower salinity and more faecal coliform and 2) a regime where dissolved organic nitrogen is dominant, associated with higher salinity and fewer faecal coliform. While the impact of the Pearl River on Hong Kong coastal waters is well characterized, we used the sharp contrast between the nitrogen regimes to produce new evidence about the role of the Pearl River on the generation of local hypoxia in Hong Kong. The impact of nitrate originating from the Pearl River on the generation of hypoxia in Hong Kong might be less important than previously thought, as no sign of eutrophication was found within the zones dominated by dissolved organic nitrogen and an historical decoupling of surface processes and bottom water oxygenation was observed. Moreover, we measured elevated ammonium levels and rapid cycling of ammonium and dissolved organic nitrogen in Victoria Harbour suggesting local sources, such as wastewater, might be rapidly oxidized and thus play an important role in the consumption of oxygen locally. A first-order calculation highlighted the potential for wastewater to drive the observed seasonal decline in oxygen. Taken together, these evidences suggest that eutrophication might not be the primary driver in the generation of seasonal hypoxia and that oxidation of ammonium released locally might play a bigger role than initially thought.
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http://dx.doi.org/10.1016/j.scitotenv.2021.145007DOI Listing
June 2021

Functionalized Phosphonium Cations Enable Zn Metal Reversibility in Aqueous Electrolytes.

Angew Chem Int Ed Engl 2021 Feb 12. Epub 2021 Feb 12.

U.S. Army Research Laboratory, Power and Energy Division, U.S. Army Research Laboratory, Adelphi, MD 20783, 20783, Adelphi, UNITED STATES.

Aqueous rechargeable zinc metal batteries promise attractive advantages including safety, high volumetric energy density and low cost; however, such benefits cannot be unlocked unless Zn reversibility meets stringent commercial viability. Herein, we report remarkable improvements on Zn reversibility in aqueous electrolytes when phosphonium-based cations are used to reshape interfacial structures and interphasial chemistries, particularly when their ligands contain ether linkage. This novel aqueous electrolyte supports unprecedented Zn reversibility by showing dendrite-free Zn plating/stripping for over 6400 h at 0.5 mA cm -2 , or over 280 h at 2.5 mA cm -2 , with coulombic efficiency above 99% even with 20% Zn utilization per-cycle. Excellent full cell performance is demonstrated with Na 2 V 6 O 16 ·1.63H 2 O cathode, which cycles for 2000 times at 300 mA g -1 . The microscopic characterization and modeling identify the mechanism of unique interphase chemistry from phosphonium and its functionalities as the key factors responsible for dictating reversible Zn chemistry .
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http://dx.doi.org/10.1002/anie.202017020DOI Listing
February 2021

Force Field Optimization Guided by Small Molecule Crystal Lattice Data Enables Consistent Sub-Angstrom Protein-Ligand Docking.

J Chem Theory Comput 2021 Mar 12;17(3):2000-2010. Epub 2021 Feb 12.

Department of Biochemistry and Institute for Protein Design, University of Washington, Seattle, Washington 98195, United States.

Accurate and rapid calculation of protein-small molecule interaction free energies is critical for computational drug discovery. Because of the large chemical space spanned by drug-like molecules, classical force fields contain thousands of parameters describing atom-pair distance and torsional preferences; each parameter is typically optimized independently on simple representative molecules. Here, we describe a new approach in which small molecule force field parameters are jointly optimized guided by the rich source of information contained within thousands of available small molecule crystal structures. We optimize parameters by requiring that the experimentally determined molecular lattice arrangements have lower energy than all alternative lattice arrangements. Thousands of independent crystal lattice-prediction simulations were run on each of 1386 small molecule crystal structures, and energy function parameters of an implicit solvent energy model were optimized, so native crystal lattice arrangements had the lowest energy. The resulting energy model was implemented in Rosetta, together with a rapid genetic algorithm docking method employing grid-based scoring and receptor flexibility. The success rate of bound structure recapitulation in cross-docking on 1112 complexes was improved by more than 10% over previously published methods, with solutions within <1 Å in over half of the cases. Our results demonstrate that small molecule crystal structures are a rich source of information for guiding molecular force field development, and the improved Rosetta energy function should increase accuracy in a wide range of small molecule structure prediction and design studies.
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http://dx.doi.org/10.1021/acs.jctc.0c01184DOI Listing
March 2021

Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.

NPJ Vaccines 2020 Aug 5;5(1):72. Epub 2020 Aug 5.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
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http://dx.doi.org/10.1038/s41541-020-00223-1DOI Listing
August 2020