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Biology subjects

Sumner, J.

Publications and source records attributed to Sumner, J..

8 recordsLinked to original sources

Residue burial encodes a protein's fold

Protein structure is controlled by a high-dimensional energy landscape, which is a function of all of the atomic coordinates of the protein. Can this landscape be accurately described by a low-dimensional representation? We find that residue core identity, a binary N-dimensional encoding indicating whether each of the N amino acids in a protein is buried in the core or not, can predict the proteins backbone conformation more efficiently than all other representations that we tested. Core identity is 4 times more efficient than previous estimates of the bits per residue needed to encode a proteins native fold, 2 times more efficient than the C contact map, and 1.5 times more efficient than the machine-learned embeddings from FoldSeeks 3Di. Even when the folded structure is unavailable, predicting each residues burial from sequence yields a more accurate estimate of fold quality than predicting pairwise contacts from the same sequence information. Thus, this work emphasizes that the problem of determining a proteins native fold can be re-framed as predicting each residues core identity.

biophysics↗

Generalists link peaks in the shifting adaptive landscape of Australia's dragon lizards

The adaptive landscape is a flexible concept in biology which helps map entities onto an eco-evolutionary topography. A common practice is clustering species to identify adaptive peaks but the pathways that allow movement into new adaptive zones remain poorly understood. Here we integrate new sequence-capture (>5,000 nuclear markers) and multivariate morphological data to investigate the adaptive landscape of Amphibolurinae dragon lizards (Agamidae). Our well-supported, time-calibrated phylogeny includes over 360 specimens from 176 species, including iconic taxa such as the frilled-neck lizard, thorny devil, and bearded dragon. We show that arrival in Australia was followed by rapid expansion of morphological diversity facilitated by generalist habitat use. Ancestral dragons were likely arboreal, but species with broad niche breadths and intermediate morphologies provided pathways to subsequent specialization. These findings reveal an evolutionary pathway for adaptive diversification in a major continental radiation.

evolutionary biology↗

Modification of Non-photochemical Quenching Pathways in the C4 Model Plant Setaria viridis Revealed Shared and Unique Photoprotection Mechanisms as Compared to C3 Plants

Light is essential for photosynthesis; however, excess light can increase the accumulation of photoinhibitory reactive oxygen species that reduce photosynthetic efficiency. Plants have evolved photoprotective non-photochemical quenching (NPQ) pathways to dissipate excess light energy. In tobacco and soybean (C3 plants), overexpression of three NPQ genes, violaxanthin de-epoxidase (VDE), Photosystem II Subunit S (PsbS), and zeaxanthin epoxidase (ZEP), hereafter VPZ, resulted in faster NPQ induction and relaxation kinetics, and increased crop yields in field conditions. NPQ is well-studied in C3 plants; however, NPQ and the translatability of the VPZ approach in C4 plants is poorly understood. The green foxtail Setaria viridis is an excellent model to study photosynthesis and photoprotection in C4 plants. To understand the regulation of NPQ and photosynthesis in C4 plants, we performed transient overexpression in Setaria protoplasts and generated (and employed) stable transgenic Setaria plants overexpressing one of the three Arabidopsis NPQ genes or all three NPQ genes (AtVPZ lines). Overexpressing (OE) AtVDE and AtZEP in Setaria produced similar results as in C3 plants, with increased or reduced zeaxanthin (thus NPQ), respectively. However, overexpressing AtPsbS appeared to be challenging in Setaria, with largely reduced NPQ in protoplasts and under-represented homozygous AtPsbS-OE lines, potentially due to competitive and tight heterodimerization of AtPsbS and SvPsbS proteins. Furthermore, Setaria AtVPZ lines had increased zeaxanthin, faster NPQ induction, higher NPQ level, but slower NPQ relaxation. Despite this, AtVPZ lines had improved growth as compared to wildtype under several conditions, especially high temperatures, which is not related to the faster relaxation of NPQ but may be attributable to increased zeaxanthin and NPQ in C4 plants. Our results identified shared and unique characteristics of the NPQ pathway in C4 model Setaria as compared to C3 plants and provide insights to improve C4 crop yields under fluctuating environmental conditions.

plant biology↗

An aptamer-mediated base editing platform for simultaneous knock-in and multiple gene knockout for allogeneic CAR-T cells generation

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply the novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

molecular biology↗

Simultaneous inhibition of DNA-PK and Pol{Theta} improves integration efficiency and precision of genome editing

Genome editing tools, especially CRISPR/Cas9-based strategies, have transformed biomedical research and opened opportunities for developing curative treatments for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of undesired mutations represent major limitations for genome editing applications. Both issues arise from the interplay between the main DNA Double-Strand Break (DSB) repair pathways, Homology-Directed Repair (HDR), Non-Homologous End Joining (NHEJ), and Microhomology-Mediated End Joining (MMEJ). To improve efficiencies of targeted CRISPR-Cas9 genome editing, we screened a large compound library. This led to the discovery of AZD7648, a DNA-dependent protein kinase (DNA-PK) inhibitor and potent enhancer of CRISPR-Cas9-mediated integration. We demonstrated that AZD7648 increased HDR and decreased mutagenic NHEJ repair, thus resulting in improved performance of precise gene editing. Furthermore, we observed additional improvement of integration efficiency by impairing MMEJ repair through DNA polymerase {ominus} (Pol{ominus}) inhibition. Combined treatment with AZD7648 and Pol{ominus} inhibitors (which we named 2iHDR) substantially increased precision of templated insertions, with efficiencies of up to 80%, and nearly no formation of undesired Insertion-Deletions (InDels). Importantly, 2iHDR also decreased Cas9-associated off-target activity, dramatically improving the performance and fidelity of CRISPR-Cas9 gene editing.

molecular biology↗

Differential dysregulation of β-TrCP1 and -2 by HIV-1 Vpu leads to inhibition of canonical and non-canonical NF-κB pathways in infected cells

The HIV-1 Vpu protein is expressed late in the virus lifecycle to promote infectious virus production and avoid innate and adaptive immunity. This includes the inhibition of the NF-{kappa}B pathway which, when activated, leads to the induction of inflammatory responses and the promotion of antiviral immunity. Here we demonstrate that Vpu can inhibit both canonical and non-canonical NF-{kappa}B pathways, through the direct inhibition of the F-box protein {beta}-TrCP, the substrate recognition portion of the Skp1-Cul1-F-box (SCF){beta}-TrCP ubiquitin ligase complex. There are two paralogues of {beta}-TrCP ({beta}-TrCP1/BTRC and {beta}-TrCP2/FBXW11), encoded on different chromosomes, which appear to be functionally redundant. Vpu, however, is one of the few {beta}-TrCP substrates to differentiate between the two paralogues. We have found that patient-derived alleles of Vpu, unlike those from lab-adapted viruses, trigger the degradation of {beta}-TrCP1 while co-opting its paralogue {beta}-TrCP2 for the degradation of cellular targets of Vpu, such as CD4. The potency of this dual inhibition correlates with stabilisation of the classical I{kappa}B and the phosphorylated precursors of the mature DNA-binding subunits of canonical and non-canonical NF-{kappa}B pathways, p105/NF{kappa}B1 and p100/NF{kappa}B2, in HIV-1 infected CD4+ T cells. Both precursors act as alternative I{kappa}Bs in their own right, thus reinforcing NF-{kappa}B inhibition at steady state and upon activation with either selective canonical or non-canonical NF-{kappa}B stimuli. These data reveal the complex regulation of NF-{kappa}B late in the viral replication cycle, with consequences for both the pathogenesis of HIV/AIDS and the use of NF-{kappa}B-modulating drugs in HIV cure strategies.

microbiology↗

Natural Variation in Brachypodium distachyon Responses to Combined Abiotic Stresses

The growing world population increases demand for agricultural production, which is more challenging as climate change increases global temperature and causes more extreme weather events. High-throughput phenotyping tools can be used to measure plant responses to the environment to identify genomic regions associated with response to stress. This study examines the phenotypic variation of 149 accessions of Brachypodium distachyon under drought, heat, and the combination of both stresses. Heat alone causes the largest amounts of tissue damage and the combination of heat and drought causes the largest decrease in plant biomass compared to other treatments. Notably, Bd21-0, the reference line for B. distachyon, was identified as not having very robust growth under stress conditions, especially in the heat-drought combined treatment. Climate data from the collection locations of these accessions (climate of origin) was used to assess whether climate of origin was correlated with responses to stresses and it was found to be significantly associated with height and percent of plant tissue damage. Additionally, genome wide association mapping found a number of genetic loci associated with changes in plant height, biomass, and the amount of damaged tissue under stress. Some SNPs found to be significantly associated with a response to heat or drought are also significantly associated in the combination of stresses, while others are not, and some significantly associated SNPs were only identified in the combined stress treatment. This, combined with the phenotypic data, indicates that the effects of these abiotic stresses are not simply additive, and the responses of B. distachyon to the combined stresses differ from drought and heat alone. Significant SNPs were closely located to genes known to be involved in plant responses to abiotic stresses.

plant biology↗

DENSS-Multiple: A Structure Reconstruction Method using Multiple Contrast Variation of Small-Angle Neutron Scattering Based on the DENSS Algorithm

Small-angle neutron scattering (SANS) provides easily manipulable neutron contrast variation that has been widely exploited to study the structure and function of biological macromolecules and their complex in solution. Here, we developed a method called DENSS-Multiple based on the DENSS (DENsity from Solution Scattering) algorithm to provide ab initio structure reconstruction with SANS contrast variation data. This new tool can exploit additional information in different SANS contrasts for improved ab initio reconstruction results.

biophysics↗