Search bioRxiv⌕ Search

Biology subjects

Marks, D.

Publications and source records attributed to Marks, D..

32 records · Page 2Linked to original sources

Learning from pre-pandemic data to forecast viral antibody escape

Effective pandemic preparedness relies on anticipating viral mutations that are able to evade host immune responses in order to facilitate vaccine and therapeutic design. However, current strategies for viral evolution prediction are not available early in a pandemic - experimental approaches require host polyclonal antibodies to test against and existing computational methods draw heavily from current strain prevalence to make reliable predictions of variants of concern. To address this, we developed EVEscape, a generalizable, modular framework that combines fitness predictions from a deep learning model of historical sequences with biophysical structural information. EVEscape quantifies the viral escape potential of mutations at scale and has the advantage of being applicable before surveillance sequencing, experimental scans, or 3D structures of antibody complexes are available. We demonstrate that EVEscape, trained on sequences available prior to 2020, is as accurate as high-throughput experimental scans at anticipating pandemic variation for SARS-CoV-2 and is generalizable to other viruses including Influenza, HIV, and understudied viruses with pandemic potential such as Lassa and Nipah. We provide continually updated escape scores for all current strains of SARS-CoV-2 and predict likely additional mutations to forecast emerging strains as a tool for ongoing vaccine development (evescape.org).

immunology↗

Non-identifiability and the Blessings of Misspecification in Models of Molecular Fitness and Phylogeny

Understanding the consequences of mutation for molecular fitness and function is a fundamental problem in biology. Recently, generative probabilistic models have emerged as a powerful tool for estimating fitness from evolutionary sequence data, with accuracy sufficient to predict both laboratory measurements of function and disease risk in humans, and to design novel functional proteins. Existing techniques rest on an assumed relationship between density estimation and fitness estimation, a relationship that we interrogate in this article. We prove that fitness is not identifiable from observational sequence data alone, placing fundamental limits on our ability to disentangle fitness landscapes from phylogenetic history. We show on real datasets that perfect density estimation in the limit of infinite data would, with high confidence, result in poor fitness estimation; current models perform accurate fitness estimation because of, not despite, misspecification. Our results challenge the conventional wisdom that bigger models trained on bigger datasets will inevitably lead to better fitness estimation, and suggest novel estimation strategies going forward.

evolutionary biology↗

Optimal Design of Stochastic DNA Synthesis Protocols based on Generative Sequence Models

Generative probabilistic models of biological sequences have widespread existing and potential applications in analyzing, predicting and designing proteins, RNA and genomes. To test the predictions of such a model experimentally, the standard approach is to draw samples, and then synthesize each sample individually in the laboratory. However, often orders of magnitude more sequences can be experimentally assayed than can affordably be synthesized individually. In this article, we propose instead to use stochastic synthesis methods, such as mixed nucleotides or trimers. We describe a black-box algorithm for optimizing stochastic synthesis protocols to produce approximate samples from any target generative model. We establish theoretical bounds on the methods performance, and validate it in simulation using held-out sequence-to-function predictors trained on real experimental data. We show that using optimized stochastic synthesis protocols in place of individual synthesis can increase the number of hits in protein engineering efforts by orders of magnitude, e.g. from zero to a thousand.

synthetic biology↗

Dormant spores sense amino acids through the B subunits of their germination receptors

Bacteria from the orders Bacillales and Clostridiales differentiate into stress-resistant spores that can remain dormant for years, yet rapidly germinate upon nutrient sensing. How spores monitor nutrients is poorly understood but in most cases requires putative membrane receptors. The prototypical receptor from Bacillus subtilis consists of three proteins (GerAA, GerAB, GerAC) required for germination in response to L-alanine. GerAB belongs to the Amino Acid-Polyamine-Organocation superfamily of transporters. Using evolutionary co-variation analysis, we provide evidence that GerAB adopts a structure similar to an L-alanine transporter from this superfamily. We show that mutations in gerAB predicted to disrupt the ligand-binding pocket impair germination, while mutations predicted to function in L-alanine recognition enable spores to respond to L-leucine or L-serine. Finally, substitutions of bulkier residues at these positions cause constitutive germination. These data suggest that GerAB is the L-alanine sensor and that B subunits in this broadly conserved family function in nutrient detection.

microbiology↗

Fast and reliable sgRNA efficiency testing using HIReff

CRISPR/Cas9 is the method of choice for gene editing like the endogenous knock-in of sequences in order to investigate protein function, abundance or intracellular localization. One of the crucial steps in the preparation of CRISPR/Cas9-mediated knock-ins is the design of sgRNAs, which need to be tested carefully in order to minimize off-target binding and reach highest cleavage efficiency. Usually, sgRNA is evaluated via mismatch cleavage assays, like Surveyor or T7 endonuclease 1 assay. We demonstrate that these methods are often highly cost- and time-intensive with a low sensitivity and high fail rate. As an alternative, we present a new HITI-based sgRNA efficiency (HIReff) test to precisely evaluate sgRNA efficiency. HIReff is based on a sophisticated integration vector with on-site generation of a linear donor fragment that allows a comparably easy read-out via fluorescence signal and integrates several internal controls. Next to a quantifiable sgRNA assessment, HIReff provides additional information on the gene/protein to be studied abundance, subcellular localization and promoter activity and allows derivation of fluorescence protein labeled clonal lines. We highlight benefits of HIReff in comparison to commonly used enzyme-based assays and demonstrate improved practicability and high sensitivity, while being less time-, labor- and costintensive at the same time. Our results suggest HIReff as a fast and easy-to-use alternative for sgRNA efficiency testing.

molecular biology↗

Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates

Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation, and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe small RNAs, pseudogenes, and transposable elements, and highlight tissue specific expression and methylation patterns. Resequencing of forty wild accessions provides insights into genome-wide genetic variation as well as QTL regions for flowering time and a seedling color phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

plant biology↗

The method of choice to knock-in large inserts via CRISPR

CRISPR/Cas9 gene editing is a revolutionary method used to study gene function by transcript silencing, knock-out, or activation. The knock-in of DNA fragments to endogenous genes of interest is another promising approach to study molecular pathways but is technically challenging. Many approaches have been suggested, but the proof of correct integration has often been relied on less convenient validation experiments. Within this work, we investigated homology-directed repair (HDR), non-homologous end joining (NHEJ), and PCRextension (PCRext) based approaches as three different methods to knock-in large DNA fragments (>1000 bp), and compared feasibility, cost effectiveness, and reliability. As a knock-in fragment, we used a fluorescent reporter sequence in order to directly assess successful integration by microscopy, subsequently proven by sequencing. For NHEJ and PCRext, we demonstrate that it is insufficient to rely on the fluorescent reporter due to false positive results. Both NHEJ and PCRext failed to reliably knock-in large DNA sequences, they were accompanied by massive generation of InDels driving the methodology cost-intensive and non-reliable. In contrast, combination of CRISPR/Cas9 and HDR revealed correct integration, proven by correct fluorescence of the subcellular localization and sequencing, and thus, corresponds to the method of choice for large fragment integration. Next to HEK293T, we demonstrate successful HDR based knock-in in human induced pluripotent stem cells (hiPSCs). Subsequent differentiation of gene-edited hiPSCs into cerebral organoids showed relevance of the approach to study subcellular protein localization and abundance in 3D tissue.

molecular biology↗

High-content screening of coronavirus genes for innate immune suppression revealsenhanced potency of SARS-CoV-2 proteins

Suppression of the host intracellular innate immune system is an essential aspect of viral replication. Here, we developed a suite of medium-throughput high-content cell-based assays to reveal the effect of individual coronavirus proteins on antiviral innate immune pathways. Using these assays, we screened the 196 protein products of seven coronaviruses (SARS-CoV-2, SARS-CoV-1, 229E, NL63, OC43, HKU1 and MERS). This includes a previously unidentified gene in SARS-CoV-2 encoded within the Spike gene. We observe immune-suppressing activity in both known host-suppressing genes (e.g., NSP1, Orf6, NSP3, and NSP5) as well as other coronavirus genes, including the newly identified SARS-CoV-2 protein. Moreover, the genes encoded by SARS-CoV-2 are generally more potent immune suppressors than their homologues from the other coronaviruses. This suite of pathway-based and mechanism-agnostic assays could serve as the basis for rapid in vitro prediction of the pathogenicity of novel viruses based on provision of sequence information alone.

systems biology↗

Large-scale clinical interpretation of genetic variants using evolutionary data and deep learning

Quantifying the pathogenicity of protein variants in human disease-related genes would have a profound impact on clinical decisions, yet the overwhelming majority (over 98%) of these variants still have unknown consequences1-3. In principle, computational methods could support the large-scale interpretation of genetic variants. However, prior methods4-7 have relied on training machine learning models on available clinical labels. Since these labels are sparse, biased, and of variable quality, the resulting models have been considered insufficiently reliable8. By contrast, our approach leverages deep generative models to predict the clinical significance of protein variants without relying on labels. The natural distribution of protein sequences we observe across organisms is the result of billions of evolutionary experiments9,10. By modeling that distribution, we implicitly capture constraints on the protein sequences that maintain fitness. Our model EVE (Evolutionary model of Variant Effect) not only outperforms computational approaches that rely on labelled data, but also performs on par, if not better than, high-throughput assays which are increasingly used as strong evidence for variant classification11-23. After thorough validation on clinical labels, we predict the pathogenicity of 11 million variants across 1,081 disease genes, and assign high-confidence reclassification for 72k Variants of Unknown Significance8. Our work suggests that models of evolutionary information can provide a strong source of independent evidence for variant interpretation and that the approach will be widely useful in research and clinical settings.

genetics↗

Gene edited fluorescent cerebral organoids to study human brain function and disease

Cerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different hiPSC lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to {beta}-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.

cell biology↗

Amyloid precursor protein causes fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates comprising its adapter protein FE65 and the tumour suppressor proteins p53 and PML. The PML nuclear body generation, known to be of relevance in virus defence and cell division, is induced and fusion occurs over time depending on APP signalling. We further show that the nuclear aggregates of APP C-terminal (APP-CT) fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimers disease brains reveal a highly significant loss of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT signalling to the nucleus takes place in the aged human brain and is potentially involved in the pathophysiology of AD. Taken the current knowledge on PML bodies into account, we hypothesize a new role for APP as a twofold virus response protein. The APP-dependent defence strategy includes A{beta}-virus interaction at the extracellular matrix and APP-CT driven PML aggregation in the nucleus to encapsulate the viral nucleic acid. This defence strategy preferentially occurs in high-plaque regions of the human brain and overstimulation of this pathway results in a pyrrhic victory.

neuroscience↗

Interpretable Machine Learning for Perturbation Biology

Systematic perturbation of cells followed by comprehensive measurements of molecular and phenotypic responses provides an informative data resource for constructing computational models of cell biology. Models that generalize well beyond training data can be used to identify combinatorial perturbations of potential therapeutic interest. Major challenges for machine learning on large biological datasets are to find global optima in an enormously complex multi-dimensional solution space and to mechanistically interpret the solutions. To address these challenges, we introduce a hybrid approach that combines explicit mathematical models of dynamic cell biological processes with a machine learning framework, implemented in Tensorflow. We tested the modeling framework on a perturbation-response dataset for a melanoma cell line after drug treatments. The models can be efficiently trained to accurately describe cellular behavior, as tested by cross-validation. Even though completely data-driven and independent of prior knowledge, the resulting de novo network models recapitulate some known interactions. The main predictive application is the identification of combinatorial candidates for cancer therapy. The approach is readily applicable to a wide range of kinetic models of cell biology.

systems biology↗

Interaction specificity of clustered protocadherins inferred from sequence covariation and structural analysis

Clustered protocadherins are a large family of paralogous proteins that play important roles in neuronal development. The more than 50 clustered protocadherin isoforms have remarkable homophilic specificity for interactions between cellular surfaces that is controlled by a large antiparallel dimer interface formed by the first four extracellular cadherin (EC) domains. To understand how specificity is achieved between the numerous paralogs, we used a combination of structural and computational approaches. Molecular dynamics simulations revealed that individual EC interactions are weak and go through binding and unbinding events, but together they form a stable complex through polyvalency. Using sequence coevolution, we generated a statistical model of interaction energy for the clustered protocadherin family that measures the contributions of all amino acid pairs in the interface. Our interaction energy model assesses specificity for all possible pairs of isoforms, recapitulating known pairings and predicting the effects of experimental changes in isoform specificity that are consistent with literature results. Our results show that sequence coevolution can be used to understand specificity determinants in a protein family and prioritize interface amino acid substitutions to reprogram specific protein-protein interactions.

biophysics↗

Cryo-electron microscopy structure of the lipid droplet-formation protein seipin

Sui et al. report the cryo-EM structure of the conserved luminal domain of the lipid droplet (LD)-formation protein seipin. The structure reveals key features of this domain and suggest a new model for seipins role in LD formation.\n\nMetabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. Here, we report a cryo-electron microscopy structure and functional characterization of D. melanogaster seipin. The structure reveals a ring-shaped dodecamer, with the luminal domain of each monomer resolved at [~]4.0 [A]. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix positioned towards the ER bilayer, and a {beta}-sandwich domain that has structural similarity with lipid-binding proteins. This structure, and our functional testing in cells, suggest a model in which seipin oligomers initially detect forming LDs in the ER via hydrophobic helices and subsequently act as membrane anchors to enable lipid transfer and LD growth.

cell biology↗