bluuebunny/crossref_metadata_2025 huggingface dataset:
Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing
Authors: Dahn Hollis A. , Mountcastle Jacquelyn , Balacco Jennifer , Winkler Sylke , Bista Iliana , Schmitt Anthony D. , Pettersson Olga Vinnere , Formenti Giulio , Oliver Karen , Smith Michelle , Tan Wenhua , Kraus Anne , Mac Stephen , Komoroske Lisa M. , Lama Tanya , Crawford Andrew J. , Murphy Robert W. , Brown Samara , Scott Alan F. , Morin Phillip A. , Jarvis Erich D. , Fedrigo Olivier
Published: July 2021

Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. We find that no single method is best for all cases. Instead, the optimal storage and extraction methods vary by taxa, by tissue, and by down-stream application. Therefore, we provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for Phase 1 of the Vertebrate Genomes Project (VGP), whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.
A Plasmid System with Tunable Copy Number
Authors: Rouches Miles V. , Xu Yasu , Cortes Louis , Lambert Guillaume
Published: July 2021

Plasmids are one of the most commonly used and time-tested molecular biology platforms for genetic engineering and recombinant gene expression in bacteria. Despite their ubiquity, little consideration is given to metabolic effects and fitness costs of plasmid copy numbers on engineered genetic systems. Here, we introduce two systems that allow for the finely-tuned control of plasmid copy number: a plasmid with an anhydrotetracycline-controlled copy number, and a massively parallel assay that is used to generate a continuous spectrum of ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our massively parallel assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our plasmid system with tunable copy number should allow for a precise control of gene expression and highlight the importance of tuning plasmid copy number as tool for the optimization of synthetic biological systems.
Effects of sub-threshold transcutaneous auricular vagus nerve stimulation on cerebral blood flow
Authors: Chen Conan , Mao Yixiang , Falahpour Maryam , MacNiven Kelly H. , Heit Gary , Sharma Vivek , Alataris Konstantinos , Liu Thomas T.
Published: July 2021

Background

Transcutaneous auricular vagus nerve stimulation (taVNS) has shown promise as a non-invasive alternative to vagus nerve stimulation (VNS) with implantable devices, which has been used to treat drug-resistant epilepsy and treatment-resistant depression. Prior work has used functional MRI to investigate the brain response to taVNS, and more recent work has also demonstrated potential therapeutic effects of high-frequency sub-threshold taVNS in rheumatoid arthritis. However, no studies to date have measured the effects of high-frequency sub-threshold taVNS on cerebral blood flow (CBF).

Objective/Hypothesis

The objective of this study was to determine whether high-frequency (20 kHz) sub-threshold taVNS induces significant changes in CBF.

Methods

Arterial spin labeling (ASL) MRI scans were performed on 20 healthy subjects in a single-blind placebo-controlled repeated measures experimental design. The ASL scans were performed before and after 15 minutes of either sub-threshold taVNS treatment or a sham control.

Results

taVNS induced significant changes in CBF in the superior posterior cerebellum that were largely localized to bilateral Crus I and Crus II. Post hoc analyses showed that the changes were driven by a treatment-related decrease in CBF.

Conclusions

Fifteen minutes of high-frequency sub-threshold taVNS can induce sustained CBF decreases in the bilateral posterior cerebellum in a cohort of healthy subjects. This study lays the foundation for future studies in clinical popluations to assess whether similar effects can be observed and are related to treatment outcomes.
Bounded rational decision-making models suggest capacity-limited concurrent motor planning in human posterior parietal and frontal cortex
Authors: Schach Sonja , Lindner Axel , Braun Daniel Alexander
Published: July 2021

While traditional theories of sensorimotor processing have often assumed a serial decision-making pipeline, more recent approaches have suggested that multiple actions may be planned concurrently and vie for execution. Evidence for the latter almost exclusively stems from electrophysiological studies in posterior parietal and premotor cortex of monkeys. Here we study concurrent prospective motor planning in humans by recording functional magnetic resonance imaging (fMRI) during a delayed response task engaging movement sequences towards multiple potential targets. We find that also in human posterior parietal and premotor cortex delay activity modulates both with sequence complexity and the number of potential targets. We tested the hypothesis that this modulation is best explained by concurrent prospective planning as opposed to the mere maintenance of potential targets in memory. We devise a bounded rationality model with information constraints that optimally assigns information resources for planning and memory for this task and determine predicted information profiles according to the two hypotheses. When regressing delay activity on these model predictions, we find that the concurrent prospective planning strategy provides a significantly better explanation of the fMRI-signal modulations. Moreover, we find that concurrent prospective planning is more costly and thus limited for most subjects, as expressed by the best fitting information capacities. We conclude that bounded rational decision-making models allow relating both behavior and neural representations to utilitarian task descriptions based on bounded optimal information-processing assumptions.

Author summary

When the future is uncertain, it can be beneficial to concurrently plan several action possibilities in advance. Electrophysiological research found evidence in monkeys that brain regions in posterior parietal and promotor cortex are indeed capable of planning several actions in parallel. We now used fMRI to study brain activity in these brain regions in humans. For our analyses we applied bounded rationality models that optimally assign information resources to fMRI activity in a complex motor planning task. We find that theoretical information costs of concurrent prospective planning explained fMRI activity profiles significantly better than assuming alternative memory-based strategies. Moreover, exploiting the model allowed us to quantify the individual capacity limit for concurrent planning and to relate these individual limits to both subjects’ behavior and to their neural representations of planning.
Aberrant enteric neuromuscular system and dysbiosis in amyotrophic lateral sclerosis
Authors: Zhang Yongguo , Ogbu Destiny , Garrett Shari , Xia Yinglin , Sun Jun
Published: July 2021

Background

Emerging evidence has demonstrated that microbiota directly affects the enteric neuron system (ENS) and smooth muscle cell functions via metabolic products or endogenous bacterial components. Amyotrophic Lateral Sclerosis is a neuromuscular disease characterized by the progressive death of motor neurons and muscle atrophy. The GI symptoms in patients were largely ignored or underestimated, especially before the diagnosis of ALS. The relationship between enteric neuromuscular system and microbiome in ALS progression is unknown.

Methods

We performed longitudinal studies on the ENS and microbiome in the ALS human-SOD1G93A transgenic G93A mice. We treated age-matched wild-type and ALS mice with bacterial product butyrate or antibiotics to investigate microbiome and neuromuscular functions. Intestinal motility, microbiome, an ENS marker GFAP, a smooth muscle marker (SMMHC), and human colonoids have been examined. The distribution of human-G93A-SOD1 (Superoxide Dismutase 1) protein was tested as an indicator of ALS progression.

Results

At 2-month-old before ALS onset, G93A mice had significant lower intestinal motility, decreased grip strength, and reduced time in the rotarod. We observed increased GFAP and decreased SMMHC expression. These changes correlated with consistent increased aggregation of mutated SOD1G93A in the colon, small intestine, and spinal cord. Butyrate and antibiotic treatment showed a significantly longer latency to fall in the rotarod test, reduced SOD1G93A aggregation, and enhanced ENS and muscle function. Feces from 2-month-old SOD1G93A mice significantly enhanced SOD1G93A aggregation in human colonoids transfected with a SOD1G93A-GFP plasmid. Longitudinal studies of microbiome data further showed the altered bacterial community related with autoimmunity (e.g., Clostridium sp. ASF502, Lachnospiraceae bacterium A4), inflammation (e.g., Enterohabdus Muris,), and metabolism (e.g., Desulfovibrio fairfieldensis) at 1- and 2-month-old SOD1G93A mice, suggesting the early microbial contribution to the pathological changes.

Conclusions

We have demonstrated a novel link between microbiome, hSOD1G93A aggregation, and intestinal mobility. Dysbiosis occurred at the early stage of the ALS mice before observed mutated-SOD1 aggregation, slow intestinal motility, and dysfunction of ENS. Manipulating the microbiome improves the muscle performance of SOD1G93A mice. Our study provides insights into fundamentals of intestinal neuromuscular structure/function and microbiome in ALS.
Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila
Authors: Li Sam , Fernandez Jose-Jesus , Fabritius Amy S. , Agard David A. , Winey Mark
Published: July 2021

Doublet microtubules (DMT) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMT, collectively named the microtubule inner proteins (MIPs). We used electron cryo-tomography to study axoneme DMT isolated from Tetrahymena thermophila. We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP Rib72A/B binds to the luminal wall of the DMT by multiple DM10 domains, likely by recognizing the acetylated K40 residue of α-tubulin. We found Fap115, a MIP containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of the A-tubule. It functions as a “molecular staple” stabilizing both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. Defects caused by the depletion of Fap115 propagate along the axoneme due to extensive structural changes in the DMT at and beyond the Fap115 binding site. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.
Active enhancers strengthen insulation by RNA-mediated CTCF binding at TAD boundaries
Authors: Islam Zubairul , Saravanan Bharath , Walavalkar Kaivalya , Thakur Jitendra , Farooq Umer , Singh Anurag Kumar , Sabarinathan Radhakrishnan , Pandit Awadhesh , Henikoff Steven , Notani Dimple
Published: July 2021

Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.
Memory-paced tapping to auditory rhythms: Effects of rate, speech and motor-engagement
Authors: Amrani Anat Kliger , Golumbic Elana Zion
Published: July 2021

Humans have a near-automatic tendency to entrain their motor actions to rhythms in the environment. Entrainment is hypothesized to play an important role in processing naturalistic stimuli, such as speech and music, which have intrinsically rhythmic properties. Here we studied two facets of entraining one’s rhythmic motor actions to an external stimulus: (1) synchronized finger tapping to auditory rhythmic stimuli, and (2) memory-paced reproduction of a previously heard rhythm. Using modifications of the Synchronization-Continuation tapping paradigm, we studied how these two rhythmic behaviours were affected by different stimulus and task features. We tested synchronization and memory-paced tapping for a broad range of rates, from sub-second to supra-second, both for isochronous tone-sequences and for rhythmic speech stimuli (counting from one to ten), which are more ecological yet less strictly isochronous. We also asked what role motor engagement plays in forming a stable internal representation for rhythms and guiding memory-paced tapping.

Results show that individuals can flexibly synchronize their motor actions to a very broad range of rhythms. However, this flexibility does not extend to memory-paced tapping, which is accurate only in a narrower range of rates, around ~1.5Hz. This pattern suggests that intrinsic rhythmic-defaults in the auditory/motor systems influence the internal representation of rhythms, in the absence of an external pace-maker. Interestingly, memory-paced tapping for speech rhythms and simple tones shared similar ‘optimal rates’, although with reduced accuracy, suggesting that internal constraints on rhythmic entrainment may generalize to more ecological stimuli. Last, active synchronization led to more accurate memory-paced tapping vs. passive listening, which emphasizes the importance of action-perception interactions in forming stable entrainment to external rhythms.
Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans
Authors: Musa Marina , Milosevic Ira , Raimundo Nuno , Krisko Anita
Published: July 2021

Exact mechanisms of heat shock induced lifespan extension, while documented across species, are still not well understood. Here we put forth evidence that fully functional peroxisomes are required for the activation of the canonical heat shock response and heat-induced hormesis in C. elegans. While during heat shock the HSP-70 chaperone is strongly upregulated in the wild-type (WT) as well as in the absence of peroxisomal catalase (Δctl-2 mutant), the small heat shock proteins display modestly increased expression in the mutant. Nuclear localization of HSF-1 is reduced in the Δctl-2 mutant. In addition, heat-induced lifespan extension, observed in the WT, is absent in the Δctl-2 mutant. Activation of the antioxidant response, the pentose phosphate pathway and increased triglyceride content are the most prominent changes observed during heat shock in the WT worm, but not in the Δctl-2 mutant. Involvement of peroxisomes in the cell-wide response to transient heat shock reported here gives new insight into the role of organelle communication in the organisms stress response.
Whole animal modelling reveals neuronal mechanisms of decision-making and reproduces unpredictable swimming in frog tadpoles
Authors: Ferrario Andrea , Palyanov Andrey , Koutsikou Stella , Li Wenchang , Soffe Steve , Roberts Alan , Borisyuk Roman
Published: July 2021

Animal behaviour is based on interaction between nervous, musculoskeletal and environmental systems. How does an animal process sensory stimuli, use it to decide whether and how to respond, and initiate the locomotor behaviour? We build the whole body computer models of a simple vertebrate with a complete chain of neural circuits and body units for sensory information processing, decision-making, generation of spiking activities, muscle innervation, body flexion, body-water interaction, and movement. Our Central Nervous System (CNS) model generates biologically-realistic spiking and reveals that sensory memory populations on two hindbrain sides compete for swimming initiation and first body flexion. Biomechanical 3-dimensional “Virtual Tadpole” (VT) model is constructed to evaluate if motor outputs of CNS model can produce swimming-like movements in a volume of “water”. We find that whole animal modelling generates reliable and realistic swimming. The combination of CNS and VT models opens a new perspective for experiments with immobilised tadpoles.
The Bardet-Biedl protein Bbs1 controls photoreceptor outer segment protein and lipid composition
Authors: Masek Markus , Etard Christelle , Hofmann Claudia , Hülsmeier Andreas , Zang Jingjing , Takamiya Masanari , Gesemann Matthias , Neuhauss Stephan , Hornemann Thorsten , Strähle Uwe , Bachmann-Gagescu Ruxandra
Published: July 2021

Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a new bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on isolated OSs show that Bbs1 is required for BBSome-entry into OSs and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a new role for Bbs1/BBSome in OS lipid homeostasis and suggest a new pathomechanism underlying retinal degeneration in BBS.
Individual beliefs about temporal continuity explain variation of perceptual biases
Authors: Glasauer Stefan , Shi Zhuanghua
Published: July 2021

Perception of magnitudes such as duration or distance is often found to be systematically biased. The biases, which result from incorporating prior knowledge in the perceptual process, can vary considerably between individuals. The variations are commonly attributed to differences in sensory precision and reliance on priors. However, another factor not considered so far is the implicit belief about how successive sensory stimuli are generated: independently from each other or with certain temporal continuity. The main types of explanatory models proposed so far – static or iterative – mirror this distinction but cannot adequately explain individual biases. Here we propose a new unifying model that explains individual variation as combination of sensory precision and beliefs about temporal continuity and predicts the experimentally found changes in biases when altering temporal continuity. Thus, according to the model, individual differences in perception depend on beliefs about how stimuli are generated in the world.
A Structure-based B-cell Epitope Prediction Model Through Combing Local and Global Features
Authors: Lu Shuai , Li Yuguang , Ma Qiang , Nan Xiaofei , Zhang Shoutao
Published: July 2021

B-cell epitopes (BCEs) are a set of specific sites on the surface of an antigen that binds to an antibody produced by B-cell. The recognition of BCEs is a major challenge for drug design and vaccines development. Compared with experimental methods, computational approaches have strong potential for BCEs prediction at much lower cost. Moreover, most of the currently methods focus on using local information around target residue without taking the global information of the whole antigen sequence into consideration. We propose a novel deep leaning method through combing local features and global features for BCEs prediction. In our model, two parallel modules are built to extract local and global features from the antigen separately. For local features, we use Graph Convolutional Networks(GCNs) to capture information of spatial neighbors of a target residue. For global features, Attention-Based Bidirectional Long Short-Term Memory(Att-BLSTM) networks are applied to extract information from the whole antigen sequence. Then the local and global features are combined to predict BCEs. The experiments show that the proposed method achieves superior performance over the state-of-the-art BCEs prediction methods on benchmark datasets. Also, we compare the performance differences between data with or without global features. The experimental results show that global features play an important role in BCEs prediction. Our detailed case study on the BCEs prediction for SARS-Cov-2 receptor binding domain confirms that our method is effective for predicting and clustering true BCEs.
Multiple lines of evidence combine to understand tree species coexistence
Authors: McNickle Gordon G. , Ritzi Morgan V. , Blackstone Kliffi M.S. , Couture John J. , Nelson Taylor , Hardiman Brady S. , Montague Madeline S. , Jacobs Douglass F.
Published: July 2021

Understanding drivers of species coexistence is a central challenge in ecology (1,2). Coexistence cannot be observed directly, and while species co-occurrence in time and space is necessary for coexistence, it is not sufficient to prove coexistence (3,4). Species exclusion from a region is potentially observable, but can take decades to occur, and still might just occur stochastically (5,6). Thus, ecologists generally use theory to identify indirect observations that are indicative of mechanisms driving coexistence or exclusion (7–9). Various methods have been developed to indirectly infer coexistence, each of which requires different data, none of which are usually conclusive on their own, and are not typically combined (e.g. (9–12)). Here, we combine three different approaches which are normally used in isolation to build a case about a lack of coexistence of multiple hardwood species. Importantly, though each method has its flaws, we find that all three point to the same conclusion which strengthens the case for a lack of coexistence. First, in an experimental planting of three mature tree species we found no relationship between productivity and species diversity, which has been hypothesized to occur due to a lack of niche differences among species. Second, we used modern coexistence theory to calculate niche and fitness differences for each pair of species, which confirmed the high niche overlap among species and competitive dominance of one species. Third, we used the United States Department of Agriculture Forest Inventory and Analysis data to examine co-occurrence patterns of our species across thousands of natural forest stands and found that, these three species were distributed randomly throughout the USA, and do not significantly co-occur as would be expected if coexistence occurred naturally. Given that these independent methods agree; we take this as strong evidence about a lack of coexistence.
Parallel processing by distinct classes of principal neurons in the olfactory cortex
Authors: Nagappan Shivathmihai , Franks Kevin M.
Published: July 2021

Understanding the specific roles that different neuron types play within a neural circuit is essential for understanding what that circuit does and how it does it. Primary olfactory (piriform, PCx) cortex contains two main types of principal neurons: semilunar (SL) and pyramidal (PYR). SLs and PYRs have different morphologies, connectivity, biophysical properties, and downstream projections, predicting distinct roles in cortical odor processing. The prevailing model is that odor processing in PCx occurs in two stages, where SLs are the primary recipients of olfactory bulb (OB) input, and PYRs receive and transform information from SLs. Here we recorded from opto-genetically-identified SLs and PYRs in awake, head-fixed mice. We found differences in SL and PYR odor-evoked activity that reflect their different connectivity profiles. But SL responses did not precede PYR responses and suppressing SL activity had little effect on PYR odor responses. These results suggest that SLs and PYRs form parallel odor processing channels.
Consistent coordination patterns provide near perfect behavior decoding in a comprehensive motor program for insect flight
Authors: Putney Joy , Angjelichinoski Marko , Ravier Robert , Ferrari Silvia , Tarokh Vahid , Sponberg Simon
Published: July 2021

Patterns of motor activity can be used to decode behavior state. Precise spike timing encoding is present in many motor systems, but is not frequently utilized to decode behavior or to examine how coordination is achieved across many motor units. Testing whether the same coordinated sets of muscles control different movements is difficult without a complete motor representation at the level of the currency of control – action potentials. Here, we demonstrate nearly perfect decoding of six hawk moth flight behaviors elicited in response to wide-field drifting visual stimuli about the flight axes – pitch, roll, and yaw – using a comprehensive, spike-resolved motor program and a simple linear decoding pipeline. A complex decoding scheme is not necessary, even if the functional patterns of control are nonlinear. We show that muscle covariation present in one pair of visual stimulus conditions can be used to decode behavior in a different pair of visual stimulus conditions, indicating the presence of conserved muscle coordination patterns at the level of motor neuronal timings in functionally distinct behaviors. We also demonstrate that as few as half the muscles can be used to retain decoding performance, linking coordination to redundancy in encoding, if not function, across the entire moth flight motor program.
Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis
Authors: Yadav Ravi , Govindan Srinivas , Daczkowski Courtney , Mesecar Andrew , Chakravarthy Srinivas , Noinaj Nicholas
Published: July 2021

Lactoferrin binding protein B (LbpB) is a lipoprotein present on the surface of Neisseria that has been postulated to serve dual functions during pathogenesis in both iron acquisition from lactoferrin, and in providing protection against the cationic antimicrobial peptide lactoferricin. Here, we present the structures of LbpB from N. meningitidis and N. gonorrhoeae in complex with human holo-lactoferrin, forming a 1:1 complex and confirmed by SEC-SAXS. LbpB consists of N- and C-lobes with the N-lobe interacting extensively with the C-lobe of lactoferrin. Our structures provides insight into LbpB’s preference towards holo-lactoferrin, and our mutagenesis and binding studies show that lactoferrin and lactoferricin bind independently. Our studies provide the molecular details for how LbpB serves to capture and preserve lactoferrin in an iron-bound state for delivery to the membrane transporter LbpA for iron piracy, and as an antimicrobial peptide sink to evade host immune defenses.
Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid
Authors: Dangerfield Tyler L. , Kirmizialtin Serdal , Johnson Kenneth A.
Published: July 2021

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3’-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.
A theoretical model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers
Authors: Mazat Jean-Pierre , Devin Anne , Yoboue Edgar , Ransac Stéphane
Published: July 2021

The m.8993T>G mutation of the mitochondrialMT-ATP6gene is associated with NARP syndrome (Neuropathy, Ataxia and Retinitis Pigmentosa). The equivalent point mutation introduced in yeastSaccharomyces cerevisiaemitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-c-oxidase preventing yeast growth on oxidative substrates. The over-expression of the mitochondrial oxodicarboxylate carrier (Odc1p) is able to rescue the growth on oxidative substrate by increasing the substrate-level phosphorylation of ADP coupled to conversion ofα-ketoglutarate (AKG) into succinate with an increase in Complex IV activity. Previous studies showed that equivalent point mutations in ATP synthase behave similarly and can be rescued by Odc1p overexpression and/or uncoupler of OXPHOS from ATP synthesis. In order to better understand the mechanism of ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as respiratory substrate. We describe the different possible metabolite output and the ATP/O ratio values as a function of ATP synthase inhibition.
Host-symbiont population genomics provide insights into partner fidelity, transmission mode and habitat adaptation in deep-sea hydrothermal vent snails
Authors: Breusing Corinna , Genetti Maximilian , Russell Shelbi L. , Corbett-Detig Russell B. , Beinart Roxanne A.
Published: July 2021

Symbiont specificity, both at the phylotype and strain level, can have profound consequences for host ecology and evolution. However, except for insights from a few model symbiosis systems, the degree of partner fidelity and the influence of host versus environmental factors on symbiont composition are still poorly understood. Nutritional symbioses between invertebrate animals and chemosynthetic bacteria at deep-sea hydrothermal vents are examples of relatively selective associations, where hosts affiliate only with particular, environmentally acquired phylotypes of gammaproteobacterial or campylobacterial symbionts. In hydrothermal vent snails of the sister genera Alviniconcha and Ifremeria this phylotype specificity has been shown to play a role in habitat distribution and partitioning among different holobiont species. However, it is currently unknown if fidelity goes beyond species level associations that might influence genetic structuring, connectivity and habitat adaptation of holobiont populations. We used metagenomic analyses to assess sequence variation in hosts and symbionts and identify correlations with geographic and environmental factors. Our analyses indicate that host populations are not differentiated across a ~800 km gradient, while symbiont populations are clearly structured between vent locations due to a combination of neutral and selective processes. Overall, these results suggest that host individuals flexibly associate with locally adapted strains of their specific symbiont phylotypes, which supports a long-standing but untested paradigm of the benefits of horizontal transmission. Strain flexibility in these snails likely enables host populations to exploit a range of habitat conditions, which might favor wide-spread genetic connectivity and ecological resilience unless physical dispersal barriers are present.

Significance Statement

Symbiont composition in horizontally transmitted symbioses is influenced by a combination of host genetics, environmental conditions and geographic barriers. Yet the relative importance of these factors and the effects of adaptive versus neutral evolutionary forces on symbiont population structure remain unknown in the majority of marine symbioses. To address these questions, we applied population genomic approaches in four species of deep-sea hydrothermal vent snails that live in obligate association with chemosynthetic bacteria. Our analyses show that host genetics plays a minor role compared to environment for symbiont strain composition despite specificity to symbiont species and corroborate a long-standing hypothesis that vent invertebrates affiliate with locally adapted symbiont strains to cope with the variable habitat conditions characterizing hydrothermal vents.
Divergence in dimerization and activity of primate APOBEC3C
Authors: Gaba Amit , Hix Mark A. , Suhail Sana , Flath Ben , Boysan Brock , Williams Danielle R. , Pelletier Tomas , Emerman Michael , Morcos Faruck , Cisneros G. Andrés , Chelico Linda
Published: July 2021

The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and the Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Multiple integrated analyses determined why rhA3C was not active against HIV-1 and how to increase this activity. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity also promoted dimerization. Although rhA3C shares a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C, establish the amino acid network for dimerization and increased activity, and track the loss and gain of A3C antiviral activity in primates. The coevolutionary analysis of the A3C dimerization interface provides a basis from which to analyze dimerization interfaces of other A3 family members.
Substrate, temperature, and geographical patterns among nearly 2,000 natural yeast isolates
Authors: Spurley William J. , Fisher Kaitlin J. , Langdon Quinn K. , Buh Kelly V. , Jarzyna Martin , Haase Max A. B. , Sylvester Kayla , Moriarty Ryan V. , Rodriguez Daniel , Sheddan Angela , Wright Sarah , Sorlie Lisa , Hulfachor Amanda Beth , Opulente Dana A. , Hittinger Chris Todd
Published: July 2021

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here we used environmental sampling and isolation to assemble a dataset of 1,962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the US, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon - substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.

Take Away

Analysis of environmental metadata of nearly 2,000 yeast isolates.

Individual yeast taxa associate with specific substrates and plant genera.

Optimal yeast isolation temperature differs depending on taxonomic rank.

Substrate type and isolation temperatures affect isolated yeast diversity.
Predicting protein domain temperature adaptation across the prokaryote-eukaryote divide
Authors: Jensen Sarah E. , Johnson Lynn C. , Casstevens Terry , Buckler Edward S.
Published: July 2021

Protein thermostability is important for fitness but difficult to measure across the proteome. Fortunately, protein thermostability is correlated with prokaryote optimal growth temperatures (OGTs), which can be predicted from genome features. Models that can predict temperature sensitivity across the prokaryote-eukaryote divide would help inform how eukaryotes adapt to elevated temperatures, such as those predicted by climate change models. In this study we test whether prediction models can cross the prokaryote-eukaryote divide to predict protein stability in both prokaryotes and eukaryotes. We compare models built using a) the whole proteome, b) Pfam domains, and c) individual amino acid residues. Proteome-wide models accurately predict prokaryote optimal growth temperatures (r2 up to 0.93), while site-specific models demonstrate that nearly half of the proteome is associated with optimal growth temperature in both Archaea and Bacteria. Comparisons with the small number of eukaryotes with temperature sensitivity data suggest that site-specific models are the most transferable across the prokaryote-eukaryote divide. Using the site-specific models, we evaluated temperature sensitivity for 323,850 amino acid residues in 2,088 Pfam domain clusters in Archaea and Bacteria species separately. 59.0% of tested residues are significantly associated with OGT in Archaea and 75.2% of tested residues are significantly associated with OGT in Bacteria species at a 5% false discovery rate. These models make it possible to identify which Pfam domains and amino acid residues are involved in temperature adaptation and facilitate future research questions about how species will fare in the face of increasing environmental temperatures.
Single Serine on TSC2 Exerts Biased Control over mTORC1 Activation by ERK1/2 but Not Akt
Authors: Dunkerly-Eyring Brittany L. , Pinilla-Vera Miguel , McKoy Desirae , Mishra Sumita , Grajeda Martinez Maria I. , Oeing Christian U. , Ranek Mark J. , Kass David A.
Published: July 2021

The mammalian target of rapamycin complex 1 (mTORC1) is tightly controlled by tuberous sclerosis complex-2 (TSC2) that is regulated by phosphorylation from kinases responding to environmental cues. Protein kinase G specifically modifies serine-1365 (S1364, human), and its phosphorylation (or phosphomimetic SE mutant) potently blocks mTORC1 co-activation by pathological stress, while a phospho-silenced (SA) mutation does the opposite. Neither alter basal mTORC1 activity. Here we show S1365 exerts biased control over mTORC1 activity (S6K phosphorylation) modifying ERK1/2 but not Akt-dependent stimulation. Whereas mTORC1 activation by endothelin-1 is potently modified by S1365 status, insulin or PDGF stimulation are unaltered. TSC2-S1365 is also phosphorylated upon ET-1 but not insulin stimulation in a PKG-dependent manner, revealing intrinsic bias. Neither energy or nutrient modulation of mTORC1 are impacted by S1365. Consistent with these results, knock-in mice with either TSC2 SA or SE mutations develop identical obesity, glucose intolerance, and fatty liver disease from a high fat diet. Thus, S1365 provides an ERK1/2-selective mTORC1 control mechanism and a genetic means to modify pathological versus physiological mTOR stimuli.
Tissue Specific Age Dependence of the Cell Receptors Involved in the SARS-CoV-2 Infection
Authors: Forst Christian V. , Zeng Lu , Wang Qian , Zhou Xianxiao , Vatansever Sezen , Tu Zhidong , Zhang Bin
Published: July 2021

The coronavirus disease 2019 (COVID-19) pandemic has affected tens of millions of individuals and caused hundreds of thousands of deaths worldwide. Due to its rapid surge, there is a shortage of information on viral behavior and host response after SARS-CoV-2 infection. Here we present a comprehensive, multiscale network analysis of the transcriptional response to the virus. We particularly focus on key-regulators, cell-receptors, and host-processes that are hijacked by the virus for its advantage. ACE2-controlled processes involve a key-regulator CD300e (a TYROBP receptor) and the activation of IL-2 pro-inflammatory cytokine signaling. We further investigate the age-dependency of such receptors and identify the adipose and the brain as potentially contributing tissues for the disease’s severity in old patients. In contrast, several other tissues in the young population are more susceptible to SARS-CoV-2 infection. In summary, this present study provides novel insights into the gene regulatory organization during the SARS-CoV-2 infection and the tissue-specific age dependence of the cell receptors involved in COVID-19.
Searching for Consistent Brain Network Topologies Across the Garden of (Shortest) Forking Paths
Authors: Luppi Andrea I. , Gellersen Helena M. , Peattie Alex R. D. , Manktelow Anne E. , Menon David K. , Dimitriadis Stavros I. , Stamatakis Emmanuel A.
Published: July 2021

The functional interactions between regions of the human brain can be viewed as a network, empowering neuroscientists to leverage tools such as graph theory to obtain insight about brain function. However, obtaining a brain network from functional neuroimaging data inevitably involves multiple steps of data manipulation, which can affect the organisation (topology) of the resulting network and its properties. Test-retest reliability is a gold standard for both basic research and clinical use: a suitable data-processing pipeline for brain networks should recover the same network topology across repeated scan sessions of the same individual. Analyzing resting-state functional Magnetic Resonance Imaging (rs-fMRI) recordings from two test-retest studies across short (45 minutes), medium (2-4 weeks) and long term delays (5-16 months), we investigated the reliability of network topologies constructed by applying 576 unique pipelines to the same fMRI data, obtained from considering combinations of atlas type and size, edge definition and thresholding, and use of global signal regression. We adopted the portrait divergence, an information-theoretic criterion to measure differences in network topology across all scales, enabling us to quantify the influence of different pipelines on the overall organisation of the resulting network. Remarkably, our findings reveal that the choice of pipeline plays a fundamental role in determining how reproducible an individual’s brain network topology will be across different scans: there is large and systematic variability across pipelines, such that an inappropriate choice of pipeline can distort the resulting network more than an interval of several months between scans. Across datasets and time-spans, we also identify specific combinations of data-processing steps that consistently yield networks with reproducible topology, enabling us to make recommendations about best practices to ensure high-quality brain networks.
High-throughput aminoacyl-tRNA synthetase engineering for genetic code expansion in yeast
Authors: Stieglitz Jessica T. , Van Deventer James A.
Published: July 2021

Protein expression with genetically encoded noncanonical amino acids (ncAAs) benefits a broad range of applications, from the discovery of biological therapeutics to fundamental biological studies. A major factor limiting the use of ncAAs is the lack of orthogonal translation systems (OTSs) that support efficient genetic code expansion at repurposed stop codons. Aminoacyl-tRNA synthetases (aaRSs) have been extensively evolved in E. coli but are not always orthogonal in eukaryotes. In this work, we use a yeast display-based ncAA incorporation reporter platform with fluorescence-activated cell sorting to screen libraries of aaRSs in high throughput for 1) incorporation of ncAAs not previously encoded in yeast; 2) improvement of the performance of an existing aaRS; 3) highly selective OTSs capable of discriminating between closely related ncAA analogs; and 4) OTSs exhibiting enhanced polyspecificity to support translation with structurally diverse sets of ncAAs. The number of previously undiscovered aaRS variants we report in this work more than doubles the total number of translationally active aaRSs available for genetic code manipulation in yeast. The success of myriad screening strategies has important implications related to the fundamental properties and evolvability of aaRSs. Furthermore, access to OTSs with diverse activities and specific or polyspecific properties is invaluable for a range of applications within chemical biology, synthetic biology, and protein engineering.

Synopsis

A range of flow cytometry-based screens yielded diverse translational machinery for genetic code expansion in yeast, facilitating access to new chemistries and tunable specificity profiles.

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