bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution of tunnels in α/β-hydrolase fold proteins – what can we learn from studying epoxide hydrolases?
Authors: Bzówka Maria , Mitusińska Karolina , Raczyńska Agata , Skalski Tomasz , Samol Aleksandra , Bagrowska Weronika , Magdziarz Tomasz , Góra Artur
Published: December 2021

The evolutionary variability of a protein’s residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to α/β-hydrolase fold proteins - one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels’ evolution analysis based on entropy values calculated for tunnels’ residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a ‘perforation’ mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzyme’s performance and can be applied widely for enzymes with buried active sites.

Author Summary

So far very little is known about proteins tunnels evolution. The goal of this study is to evaluate the evolution of tunnels in the family of soluble epoxide hydrolases - representatives of numerous α/β-hydrolase fold enzymes. As a result two types of tunnels evolution analysis were proposed (a general and a detailed approach), as well as a ‘perforation’ mechanism which can mimic native evolution in proteins and can be used as an additional strategy for enzymes redesign.
A mathematical model for cell-induced gel contraction incorporating osmotic effects
Authors: Reoch J. R. , Stokes Y. M. , Green J. E. F.
Published: December 2021

Biological tissues are composed of cells surrounded by the extracellular matrix (ECM). The ECM can be thought of as a fibrous polymer network, acting as a natural scaffolding to provide mechanical support to the cells. Reciprocal mechanical and chemical interactions between the cells and the ECM are crucial in regulating the development of tissues and maintaining their functionality. Hence, to maintain in vivo-like behaviour when cells are cultured in vitro, they are often seeded in a gel, which aims to mimic the ECM. In this paper, we present a mathematical model that incorporate cell-gel interactions together with osmotic pressure to study the mechanical behaviour of biological gels. In particular, we consider an experiment where cells are seeded within a gel, which gradually compacts due to forces exerted on it by the cells. Adopting a one-dimensional Cartesian geometry for simplicity, we use a combination of analytical techniques and numerical simulations to investigate how cell traction forces interact with osmotic effects (which can lead to either gel swelling or contraction depending on the gel’s composition). Our results show that a number of qualitatively different behaviours are possible, depending on the composition of the gel (i.e. the chemical potentials) and the strength of the cell traction forces. We observe an unusual case where the gel oscillates between swelling and contraction. We also consider on how physical parameters like drag and viscosity affect the manner in which the gel evolves.
Exact breakpoints of In(1)wm4 rearrangement
Authors: Solodovnikov A. A. , Lavrov S. A.
Published: December 2021

In(1)wm4 was known for decades as a classic example of position effect variegation-causing rearrangement and was mentioned in hundreds of publications. Nevertheless, the euchromatin breakpoint position of the rearrangement was not precisely localized. We performed nanopore sequencing of DNA from In(1)wm4 homozygous flies and determined the exact position of euchromatic (chrX:2767875) and heterochromatic breakpoints of the rearrangement.
Isolation and characterization of fucosylated extracellular vesicles based on a novel high-throughput GlyExo-Capture technique
Authors: Li Boan , Hao Kun , Ma Cuidie , Li Zhengtai , Li Hongjiang , Du Wenqian , Sun Lijuan , Jia Tianye , Liu Aixia , Li Yanzhao , Xu Lida , Gao Qi , Yang Ruifu , Lin Changqing
Published: December 2021

Owing to their diagnostic and therapeutic potential, extracellular vesicles (EVs) derived from tumour cells have recently garnered great interest. The presence of different glycosylation sites at the EV surface supports the need for efficient glycosylated EV isolation. Here, we developed a GlyExo-Capture technique for robustly capturing fucosylated EVs from sera and cell supernatants. Lens culinaris lectin (LCA)-immobilized magnetic complexes were found to capture approximately 60% of the total EVs from HepG2 cells. The capture efficiency was reduced to less than 40% in nontumorigenic MIHA cells. Notably, the cellular uptake pattern of highly fucosylated EVs was markedly different from that of EVs with low fucosylation. The unearthing of enriched fucosylated EV miRNA cargos by next-generation deep sequencing (NGS) revealed 75 differentially expressed miRNAs (DEMs) in hepatocellular carcinoma (HCC). Among them, a 4-miRNA panel was chosen and yielded an area under the ROC curve (AUC) of 0.86 and 0.84 for the detection of HCC from non-HCC controls in testing samples and independent validation samples, respectively. The 4-miRNA signature was independent of alpha-fetoprotein (AFP), and a combined model with AFP yielded an increased AUC of 0.92. In conclusion, we developed a high-throughput method for capturing fucosylated EVs efficiently and shed light on the use of fucosylated EVs as potential sources of miRNAs for cancer biomarker detection.
Preliminary Characterization of Phage-like Particles from the Male-Killing MollicuteSpiroplasma poulsonii(an Endosymbiont ofDrosophila)
Authors: Ramirez Paulino , Leavitt Justin C. , Gill Jason J. , Mateos Mariana
Published: December 2021

Bacteriophages are vastly abundant, diverse, and influential, but with few exceptions (e.g. the Proteobacteria generaWolbachiaandHamiltonella), the role of phages in heritable bacteria-arthropod interactions, which are ubiquitous and diverse, remains largely unexplored. Despite prior studies documenting phage-like particles in the mollicuteSpiroplasmaassociated withDrosophilaflies, genomic sequences of such phage are lacking, and their effects on theSpiroplasma-Drosophilainteraction have not been comprehensively characterized. We used a density step gradient to isolate phage-like particles from the male-killing bacteriumSpiroplasma poulsonii(strains NSRO and MSRO-Br) harbored byDrosophila melanogaster. Isolated particles were subjected to DNA sequencing, assembly, and annotation. Several lines of evidence suggest that we recovered phage-like particles of similar features (shape, size, DNA content) to those previously reported inDrosophila-associatedSpiroplasmastrains. We recovered three ∼19 kb phage-like contigs (two in NSRO and one in MSRO-Br) containing 21–24 open reading frames, a read-alignment pattern consistent with circular permutation, and terminal redundancy (at least in NSRO). Although our results do not allow us to distinguish whether these phage-like contigs represent infective phage-like particles capable of transmitting their DNA to new hosts, their encoding of several typical phage genes suggests that they are at least remnants of functional phage. We also recovered two smaller non-phage-like contigs encoding a knownSpiroplasmatoxin (Ribosome Inactivating Protein; RIP), and an insertion element, suggesting that they are packaged into particles. Substantial homology of our particle-derived contigs was found in the genome assemblies of members of theSpiroplasma poulsoniiclade.
InterPepScore: A Deep Learning Score for Improving the FlexPepDock Refinement Protocol
Authors: Johansson-Åkhe Isak , Wallner Björn
Published: December 2021

Motivation

Interactions between peptide fragments and protein receptors are vital to cell function yet difficult to experimentally determine the structural details of. As such, many computational methods have been developed to aid in peptide-protein docking or structure prediction. One such method is Rosetta FlexPepDock which consistently refines coarse peptide-protein models into sub-Ångström precision using Monte-Carlo simulations and statistical potentials. Deep learning has recently seen increased use in protein structure prediction, with graph neural network seeing use in protein model quality assessment.

Results

Here, we introduce a graph neural network, InterPepScore, as an additional scoring term to complement and improve the Rosetta FlexPepDock refinement protocol. InterPepScore is trained on simulation trajectories from FlexPepDock refinement starting from thousands of peptide-protein complexes generated by a wide variety of docking schemes. The addition of InterPepScore into the refinement protocol consistently improves the quality of models created, and on an independent benchmark on 109 peptide-protein complexes its inclusion results in an increase in the number of complexes for which the top-scoring model had a DockQ-score of 0.49 (Medium quality) or better from 14.8% to 26.1%.

Availability

InterPepScore is available online at http://wallnerlab.org/InterPepScore.
The role of surface adhesion on the macroscopic wrinkling of biofilms
Authors: Geisel Steffen , Secchi Eleonora , Vermant Jan
Published: December 2021

Biofilms, bacterial communities of cells encased by a self-produced matrix, exhibit a variety of three-dimensional structures. Specifically, channel networks formed within the bulk of the biofilm have been identified to play an important role in the colonies viability by promoting the transport of nutrients and chemicals. Here, we study channel formation and focus on the role of the adhesion of the biofilm matrix to the substrate in Pseudomonas aeruginosa biofilms grown under constant flow in microfluidic channels. We perform phase contrast and confocal laser scanning microscopy to examine the development of the biofilm structure as a function of the substrates surface energy. The formation of the wrinkles and folds is triggered by a mechanical buckling instability, controlled by biofilm growth rate and the film’s adhesion to the substrate. The three-dimensional folding gives rise to hollow channels that rapidly increase the overall volume occupied by the biofilm and facilitate bacterial movement inside them. The experiments and analysis on mechanical instabilities for the relevant case of a bacterial biofilm grown during flow enable us to predict and control the biofilm morphology.
Comparing Brain Asymmetries Independently of Brain Size
Authors: Williams Camille Michèle , Peyre Hugo , Toro Roberto , Ramus Franck
Published: December 2021

Studies examining cerebral asymmetries typically divide the L-R Measure (e.g., Left– Right Volume) by the L+R Measure to obtain an Asymmetry Index (AI). However, contrary to widespread belief, such a division fails to render the AI independent from the L+R Measure and/or from total brain size. As a result, variations in brain size may bias correlation estimates with the AI or group differences in AI. We investigated how to analyze brain asymmetries in to distinguish global from regional effects, and report unbiased group differences in cerebral asymmetries.

We analyzed the extent to which the L+R Measure, Total Cerebral Measure (TCM, e.g., Total Brain Volume), and L-R TCM predict regional asymmetries. As a case study, we assessed the consequences of omitting each of these predictors on the magnitude and significance of sex differences in asymmetries.

We found that the L+R Measure, the TCM, and the L-R TCM predicted the AI of more than 89% of regions and that their relationships were generally linear. Removing any of these predictors changed the significance of sex differences in 33% of regions and the magnitude of sex differences across 13-42% of regions. Although we generally report similar sex and age effects on cerebral asymmetries to those of previous large-scale studies, properly adjusting for regional and global brain size revealed additional sex and age effects on brain asymmetry.

Highlights

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The typical Asymmetry Index (AI) scales with the size of a region and brain size.

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Omitting the Left+Right Measure influences reported sex differences in asymmetries.

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Omitting brain size or asymmetry influences reported sex differences in asymmetries.

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We report sex and age effects on AIs independent of regional and global brain size.
A novel Alex3/Gαqprotein complex regulating mitochondrial dynamics, dendritic complexity, and neuronal survival
Authors: Izquierdo-Villalba Ismael , Mirra Serena , Manso Yasmina , Parcerisas Antoni , Rubio Javier , Del Valle Jaume , Gil-Bea Francisco J. , Ulloa Fausto , Herrero-Lorenzo Marina , Verdaguer Ester , Benincá Cristiane , Castro-Torres Rubén D. , Rebollo Elena , Marfany Gemma , Auladell Carme , Navarro Xavier , Enríquez José A. , de Munain Adolfo López , Aragay Anna M. , Soriano Eduardo
Published: December 2021

In neurons, mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neuronal activity. Recent studies point to GPCR and G proteins as important regulators of mitochondrial dynamics and energy metabolism. Here we show that activation of Gαqnegatively regulates mitochondrial dynamics and trafficking in neurons. Gαqinteracts with the mitochondrial trafficking protein Alex3. By generating a CNS-specificarmcx3knock-out mouse line, we demonstrate that Alex3 is required for Gαqeffects on mitochondrial dynamics and trafficking, and dendritic growth.Armcx3-deficient mice present decreased OXPHOS complex and ER stress response protein levels, which correlate with increased neuronal death, motor neuron and neuromuscular synaptic loss, and severe motor alterations. Finally, we show that Alex3 disassembles from the Miro1/Gαqcomplex upon calcium rise. These data uncover a novel Alex3/Gαqcomplex that regulates neuronal mitochondrial dynamics and neuronal death and allows the control of mitochondrial functions by GPCRs.
Variable oxygen environments and DNMT2 determine the DNA cytosine epigenetic landscape of Plasmodium falciparum
Authors: Hammam Elie , Miled Samia , Bonhomme Frédéric , Arcangioli Benoit , Arimondo Paola B. , Scherf Artur
Published: December 2021

DNA cytosine methylation and its oxidized products are important epigenetic modifications in mammalian cells. Although 5-methylcytosine (5mC) was detected in the human malaria parasite Plasmodium falciparum, the presence of oxidized 5mC forms remain to be characterized. Here we establish a protocol to optimize nuclease-based DNA digestion for the extremely AT-rich genome of P. falciparum (>80% A+T) for quantitative LC-MS/MS analysis of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We demonstrate the presence of 5hmC, 5fC and 5caC cytosine modifications in a DNMT2-only organism and observe striking ratio changes between 5mC and 5hmC during the 48-hour blood stage parasite development. Parasite-infected red blood cells cultured in different physiological oxygen concentrations revealed a shift in the cytosine modifications distribution towards the oxidized 5hmC and 5caC forms. In the absence of the canonical C5-DNA methyltransferase (DNMT1 and DNMT3A/B) in P. falciparum, we show that all cytosine modifications depend on the presence of DNMT2. We conclude that DNMT2 and oxygen levels are critical determinants that shape the dynamic cytosine epigenetic landscape in this human pathogen.
NITROGEN LIMITATION ADAPTATIONfunctions as a negative regulator of Arabidopsis immunity
Authors: Val-Torregrosa Beatriz , Bundó Mireia , Chiou Tzyy-Jen , Flors Victor , Segundo Blanca San
Published: December 2021

Background

Phosphorus is an important macronutrient required for plant growth and development. It is absorbed through the roots in the form of inorganic phosphate (Pi). To cope with Pi limitation, plants have evolved an array of adaptive mechanisms to facilitate Pi acquisition and protect them from stress caused by Pi starvation. The NITROGEN LIMITATION ADAPTION (NLA) gene plays a key role in the regulation of phosphate starvation responses (PSR), its expression being regulated by the microRNA miR827. Stress caused by Pi limiting conditions might also affect the plant’s response to pathogen infection. However, cross-talk between phosphate signaling pathways and immune responses remains unclear.

Results

In this study, we investigated whetherNLAplays a role in Arabidopsis immunity. We show that loss-of-function ofNLAandMIR827overexpression causes an increase in phosphate (Pi) content which results in resistance to infection by the fungal pathogenPlectosphaerella cucumerina. Thenlamutant plants accumulated callose in their leaves, a response that is also observed in wild-type plants that have been treated with high Pi. We also show that pathogen infection and treatment with fungal elicitors is accompanied by transcriptional activation ofMIR827and down-regulation ofNLA. Upon pathogen challenge,nlaplants exhibited higher levels of the phytoalexin camalexin compared to wild type plants. Camalexin level also increases in wild type plants treated with high Pi. Furthermore, thenlamutant plants accumulated salicylic acid (SA) and jasmonic acid (JA) in the absence of pathogen infection whose levels further increased upon pathogen.

Conclusions

This study shows thatNLAacts as a negative regulator of Arabidopsis immunity. Overaccumulation of Pi innlaplants positively affects resistance to infection by fungal pathogens. This piece of information reinforces the idea of signaling convergence between Pi and immune responses for the regulation of disease resistance in Arabidopsis.
Divalent metal ions boost effect of nucleic acids delivered by cell-penetrating peptides
Authors: Maloverjan Maria , Padari Kärt , Abroi Aare , Rebane Ana , Pooga Margus
Published: December 2021

Cell-penetrating peptides (CPPs) are promising tools for transfection of various substances, including nucleic acids, into cells. The aim of current work was to search for novel safe and effective approaches for enhancing transfection efficiency of nanoparticles formed of CPP and splice-correcting oligonucleotide (SCO) without increasing the concentration of peptide. We analyzed an effect of inclusion of calcium and magnesium ions into nanoparticles on CPP-mediated transfection in cell culture. We also studied the mechanism of such transfection as well as its efficiency, applicability in case of different cell lines, nucleic acid types and peptides, and possible limitations. We discovered a strong positive effect of these ions on transfection efficiency of SCO, that translated to enhanced synthesis of functional reporter protein. We observed significant changes in intracellular distribution and trafficking of nanoparticles formed with addition of the ions, without increasing cytotoxicity. We propose a novel strategy of preparing CPP-oligonucleotide nanoparticles with enhanced efficiency and, thus, higher therapeutic potential. Our discovery may be translated to primary cell cultures and, possibly, in vivo studies, in the aim to increase CPP-mediated transfection efficiency and likelihood of using CPPs in clinics.
BioImageIT: Open-source framework for integration of image data-management with analysis
Authors: Prigent Sylvain , Valades-Cruz Cesar Augusto , Leconte Ludovic , Maury Leo , Salamero Jean , Kervrann Charles
Published: December 2021

Open science and FAIR principles have become major topics in the field of bioimaging. This is due to both new data acquisition technologies that generate large datasets, and new analysis approaches that automate data mining with high accuracy. Nevertheless, data are rarely shared and rigorously annotated because it requires a lot of manual and tedious management tasks and software packaging. We present BioImageIT, an open-source framework for integrating data management according to FAIR principles with data processing.
Novel piece of the puzzle: ALI1 is required for oxo-C14-HSL priming in Arabidopsis
Authors: Shrestha Abhishek , Hernández-Reyes Casandra , Grimm Maja , Krumwiede Johannes , Stein Elke , Schenk Sebastian T. , Schikora Adam
Published: December 2021

Quorum sensing (QS) molecules mediate communication between bacterial cells. N-acyl homoserine lactones (AHL) are one of the best-studied groups of QS molecules. In addition to bacterial communication, AHL are involved in interactions with eukaryotes. Short side-chain AHL are readily taken up by plants. They induce root elongation and growth promotion. Hydrophobic long side-chain AHL are usually not transported over long distances although, they may prime plants for enhanced resistance. Unfortunately, studies elucidating the plant factors required for response to AHL are sparse. Here, we provide evidence of a plant protein, namely the AHL-priming protein 1 (ALI1), indispensable for enhanced resistance response induced by the N-3-oxotetradecanoyl-homoserine lactone (oxo-C14-HSL). Comparing Col-0 and the ali1 mutant, we revealed loss of AHL-priming in ali1. This phenomenon is reverted with the reintroduction of ALI1 into ali1. Additional transcriptome analysis revealed that ali1 is less sensitive to oxo-C14-HSL treatment compared to the wild-type. Our results suggest, therefore, that ALI1 is required for oxo-C14-HSL-dependent priming for enhanced resistance in Arabidopsis.
Clonal transcriptomics identifies mechanisms of chemoresistance and empowers rational design of combination therapies
Authors: Wild Sophia A , Cannell Ian G , Kania Katarzyna , Nicholls Ashley , Bressan Dario ,   CRUK IMAXT Grand Challenge Team, Hannon Gregory J , Sawicka Kirsty
Published: December 2021

Tumor heterogeneity is thought to be a major barrier to successful cancer treatment due to the presence of drug resistant clonal lineages. However, identifying the characteristics of such lineages that underpin resistance to therapy has remained challenging. Here we utilize clonal transcriptomics with WILD-seq; Wholistic Interrogation of Lineage Dynamics by sequencing, in mouse models of triple-negative breast cancer (TNBC) to understand response and resistance to therapy, including BET bromodomain inhibition and taxane-based chemotherapy. This analysis revealed oxidative stress protection by NRF2 as a major mechanism of taxane resistance and led to the discovery that our tumor models are collaterally sensitive to asparagine deprivation therapy using the clinical stage drug L-asparaginase after frontline treatment with docetaxel. In summary, clonal transcriptomics with WILD-seq identifies mechanisms of resistance to chemotherapy that are also operative in patients and pin points asparagine bioavailability as a druggable vulnerability of taxane resistant lineages.
A fungal plant pathogen overcomesmlo-mediated broad-spectrum disease resistance by rapid gene loss
Authors: Kusch Stefan , Frantzeskakis Lamprinos , Lassen Birthe D. , Kümmel Florian , Pesch Lina , Barsoum Mirna , Walden Kim D. , Panstruga Ralph
Published: December 2021

Hosts and pathogens typically engage in a co-evolutionary arms race. This also applies to phytopathogenic powdery mildew fungi, which can rapidly overcome plant resistance and perform host jumps. Using experimental evolution, we show that the powdery mildew pathogenBlumeria hordeiis capable of breaking the agriculturally important broad-spectrum resistance conditioned by barley loss-of-functionmlomutants. Partialmlovirulence of evolvedB. hordeiisolates is correlated with a distinctive pattern of adaptive mutations, including small-sized (8-40 kb) deletions, of which one is linked to thede novoinsertion of a transposable element. Occurrence of the mutations is associated with a transcriptional induction of effector proteinencoding genes that is absent inmlo-avirulent isolates onmlomutant plants. The detected mutational spectrum comprises the same loci in at least two independently isolatedmlo-virulent isolates, indicating convergent multigenic evolution. The mutational events emerged in part early (within the first five asexual generations) during experimental evolution, likely generating a founder population in which incipientmlovirulence was later stabilized by additional events. This work highlights the rapid dynamic genome evolution of an obligate biotrophic plant pathogen with a transposon-enriched genome.
Preserved representations and decodability of diverse cognitive functions across the cortex, cerebellum, and subcortex
Authors: Nakai Tomoya , Nishimoto Shinji
Published: December 2021

Which part of the brain contributes to our complex cognitive processes? Studies have revealed contributions of the cerebellum and subcortex to higher-order cognitive functions; however it is unclear whether such functional representations are preserved across the cortex, cerebellum, and subcortex. In this study, we used functional magnetic resonance imaging data with 103 cognitive tasks and constructed three voxel-wise encoding and decoding models independently using cortical, cerebellar, and subcortical voxels. Representational similarity analysis revealed that the structure of task representations is preserved across the three brain parts. Principal component analysis visualized distinct organizations of abstract cognitive functions in each part of the cerebellum and subcortex. More than 90% of the cognitive tasks were decodable from the cerebellum and subcortical activities, even for the novel tasks not included in model training. Furthermore, we discovered that the cerebellum and subcortex have sufficient information to reconstruct activity in the cerebral cortex.
Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity
Authors: McEvoy Caitriona M. , Murphy Julia M. , Zhang Lin , Clotet-Freixas Sergi , Mathews Jessica A. , An James , Karimzadeh Mehran , Pouyabahar Delaram , Su Shenghui , Zaslaver Olga , Röst Hannes , Arambewela Madhurangi , Liu Lewis Y. , Zhang Sally , Lawson Keith A. , Finelli Antonio , Wang Bo , MacParland Sonya A. , Bader Gary D. , Konvalinka Ana , Crome Sarah Q.
Published: December 2021

Maintaining organ homeostasis requires complex functional synergy between distinct cell types, a snapshot of which is glimpsed through the simultaneously broad and granular analysis provided by single-cell atlases. Knowledge of the transcriptional programs underpinning the complex and specialized functions of human kidney cell populations at homeostasis is limited by difficulty accessing healthy, fresh tissue. Here, we present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 high-quality cells to map healthy kidney at high resolution. Our sex-balanced dataset revealed sex-based differences in gene expression within proximal tubular cells, specifically, increased anti-oxidant metallothionein genes in females and the predominance of aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed between male and female proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. Within the immune niche, we identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions and validated findings by flow cytometry. We observed significant heterogeneity in resident myeloid populations and identified an MRC1+ LYVE1+ FOLR2+ C1QC+ population as the predominant myeloid population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, revealing novel insights into the complexity of renal parenchymal cells and kidney-resident immune populations.
Discovery of a new species of trichomonasvirus in the human parasite Trichomonas vaginalis using transcriptome mining
Authors: Manny Austin R. , Hetzel Carrie A. , Mizani Arshan , Nibert Max L.
Published: December 2021

Trichomonas vaginalis is the most common nonviral cause of sexually transmitted infections globally, with an estimated quarter of a billion people infected around the world. Infection by the protozoan parasite results in the clinical syndrome trichomoniasis, which manifests as an inflammatory syndrome with acute and chronic consequences. Half or more of these parasites are themselves infected with one or more dsRNA viruses which can exacerbate the inflammatory disease. Four distinct viruses have been found in T. vaginalis to date, Trichomonas vaginalis virus 1 through 4 (or TVVs). Despite the global prevalence of these viruses, few coding-complete genome sequences have been determined. We conducted viral sequence mining in publicly available transcriptomes across 60 RNA-seq datasets representing 13 distinct T. vaginalis isolates. We assembled sequences for 27 new trichomonasvirus strains across all known TVV species, with 17 of these assemblies representing coding-complete genomes. Using a strategy of de novo sequence assembly followed by taxonomic classification, we discovered a fifth species of TVV that we term Trichomonas vaginalis virus 5 (TVV5). Six strains of TVV5 were assembled, including two coding-complete genomes. These TVV5 sequences exhibit high sequence identity to each other, but low identity to any strains of TVV1-4. Phylogenetic analysis corroborates the species-level designation. These results substantially increase the number of coding-complete TVV genome sequences and demonstrate the utility of mining publicly available transcriptomes for the discovery of RNA viruses in a critical human pathogen.
The Landscape-Based Protein Stability Analysis and Network Modeling of Multiple Conformational States of the SARS-CoV-2 Spike D614 Mutant: Conformational Plasticity and Frustration-Driven Allostery as Energetic Drivers of Highly Transmissible Spike Variant
Authors: Verkhivker Gennady , Agajanian Steve , Kassab Ryan , Krishnan Keerthi
Published: December 2021

The structural and functional studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across many variants of concern(VOCs), suggesting the effect of this mutation on the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies provided important evidence about multiple conformational substates of the D614G spike protein. The development of a plausible mechanistic model which can explain the experimental observations from a more unified thermodynamic perspective is an important objective of the current work. In this study, we employed efficient and accurate coarse-grained simulations of multiple structural substates of the D614G spike trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration profiling of the conformational states with residue-based mutational scanning of protein stability and network analysis of allosteric interactions and communications, we determine the patterns of mutational sensitivity in the functional regions and sites of variants. We found that the D614G mutation may induce a considerable conformational adaptability of the open states in the SARS-CoV-2 spike protein without compromising folding stability and integrity of the spike protein. The results suggest that the D614G mutant may employ a hinge-shift mechanism in which the dynamic couplings between the site of mutation and the inter-protomer hinge modulate the inter-domain interactions, global mobility change and the increased stability of the open form. This study proposes that mutation-induced modulation of the conformational flexibility and energetic frustration at the inter-protomer interfaces may serve as an efficient mechanism for allosteric regulation of the SARS-CoV-2 spike proteins.
A correlation map of genome-wide DNA methylation patterns between paired human brain and buccal samples
Authors: Sommerer Yasmine , Ohlei Olena , Dobricic Valerija , Oakley Derek H. , Wesse Tanja , Sabet Sanaz Sedghpour , Demuth Ilja , Franke Andre , Hyman Bradley T. , Lill Christina M. , Bertram Lars
Published: December 2021

Epigenome-wide association studies (EWAS) assessing the link between DNA methylation (DNAm) and phenotypes related to structural brain measures, cognitive function, and neurodegenerative diseases are becoming increasingly more popular. Due to the inaccessibility of brain tissue in humans, several studies use peripheral tissues such as blood, buccal swabs, and saliva as surrogates. To aid the functional interpretation of EWAS findings in such settings, there is a need to assess the correlation of DNAm variability across tissues in the same individuals. In this study, we performed a correlation analysis between DNAm data of a total of n=120 matched post-mortem buccal and prefrontal cortex samples. We identified nearly 25,000 (3% of approximately 730,000) cytosine-phosphate-guanine (CpG) sites showing significant (False Discovery Rate q < 0.05) correlations between buccal and PFC samples. Correlated CpG sites showed a preponderance to being located in promoter regions and showed a significant enrichment of being determined by genetic factors, i.e. methylation quantitative trait loci (mQTL), based on buccal and dorsolateral prefrontal cortex mQTL databases. Our novel buccal-brain DNAm correlation map will provide a valuable resource for future EWAS using buccal samples for studying DNAm effects on phenotypes relating to the brain. All correlation results are made freely available to the public online.
Aggregate trait evolvability and macroevolution in two sister species of the bryozoanStylopoma
Authors: Leventhal Sarah , Jamison-Todd Sarah , Simpson Carl
Published: December 2021

The study of trait evolution in modular animals is more complicated than that in solitary animals, because a single genotype of a modular colony can express an enormous range of phenotypic variation. Furthermore, traits can occur either at the module level or at the colony level. However, it is unclear how the traits at the colony level evolve. We test whether colony-level aggregate traits, defined as the summary statistics of a phenotypic distribution, can evolve. To quantify this evolutionary potential, we use parent-offspring pairs in two sister species of the bryozoanStylopoma, grown and bred in a common garden breeding experiment. We find that the medians of phenotypic distributions are evolvable between generations of colonies. We also find that the structure of this evolutionary potential differs between these two species. Ancestral species align more closely with the direction of species divergence than the descendent species. This result indicates that aggregate trait evolvability can itself evolve.
Macroevolutionary dynamics of climatic niche space
Authors: Quintero Ignacio , Suchard Marc A. , Jetz Walter
Published: December 2021

How and why lineages evolve along niche space as they diversify and adapt to different environments is fundamental to evolution. Progress has been hampered by the difficulties of linking a comprehensive empirical characterization of species niches with flexible evolutionary models that describe their evolution. Consequently, the relative influence of external episodic and biotic factors remains poorly understood. Here we characterize species’ two-dimensional temperature and precipitation niche space occupied (i.e., species niche envelope) as complex geometries and assess their evolution across a large vertebrate radiation (all Aves) using a model that captures heterogeneous evolutionary rates on time-calibrated phylogenies. We find that extant birds coevolved from warm, mesic climatic niches into colder and drier environments and responded to the K-Pg boundary with a dramatic increase in disparity. Contrary to expectations of subsiding rates of niche evolution as lineages diversify, our results show that overall rates have increased steadily, with some lineages experiencing exceptionally high evolutionary rates, associated with colonization of novel niche spaces, and others showing niche stasis. Both competition- and environmental change-driven niche evolution transpire and result in highly heterogeneous rates near the present. Our findings share the limitations of all work based purely on extant taxa but highlight the growing ecological and conservation insights arising from the model-based integration of increasingly comprehensive and robust environmental and phylogenetic information.
Chronic AMPK inactivation slows SHH medulloblastoma progression by inhibiting mTORC1 signaling and depleting tumor stem cell populations
Authors: Dismuke Taylor , Malawsky Daniel Shiloh , Liu Hedi , Brenman Jay , Dasgupta Biplab , Tikunov Andrey , Gershon Timothy R.
Published: December 2021

We show that inactivating AMPKin vivoin a genetic model of medulloblastoma depletes tumor stem cell populations and slows tumor progression. Medulloblastoma, the most common malignant pediatric brain tumor, grows as heterogenous communities comprising diverse types of tumor and stromal cells. Previously, we showed that different populations in medulloblastomas show different sensitivities to specific targeted therapies. To determine if specific populations depend on AMPK, we analyzed mice with AMPK-inactivated medulloblastomas. We engineered mice with conditional deletion of the AMPK catalytic subunitsPrkaa1andPrkaa2and conditional expressionSmoM2, an oncogenicSmoallele that hyperactivates Sonic Hedgehog (SHH) signaling. We compared these medulloblastomas to SmoM2-driven medulloblastomas in AMPK-intact mice. AMPK-inactivation slowed tumor growth and progression, allowing longer event-free survival (EFS). scRNA-seq showed that AMPK inactivation altered cellular heterogeneity, increasing differentiation, decreasing tumor stem cell populations and reducing glio-neuronal multipotency. Surprisingly, AMPK-inactivated tumors showed decreased mTORC1 activation andHk2expression. GeneticHk2deletion in SmoM2-medulloblastomas similarly decreased stem cell populations, implicating reduced aerobic glycolysis in the tumor-suppressive effect of AMPK inactivation. Our results show that AMPK inactivation impairs tumor growth through mechanisms that disproportionately affect tumor stem cell populations that have proved refractory to conventional therapies.
Pharmacologic analysis of non-synonymous coding 5-HT2A SNPs reveals alterations psychedelic drug potencies and efficacies
Authors: Schmitz Gavin P. , Jain Manish K. , Slocum Samuel T. , Roth Bryan L.
Published: December 2021

Serotonin (5-Hydroxytryptamine; 5-HT) 2A receptor (5-HT2AR) signaling is essential for the actions of classical psychedelic drugs. In this study, we examined whether random sequence variations in the gene (single nucleotide polymorphisms, SNPs) encoding the 5-HT2AR affect the signaling of four commonly used psychedelic drugs. We examined the in vitro pharmacology of seven non-synonymous SNPs, which give rise to S12N, T25N, D48N, I197V, A230T, A447V, and H452Y variant 5-HT2A serotonin receptors. We found that these non-synonymous SNPs exert statistically significant, although modest, effects on the efficacy and potency of four therapeutically relevant hallucinogens. Significantly, the in vitro pharmacological effects of the SNPs drug actions at 5-HT2AR are drug specific.

Table of Contents/Abstract Graphic
Quantitative Structural Analysis of Influenza Virus by Cryo-electron Tomography and Convolutional Neural Networks
Authors: Huang Qiu Yu , Song Kangkang , Xu Chen , Bolon Daniel N.A. , Wang Jennifer P. , Finberg Robert W. , Schiffer Celia A. , Somasundaran Mohan
Published: December 2021

Influenza viruses pose severe public health threats; they cause millions of infections and tens of thousands of deaths annually in the US. Influenza viruses are extensively pleomorphic, in both shape and size as well as organization of viral structural proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships as well as aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and extensive viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we developed a cryoET processing pipeline leveraging convolutional neural networks (CNNs) to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza viral morphology, glycoprotein density, and conduct subtomogram averaging of HA glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.

Graphical abstract
Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing
Authors: Schmidt Matthias , Pearson Allison N. , Incha Matthew R. , Thompson Mitchell G. , Baidoo Edward E. K. , Kakumanu Ramu , Mukhopadhyay Aindrila , Shih Patrick M. , Deutschbauer Adam M. , Blank Lars M. , Keasling Jay D.
Published: December 2021

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop- out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers.

IMPORTANCE

Understanding the genetic basis of P. putida’s diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators--which are of particular interest for metabolic engineering.
Rational domestication of a plant-based recombinant expression system expands its biosynthetic range
Authors: Jackson Mark A. , Chan Lai Yue , Harding Maxim D. , Craik David J. , Gilding Edward K.
Published: December 2021

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.
Identification of genome-wide distribution of cyanobacterial group 2 sigma factor SigE accountable for its regulon
Authors: Kariyazono Ryo , Osanai Takashi
Published: December 2021

Sigma factors are the subunits of bacterial RNA polymerase that govern the expression of genes by recognizing the promoter sequence. Cyanobacteria, which are oxygenic phototrophic eubacteria, have multiple alternative sigma factors that respond to various environmental stresses. The subgroup highly homologous to the primary sigma factor (SigA) is called the group 2 sigma factor. The model cyanobacterium, Synechocystis sp. PCC 6803, has four group 2 sigma factors (SigB-E) conserved within the phylum Cyanobacteria. Among the group 2 sigma factors in Synechocystis sp. PCC 6803, SigE is unique because it alters metabolism by inducing the expression of genes related to sugar catabolism and nitrogen metabolism. However, the features of promoter sequence of the SigE regulon remains elusive. Here, we identified the direct targets of SigA and SigE by chromatin immunoprecipitation sequencing (ChIP-seq). We then showed that the binding sites of SigE and SigA overlapped substantially, but SigE exclusively localized to SigE-dependent promoters. We also found consensus sequences from SigE-dependent promoters and confirmed their importance. ChIP-seq analysis showed both the redundancy and specificity of SigE compared with SigA, integrating information obtained from a previously adopted genetic approach and in vitro assays. The features of SigE elucidated in our study indicate its similarity with group 2 sigma factors of other bacteria, even though they are evolutionally irrelevant. Our approach is also applicable to other organisms and organelles, such as plant plastids, which have multiple group 2 sigma factors.

Importance

Group 2 sigma factors are alternative sigma factors highly homologous to primary sigma factors. Cyanobacteria, which are photosynthetic eubacteria, are unique because they have multiple group 2 sigma factors. Although each sigma factor induces the expression of specific genes, the redundancy and complicated network of the primary and group 2 sigma factors hinder the identification of their regulons via a genetic approach. Here, we identified the binding sites of SigE (group 2 sigma factor) and SigA (primary sigma factor) using chromatin immunoprecipitation sequencing and identified the minimal element of SigE-dependent promoters by subsequent promoter assays. Our study provides insights into the common features of group 2 sigma factors, which, though evolutionarily irrelevant, are widespread among eubacteria and plant plastids.