bluuebunny/crossref_metadata_2025 huggingface dataset:
Detecting bat environmental DNA from water-filled road-ruts in upland forest
Authors: Marshall Nathaniel T. , Symonds Daniel E. , Walker Faith M , Sanchez Daniel E , Couch Zachary L. , Kiser James D.
Published: June 2022

Declines in population and diversity of North American bats are rapidly occurring due to habitat loss, incidental take from various industry projects, and lethal White-nose Syndrome disease. It is critical to accurately census habitat for appropriate conservation measures, yet traditional sampling methodology, such as mist netting and acoustic recordings, can be time-intensive and biased. Instead, a passive sampling tool that does not rely on the a priori knowledge of bat roosts may provide crucial information on bat communities. In the water-limited habitats of forested uplands of the Appalachian Plateau, water-filled road-ruts are important resources for bats. Therefore, we developed an environmental DNA (eDNA) protocol to sample isolated road-ruts that may have the presence of sloughed cellular material from actively drinking bats. The detection of bat eDNA was investigated from a positive control experiment, and across 47 water samples collected in Kentucky and Ohio. Water samples were analyzed using both species-specific quantitative polymerase chain reaction (qPCR) and community metabarcoding methodologies. Using qPCR analysis, we detected eDNA from big brown bat (Eptesicus fuscus) and eastern red bat (Lasiurus borealis) from water-filled road-ruts. While the community metabarcoding approach failed to detect any bat eDNA, many non-target amphibians, birds, and mammals were identified. These results suggest eDNA found within road-ruts provides an additional detection tool for surveying biodiversity across upland forests. Additionally, the use of qPCR increased the detection of rare eDNA targets, which will be crucial for properly implementing future eDNA applications for improving bat conservation efforts across the landscape.

Article impact statement

Environmental DNA provides detection of bats from drinking sources offering a novel survey method for management and conservation efforts
Spatio-temporal dynamics of nuclear CREB1: what does it mean?
Authors: Farias Altamirano Luz E. , Vásquez Elena , Freites Carlos L. , Ibañez Jorge E. , Guido Mario E. , Muñoz Estela M.
Published: June 2022

In the mammalian pineal gland (PG), cyclic AMP responsive element-binding protein 1 (CREB1) participates in the nocturnal melatonin synthesis that rhythmically modulates physiology and behavior. Phosphorylation of CREB1 present in pinealocyte nuclei is one of the key regulatory steps that drives pineal transcription. The spatio-temporal dynamics of CREB1 itself within PG cell types have not yet been documented. In this study we analyzed total CREB1 via Western blot, and the dynamism of CREB1 nuclear distribution in individual rat pinealocytes using fluorescence immunohistochemistry followed by confocal laser-scanning microscopy and quantitative analysis. Total CREB1 levels remained constant in the PG throughout the light:dark cycle. The distribution pattern of nuclear CREB1 did vary, however, among different PG cells. Pinealocytes emerged as having discrete CREB1 domains within their nucleoplasm that were especially distinct. The number, size, and location of CREB1 foci fluctuated among pinealocytes, within the same PG and amongZeitgebertimes. A significantly larger dispersion of CREB1-immunoreactive nuclear sites was found at night. This was not accompanied by changes in the overall transcription activity, which was mostly conserved between the light and dark phases, as shown by the expression of a particular phosphorylated form of the RNA polymerase II (RNAPII-pSer5CTD). Suppression of the nocturnal norepinephrine pulse by chronic bilateral superior cervical ganglionectomy increased CREB1 dispersion in pinealocyte nuclei, as compared to sham-derived cells. In addition, differences in CREB1 distribution were found between sham-operated and non-operated rats at early night. Together, these data suggest that in mature pinealocytes nuclear CREB1 is subjected to a dynamic spatio-temporal distribution. Further studies are necessary to elucidate the underlying mechanisms, including the role of chromatin and interchromatin elements, and to understand the impact of CREB1 reorganization in the pineal transcriptome.
Integration of synthetic microbial consortia based bioprocessing with pyrolysis for efficient conversion of cellulose to valuables
Authors: Joshi Chandrakant , Kumar Mahesh , Bennett Martyn , Thakur Jyotika , Leak David J. , Sharma Swati , MacKinnon Neil , Masakapalli Shyam Kumar
Published: June 2022

Improved technologies are needed for sustainable conversion of cellulosic waste to valuable products. Here we demonstrate the successful integration of a synthetic microbial consortium (SynCONS) based consolidated bioprocessing with pyrolysis to produce commodity chemicals from cellulose. Promising microbial partners were rationally identified from 7626 organisms via comparative metabolic mapping which led to establishing two promising SynCONS with abilities to convert cellulose to ethanol and lactate in bioreactors. The partners in the two SynCONS were a) the mesophilic fungus Trichoderma reesei grown sequentially with the thermophilic bacterium Parageobacillus thermoglucosidasius NCIMB 11955 (TrPt) and b) a thermophilic bacterium Thermobifida fusca grown together with Parageobacillus thermoglucosidasius NCIMB 11955 (TfPt). TrPt sequential bioprocessing resulted in 39% (g/g) cellulose consumption with product yields up to 9.3% g/g (ethanol + lactate). The TfPt co-cultures demonstrated a cellulose consumption of 30% (g/g) and combined yields of ethanol and lactic acid up to 23.7% g/g of consumed cellulose. The total product yields were further enhanced (51% g/g cellulose) when commercially available cellulases were used in place of T. fusca. Furthermore, when the metabolically engineered ethanol-producing strain of P. thermoglucosidasius TM242 (TfPt242) was substituted in the thermophilic TfPt co-culture consortium, ethanol yields were substantially higher (32.7% g/g of consumed cellulose). Finally, subjecting the residual cellulose and microbial biomass to pyrolysis resulted in carbon material with physicochemical properties similar to commercially available activated carbon as analysed using Scanning Electron Microscopy, X-Ray Diffraction and Raman spectroscopy. Overall, the integration of this synthetic microbial consortia-based bioprocessing strategy with pyrolysis demonstrated a promising strategy for conversion of waste cellulose to chemicals, biofuels, and industrial carbon potentially suitable for several industrial applications.
Genotype-environment interactions determine microbiota plasticity inNematostella vectensis
Authors: Baldassarre Laura , Reitzel Adam M. , Fraune Sebastian
Published: June 2022

Most multicellular organisms harbor microbial colonizers that provide various benefits to their hosts. Although these microbial communities may be host species- or even genotype-specific, the associated bacterial communities can respond plastically to environmental changes. In this study, we estimated the relative contribution of environment and host genotype to bacterial community composition inNematostella vectensis, an estuarine cnidarian. We isolatedN. vectensispolyps from five different populations along a north-south gradient on the Atlantic coast of the United States and Canada at three different times of the year. While half of the polyps were immediately analyzed for their bacterial composition by 16S rRNA gene sequencing, the remaining polyps were cultured under laboratory conditions for one month. Bacterial community comparison analyses revealed that laboratory maintenance reduced bacterial diversity by fourfold, but maintained a population-specific bacterial colonization. Interestingly, the differences between bacterial communities correlated strongly with seasonal variations, especially with ambient water temperature. To decipher the contribution of both ambient temperature and host genotype to bacterial colonization, we generated 12 clonal lines from six different populations in order to maintain each genotype at three different temperatures for three months. The bacterial community composition of the sameN. vectensisclone differed greatly between the three different temperatures, highlighting the contribution of ambient temperature to bacterial community composition. To a lesser extent, bacterial community composition varied between different genotypes under identical conditions, indicating the influence of host genotype. In addition, we identified a significant genotype x environment interaction determining microbiota plasticity inN. vectensis. From our results we can conclude thatN. vectensis-associated bacterial communities respond plastically to changes in ambient temperature, with the association of different bacterial taxa depending in part on the host genotype. Future research will reveal how this genotype-specific microbiota plasticity affects the ability to cope with changing environmental conditions.
Improving the workflow to crack Small, Unbalanced, Noisy, but Genuine (SUNG) datasets in bioacoustics: the case of bonobo calls
Authors: Arnaud Vincent , Pellegrino François , Keenan Sumir , St-Gelais Xavier , Mathevon Nicolas , Levréro Florence , Coupé Christophe
Published: June 2022

Despite the accumulation of data and studies, deciphering animal vocal communication remains highly challenging. While progress has been made with some species for which we now understand the information exchanged through vocal signals, researchers are still left struggling with sparse recordings composing Small, Unbalanced, Noisy, but Genuine (SUNG) datasets. SUNG datasets offer a valuable but distorted vision of communication systems. Adopting the best practices in their analysis is therefore essential to effectively extract the available information and draw reliable conclusions. Here we show that the most recent advances in machine learning applied to a SUNG dataset succeed in unraveling the complex vocal repertoire of the bonobo, and we propose a workflow that can be effective with other animal species. We implement acoustic parameterization in three feature spaces along with three classification algorithms (Support Vector Machine, xgboost, neural networks) and their combination to explore the structure and variability of bonobo calls, as well as the robustness of the individual signature they encode. We underscore how classification performance is affected by the feature set and identify the most informative features. We highlight the need to address data leakage in the evaluation of classification performance to avoid misleading interpretations. Finally, using a Uniform Manifold Approximation and Projection (UMAP), we show that classifiers generate parsimonious data descriptions which help to understand the clustering of the bonobo acoustic space. Our results lead to identifying several practical approaches that are generalizable to any other animal communication system. To improve the reliability and replicability of vocal communication studies with SUNG datasets, we thus recommend: i) comparing several acoustic parameterizations; ii) adopting Support Vector Machines as the baseline classification approach; iii) explicitly evaluating data leakage and possibly implementing a mitigation strategy; iv) visualizing the dataset with UMAPs applied to classifier predictions rather than to raw acoustic features.
Comparative analysis of piRNA sequences, targets and functions in nematodes
Authors: Hertz Hannah L. , Pastore Benjamin , Tang Wen
Published: June 2022

Piwi proteins and Piwi-interacting RNAs (piRNAs) are best known for their roles in suppressing transposons and promoting fertility. Yet piRNA biogenesis and its mechanisms of action differ widely between distantly related species. To better understand the evolution of piRNAs, we characterized the piRNA pathway in C. briggsae, a sibling species of the model organism C. elegans. Our analyses define 25,883 piRNA producing-loci in C. briggsae. piRNA sequences in C. briggsae are extremely divergent from their counterparts in C. elegans, yet both species adopt similar genomic organization that drive piRNA expression. By examining production of Piwi-mediated secondary small RNAs, we identified a set of protein-coding genes that are evolutionarily conserved piRNA targets. In contrast to C. elegans, small RNAs targeting ribosomal RNAs or histone transcripts are not hyper-accumulated in C. briggsae Piwi mutants. Instead, we found that transcripts with few introns are prone to small RNA overamplification. Together our work highlights evolutionary conservation and divergence of the nematode piRNA pathway and provides insights into its role in endogenous gene regulation.
Binding Free Energies of Piezo1 Channel Agonists at Protein-Membrane Interface
Authors: Jiang Wenjuan , Zhang Han , Yichun-Lin  , Im Wonpil , Lacroix Jerome J. , Luo Yun Lyna
Published: June 2022

Mechanosensitive Piezo channels convert mechanical stimuli into biological signals in vertebrates. Piezo1 chemical modulators are anticipated to yield many clinical benefits. To date, Yoda1 is the most potent and widely used Piezo1-selective agonist, yet how Yoda1 interacts with Piezo1 at the protein-membrane interface and stabilizes Piezo1’s open state remains elusive. Here, using a previously identified putative Yoda1 binding site and three molecular dynamics (MD)-based methods, we computed the binding free energies of Yoda1 and its analogs in a Piezo1 cryo-EM closed state and anin silicoopen state. Our computed absolute binding free energy of Yoda1 in the closed state agrees well with the experimentalKdin which Piezo1 is expected to be in a closed state. More importantly, Yoda1 binds the open state better than the closed state, in agreement with its agonist effects. All three methods predicted that Dooku1, a Yoda1 analog, binds the closed state stronger than Yoda1, but binds the open state weaker than Yoda1. These results are consistent with the fact that Dooku1 antagonizes the effects of Yoda1 but lacks the ability to activate Piezo1. The relative binding free energies of seven Yoda1 analogs recapitulate key experimental structure-activity-relationships (SAR). Based on the state-dependent binding free energies, we were able to predict whether a molecule is an agonist or inhibitor and whether a chemical modification will lead to a change in affinity or efficacy. These mechanistic insights and computational workflow designed for transmembrane binders open an avenue to structural-based screening and design of novel Piezo1 agonists and inhibitors.
Geometric effects in gas vesicle buckling under ultrasound
Authors: Salahshoor Hossein , Yao Yuxing , Dutka Przemysław , Nyström Nivin N. , Jin Zhiyang , Min Ellen , Malounda Dina , Jensen Grant J. , Ortiz Michael , Shapiro Mikhail G.
Published: June 2022

Acoustic reporter genes based on gas vesicles (GVs) have enabled the use of ultrasound to noninvasively visualize cellular function in vivo. The specific detection of GV signals relative to background acoustic scattering in tissues is facilitated by nonlinear ultrasound imaging techniques taking advantage of the sonomechanical buckling of GVs. However, the effect of geometry on the buckling behavior of GVs under exposure to ultrasound has not been studied. To understand such geometric effects, we developed computational models of GVs of various lengths and diameters and used finite element simulations to predict their threshold buckling pressures and post-buckling deformations. We demonstrated that the GV diameter has an inverse cubic relation to the threshold buckling pressure, whereas length has no substantial effect. To complement these simulations, we experimentally probed the effect of geometry on the mechanical properties of GVs and the corresponding nonlinear ultrasound signals. The results of these experiments corroborate our computational predictions. This study provides fundamental insights into how geometry affects the sonomechanical properties of GVs, which, in turn, can inform further engineering of these nanostructures for high-contrast, nonlinear ultrasound imaging.

STATEMENT OF SIGNIFICANCE

Gas vesicles (GVs) are an emerging class of genetically encodable and engineerable imaging agents for ultrasound whose sonomechanical buckling generates nonlinear contrast to enable sensitive and specific imaging in highly scattering biological systems. Though the effect of protein composition on GV buckling has been studied, the effect of geometry has not previously been addressed. This study reveals that geometry, especially GV diameter, significantly alters the threshold acoustic pressures required to induce GV buckling. Our computational predictions and experimental results provide fundamental understanding of the relationship between GV geometry and buckling properties and underscore the utility of GVs for nonlinear ultrasound imaging. Additionally, our results provide suggestions to further engineer GVs to enable in vivo ultrasound imaging with greater sensitivity and higher contrast.
Turicibacterstrains differentially modify bile acids and host lipids
Authors: Lynch Jonathan B. , Gonzalez Erika L. , Choy Kayli , Faull Kym F. , Jewell Talia , Arellano Abelardo , Liang Jennifer , Yu Kristie B. , Paramo Jorge , Hsiao Elaine Y.
Published: June 2022

Bacteria from theTuricibactergenus are prominent members of the mammalian gut microbiota and are associated with alterations in dietary fat and body weight, but the specific connections between these symbionts and host physiology are poorly understood. We genomically and phenotypically characterized a diverse set of mouse-and human-derivedTuricibacterstrains, and found they group into three clades that differ in their transformations of bile acids. We identifiedTuricibacterbile salt hydrolases that confer strain-specific differences in bile deconjugation. Colonization with individualTuricibacterstrains led to changes in host bile acid profiles, generally aligning with those producedin vitro. Further, colonizing mice with another bacterium expressing bile-modifying genes from these strains decreased serum cholesterol and triglycerides, as well as adipose tissue mass. This work identifies genes that enable diverseTuricibacterstrains to differentially modify host bile acids and lipid metabolism, and positions multipleTuricibacterstrains as candidates for altering host fat biology.

MAIN TEXT
Angular speed should be avoided when estimating the speed-curvature power law in movement
Authors: Matić Adam
Published: June 2022

The speed-curvature power law is one of the most studied constraints in biological movement. In many types of movements, there is a strong relationship between instantaneous speed and local curvature. For example, in elliptical trajectories, tangential speed is proportional to curvature raised to the power -1/3, (V∼C-1/3). This phenomenon is known as the “one-thirds power law” and is generally considered to be mathematically equivalent to the “two-thirds power law” that describes the relationship between angular speed and curvature (A∼C2/3); the two formulations are used interchangeably. However, in this paper, analysis of empirical and synthetic data demonstrates that using angular speed instead of tangential speed to estimate the power law tends to result in much stronger correlations, impacting the interpretation of the strength of the relationship, and therefore the existence of the law. Further analysis shows that angular speed and curvature are often trivially correlated, since angular speed is not a purely kinematic variable and depends on curvature. In conclusion, two forms of the law are not equivalent, angular speed should be avoided when expressing the speed-curvature power law.
Ideal free distribution of unequal competitors: spatial assortment and evolutionary diversification of competitive ability
Authors: Netz Christoph , Ramesh Aparajitha , Weissing Franz J.
Published: June 2022

Ideal free distribution theory attempts to predict the distribution of well-informed (‘ideal’) and unconstrained (‘free’) foragers in space based on adaptive individual decisions. When individuals differ in competitive ability, a whole array of equilibrium distributions is possible, and it is unclear which of these distributions are most likely. In the first part of our study, we show that strong competitors have an intrinsically stronger preference for highly productive habitat patches than poor competitors. This leads to an equilibrium distribution where the average competitive ability on a patch is strongly correlated with the productivity of the patch. In the second part of our study, we consider what happens if differences in competitive ability are heritable and, hence, subject to natural selection. Under constant environmental conditions, selection eliminates such differences: a single strategy prevails that optimally balances the costs and benefits associated with competitive ability. If the productivity of patches changes during the lifetime of individuals, the spatial assortment of competitors of equal competitive ability gives poor competitors a systematic advantage in times of environmental change, while good competitors benefit from equilibrium conditions. Using evolutionary individual-based simulations, we demonstrate that environmental change may then lead to the diversification of competitive ability.
Following cell type transitions in space and time by combining live-cell tracking and endpoint cell identity in intestinal organoids
Authors: Zheng Xuan , Betjes Max A. , Goos Yvonne J. , Huelsz-Prince Guizela , Clevers Hans , van Zon Jeroen S. , Tans Sander J.
Published: June 2022

Elucidating the dynamics of cellular differentiation in space and time is key to advancing organoid biology and technology. Direct visualization of differentiation patterns is challenging, however, in part because of the difficulty of simultaneously detecting all relevant cell types by fluorescence imaging. Here we present TypeTracker, which determines the differentiation trajectories of all cells within a region of interest, including their type transitions, growth, divisions, and changes in spatial organization. We show how the lineage tree topology, as determined by automated cell tracking, allows for the retrospective identification of cell types across multiple generations. The data revealed various surprising aspects of intestinal organoid differentiation, including symmetric fate adoption by sister cells, cell type abundance regulated by division, and type commitment of cells occurring prior to their spatial reorganization. Our method can be applied broadly to study other organoid systems and to screen for compounds that affect differentiation programs.
Mapping nanoscale forces and potentials in live cells with microsecond 3D single-particle tracking
Authors: Hou Shangguo , Zhang Chen , Niver Anastasia , Welsher Kevin
Published: June 2022

3D single-particle tracking has the potential to resolve the molecular level forces which dictate particle motion in biological systems. However, the information gleaned from 3D single-particle tracking often cannot resolve underlying nanoscale potentials due to limited spatiotemporal resolution. To this end, we introduce an active-feedback 3D tracking microscope that utilizes silver nanoparticles (AgNPs) as probes to study intricate biophysical events in live cells at the nanometer and microsecond scales. Due to this extremely high and durable scattering photon flux of the plasmonic particles, 1 MHz sampling frequency at nanometer precision in all three dimensions can be achieved over an unlimited observation times. In this work, we applied microsecond-sampling, active-feedback 3D single-particle tracking to investigate the interaction between AgNPs and nanoscale filopodium on the live-cell surface. The nanometer precision and microsecond sampling revealed that TAT peptide modified particles visit and dwell at local “hot spots” on the filopodium surface. The high sampling rate further enabled the calculation of the local forces and potentials within these nanoscale hotspots on the cylindrical surface of live cell filopodia. This study presents a promising tool to investigate intracellular biophysical events with unprecedented spatiotemporal resolution and a pipeline to study nanoscale potentials on three-dimensional cellular structures.
A Telltale Sign of Irreversibility in Transcriptional Regulation
Authors: Shelansky Robert , Abrahamsson Sara , Doody Michael , Brown Christopher R. , Patel Heta P. , Lenstra Tineke L. , Larson Daniel R. , Boeger Hinrich
Published: June 2022

Transcription occurs in stochastic bursts, i.e., transcription events are temporally clustered. The clustering does not ensue from environmental fluctuations but springs from the intrinsically stochastic behavior of the regulatory process that controls transcription. Based on microscopic observations of transcription at a single gene copy of yeast, we show that the regulatory process is cyclic and irreversible, i.e., the process violates the detailed balance conditions for thermodynamic equilibrium. The theoretical significance of this finding is discussed.
WTAP targets the METTL3 m6A-methyltransferase complex to cytoplasmic hepatitis C virus RNA to regulate infection
Authors: Sacco Matthew T. , Bland Katherine M. , Horner Stacy M.
Published: June 2022

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral lifecycle. This lifecycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and for m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production were independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm.

IMPORTANCE

Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described how HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV lifecycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic lifecycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.
Predicting the locations of cryptic pockets from single protein structures using the PocketMiner graph neural network
Authors: Meller Artur , Ward Michael , Borowsky Jonathan , Lotthammer Jeffrey M. , Kshirsagar Meghana , Oviedo Felipe , Ferres Juan Lavista , Bowman Gregory R.
Published: June 2022

Cryptic pockets expand the scope of drug discovery by enabling targeting of proteins currently considered undruggable because they lack pockets in their ground state structures. However, identifying cryptic pockets is labor-intensive and slow. The ability to accurately and rapidly predict if and where cryptic pockets are likely to form from a protein structure would greatly accelerate the search for druggable pockets. Here, we present PocketMiner, a graph neural network trained to predict where pockets are likely to open in molecular dynamics simulations. Applying PocketMiner to single structures from a newly-curated dataset of 39 experimentally-confirmed cryptic pockets demonstrates that it accurately identifies cryptic pockets (ROC-AUC: 0.87) >1,000-fold faster than existing methods. We apply PocketMiner across the human proteome and show that predicted pockets open in simulations, suggesting that over half of proteins thought to lack pockets based on available structures are likely to contain cryptic pockets, vastly expanding the druggable proteome.
Structural basis for host recognition and superinfection exclusion by bacteriophage T5
Authors: van den Berg Bert , Silale Augustinas , Baslé Arnaud , Mader Sophie L. , Khalid Syma
Published: June 2022

A key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent a secondary viral infection by the same or a closely related phage, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here we present the cryo-EM structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug, but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a non-native conformation of the plug of FhuA, causing the inward folding of two extracellular loops via “reverse” allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of E. coli by phage T5, and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.
Peptide from NSP7 is able to form amyloid-like fibrils: artifact or challenge to drug design?
Authors: Garmay Yuri , Rubel Aleksandr , Egorov Vladimir
Published: June 2022

Using a set of programs for analyzing the primary structure of amyloidogenic proteins, we analyzed the primary structure of the of the COVID-19 NSP7 protein. Peptides containing potential amyloidogenic determinants in the primary structure have been synthesized and shown to be capable of forming amyloid-like fibrils in vitro. In the future, this peptide can be used as a basis for creating antiviral agents that act directly on the spatial structure of the NSP7 subunit of the COVID-19 polymerase.
Short communication: Antibiotic resistance in Gram-negative bacteria isolated from street-vended foods in Bangladesh
Authors: Meem Fariha Chowdhury , Raihan Topu , Shourove Jahid Hasan , Azad Abul Kalam , Islam GM Rabiul
Published: June 2022

Antibiotic-resistant pathogens disseminated through food are a public health concern. Although a significant proportion of the urban population in developing countries consume street-vended foods, their role in spreading antibiotic resistance has been rarely investigated. In this study, we evaluated the antibiotic resistance patterns of bacterial isolates (n = 50) collected from five categories of street-vended foods (phuchka, chatpati, sausage, bun, and salad) in Bangladesh. Antibiotic susceptibility to twelve antibiotics was investigated by the Kirby-Bauer disk diffusion method. We found a high prevalence of E. coli (n = 32) in street-vended foods, with most isolates (65.63%) exhibiting multidrug resistance. The multiple antibiotic resistance (MAR) index showed that 22 isolates had MAR above 0.2, with resistance mostly against oxacillin, ampicillin, and cefuroxime. From the rest, three representative isolates were selected for molecular identification by DNA sequencing of 16S rDNA. Klebsiella oxytoca showed multiple drug resistance (MDR) and was resistant to ampicillin, oxacillin, cefuroxime, and kanamycin. Burkholderia fungorum showed no distinct inhibition zone against ampicillin and chloramphenicol. Additionally, the Serratia nematodiphila isolate showed no distinct inhibition zone against three antibiotics, including ampicillin, oxacillin, and cefuroxime. These findings might contribute to the knowledge of emerging antibiotic-resistant foodborne pathogens and raise concerns about the safety of street-vended foods in Bangladesh.
Mechanical loading is required for initiation of extracellular matrix deposition at the developing murine myotendinous junction
Authors: Lipp Sarah N. , Jacobson Kathryn R. , Colling Haley A. , Tuttle Tyler G. , Miles Dalton T. , McCreery Kaitlin P. , Calve Sarah
Published: June 2022

The myotendinous junction (MTJ) contributes to the generation of motion by connecting muscle to tendon. At the adult MTJ, a specialized extracellular matrix (ECM) is thought to contribute to the mechanical integrity of the muscle-tendon interface, but the factors that influence MTJ formation during mammalian development are unclear. Here, we combined 3D imaging and proteomics with murine models in which muscle contractility and patterning are disrupted to resolve morphological and compositional changes in the ECM during MTJ development. We found that MTJ-specific ECM deposition can be initiated via static loading due to growth; however, it required cyclic loading to develop a mature morphology. Furthermore, the MTJ can mature without the tendon terminating into cartilage. Based on these results, we describe a model wherein MTJ development depends on mechanical loading but not insertion into an enthesis.
Oxidative stress drives mutagenesis through transcription coupled repair in bacteria
Authors: Carvajal-Garcia Juan , Samadpour Ariana N. , Hernandez Viera Angel J. , Merrikh Houra
Published: June 2022

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one the main public health problems of the 21stcentury. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics tested and across highly divergent species, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This strongly suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies that a highly conserved pathway drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.
Optimal reliability, robustness and control of nucleus centering in fission yeast is contingent on nonequilibrium force patterning
Authors: Jain Ishutesh , Rao Madan , Tran Phong T.
Published: June 2022

Cells, such as fission yeast, center their division apparatus to ensure symmetric cell division, a challenging task when the governing dynamics is stochastic. Here we show that the spindle pole body (SPB) positioning, which defines the division septum, is controlled by the patterning of nonequilibrium polymerization forces of microtubule (MT) bundles. We define two cellular objectives, reliability, the mean SPB position relative to the geometric center, and robustness, the variance of the SPB position, which are sensitive to genetic perturbations that change cell length, MT-bundle number/orientation and MT dynamics. We show that the optimal control of reliability and robustness required to minimize septum positioning error is achieved by the wild-type (WT). A stochastic model for the MT-based nucleus centering, with parameters measured directly or estimated using Bayesian inference, recapitulates WT optimality. We use this to perform a sensitivity analysis of the parameters that control nuclear centering.
Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections
Authors: Fries Michel , Brown Thomas W. , Jolicoeur Christine , Boudreau-Pinsonneault Camille , Javed Awais , Abram Pénélope , Cayouette Michel
Published: June 2022

The balance of contralateral and ipsilateral retinogeniculate projections is critical for the establishment of binocular vision, but the transcriptional programs regulating this process remain ill-defined. In this study, we show that the Pou class homeobox protein POU3F1 is selectively expressed in developing mouse contralateral retinal ganglion cells (cRGCs). Inactivation of Pou3f1 increases the number of ipsilateral RGCs produced, leading to abnormal ipsilateral to contralateral projection ratio at the optic chiasm, whereas expression of Pou3f1 in retinal progenitors increases the production of cRGCs. Using Cut&Run and RNA sequencing in wildtype and Pou3f1 mouse knockout retinas, we demonstrate that Pou3f1 is necessary for the establishment of a cRGC gene regulatory network through activation and repression of several known contralateral and ipsilateral determinants, respectively. Finally, we report that POU3F1 is sufficient to induce production of RGC-like cells sending projections to the optic nerve, even in late-stage retinal progenitors not normally competent to generate RGCs. This work uncovers POU3F1 as a master regulator of the cRGC transcriptional program and opens new possibilities for optic nerve regenerative therapies.
Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes
Authors: Fan Chengcheng , Cohen Alexander A. , Park Miso , Hung Alfur Fu-Hsin , Keeffe Jennifer R. , Gnanapragasam Priyanthi N.P. , Lee Yu E. , Kakutani Leesa M. , Wu Ziyan , Malecek Kathryn E. , Williams John C. , Bjorkman Pamela J.
Published: June 2022

Protection from SARS-related coronaviruses with spillover potential and SARS-CoV-2 variants could prevent and/or end pandemics. We show that mice immunized with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicit cross-reactive anti-sarbecovirus antibodies against conserved class 1/4 and class 3 RBD epitopes. Monoclonal antibodies (mAbs) identified from initial screening of <10,000 single B-cells secreting IgGs binding two or more sarbecovirus RBDs showed cross-reactive binding and neutralization of SARS-CoV-2 variants and animal sarbecoviruses. Single-particle cryo-EM structures of antibody–spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes and revealed neutralization mechanisms, potentials for intra-spike trimer crosslinking by single IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticles to identify therapeutic pan-sarbecovirus and pan-variant mAbs and to elicit them by vaccination.
Quantitative prediction and measurement of Piezo’s membrane footprint
Authors: Haselwandter Christoph A. , Guo Yusong R. , Fu Ziao , MacKinnon Roderick
Published: June 2022

Piezo proteins are mechanosensitive ion channels that can locally curve the membrane into a dome shape (Y. R. Guo, R. MacKinnon, 2017). The curved shape of the Piezo dome is expected to deform the surrounding lipid bilayer membrane into a membrane footprint, which may serve to amplify Piezo’s sensitivity to applied forces (C. A. Haselwandter, R. MacKinnon, 2018). If Piezo proteins are embedded in lipid bilayer vesicles, the membrane shape deformations induced by the Piezo dome depend on the vesicle size. We employ here membrane elasticity theory to predict, with no free parameters, the shape of such Piezo vesicles outside the Piezo dome, and show that the predicted vesicle shapes agree quantitatively with the corresponding measured vesicle shapes obtained through cryo-electron tomography, for a range of vesicle sizes (Helfrich W. 1973). On this basis, we explore the coupling between Piezo and membrane shape, and demonstrate that the features of the Piezo dome affecting Piezo’s membrane footprint follow approximately a spherical cap geometry. Our work puts into place the foundation for deducing key elastic properties of the Piezo dome from membrane shape measurements and provides a general framework for quantifying how proteins deform bilayer membranes.

Classification

Biophysicss
Structural basis for FLCN RagC GAP activation in TFEB substrate-selective mTORC1 regulation
Authors: Jansen Rachel M. , Peruzzo Roberta , Fromm Simon A. , Yokom Adam L. , Zoncu Roberto , Hurley James H.
Published: June 2022

mTORC1 regulates cell growth and catabolism in response to fluctuations in nutrients through phosphorylation of key substrates. The tumor suppressor FLCN is a RagC GTPase activating protein (GAP) that regulates mTORC1 phosphorylation of TFEB, controlling lysosome biogenesis and autophagy. Here, we determined the cryo-EM structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagAGDP:RagCGDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex (LFC) structure, FLCN reorients by 90°, breaks its contacts with RagA, and makes new contacts with RagC that position its Arg164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity in vitro and led to nuclear retention of TFE3, with no effect on mTORC1 phosphorylation of S6K or 4E-BP1. The structure thus provides a roadmap to discover TFEB-selective mTORC1 antagonists.

One-Sentence Summary

The cryo-EM structure of the active FLCN RagC GAP complex provides a structural basis for TFEB/TFE3 substrate-selective targeting of mTORC1.
Connecting sequence features within the disordered C-terminal linker ofB. subtilisFtsZ to functions and bacterial cell division
Authors: Shinn Min Kyung , Cohan Megan C. , Bullock Jessie L. , Ruff Kiersten M. , Levin Petra A. , Pappu Rohit V.
Published: June 2022

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein, FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT inBacillus subtilisFtsZ (Bs-FtsZ). Molecular simulations show that the CT17 ofBs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical Coupling Analysis of more than 103sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain whereas the CTL is under independent selection. Despite this, we discover the conservation of non-random sequence patterns within CTLs across orthologs. To test how the non-random patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designedBs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysisin vitroand protein degradation, aberrant assembly, and disruption of cell divisionin vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass non-random, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

Significance Statement

Z-ring formation by the protein FtsZ controls cell division in rod-shaped bacteria. The C-terminus of FtsZ encompasses a disordered C-terminal linker (CTL) and a conserved CT17 motif. Both modules are essential for Z-ring formation and proper localization of FtsZ in cells. Previous studies suggested that generic intrinsically disordered regions (IDRs) might be suitable functional replacements for naturally occurring CTLs. Contrary to this suggestion, we find that the sequence-encoded conformational properties of CTLs help mediate autoregulatory interactions between covarying regions within FtsZ. Functional properties of the CTL are encoded via evolutionarily conserved, non-random sequence patterns. Disruption of these patterns impair molecular functions and cellular phenotypes. Our findings have broad implications for discovering functionally consequential sequence features within IDRs of other proteins.
Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via PDH regulation
Authors: Ikizawa Takeshi , Ikeda Kazutaka , Arita Makoto , Kitajima Shojiro , Soga Tomoyoshi , Ichijo Hidenori , Naguro Isao
Published: June 2022

A high-salt diet significantly impacts various diseases, including cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, the PDH phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmo-sensing mechanism. Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of PDH phosphorylation by direct osmosensing within mitochondria.