bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrated single-cell multiomic analysis of HIV latency reversal reveals novel regulators of viral reactivation
Authors: Manickam Ashokkumar , Peterson Jackson J , Harigaya Yuriko , Murdoch David M , Margolis David M , Oesterling Alex , Guo Zhicheng , Rudin Cynthia D , Jiang Yuchao , Browne Edward P
Published: July 2022

Despite the success of antiretroviral therapy, HIV cannot be cured because of a reservoir of latently infected cells that evades therapy. To understand the mechanisms of HIV latency, we employed an integrated single-cell RNA-seq/ATAC-seq approach to simultaneously profile the transcriptomic and epigenomic characteristics of ~4000 latently infected cells after reactivation using three different latency-reversing agents (LRAs). Differentially expressed genes and differentially accessible motifs were used to examine transcriptional pathways and transcription factor (TF) activities across the cell population. We identify cellular transcripts and TFs whose expression/activity was correlated with viral reactivation and demonstrate that a machine learning model trained on these data was 68% accurate at predicting viral reactivation. Finally, we validate the role of a new candidate HIV-regulating factor, GATA3, in the viral response to prostratin stimulation. These data demonstrate the power of integrated multimodal single-cell analysis to uncover novel relationships between host cell factors and HIV latency.
Gluebodies improve crystal reliability and diversity through transferable nanobody mutations that introduce constitutive close contacts
Authors: Ye Mingda , Makola Mpho , Newman Joseph A. , Fairhead Michael , Maclean Elizabeth , Wright Nathan D. , Koekemoer Lizbé , Thompson Andrew , Bezerra Gustavo A. , Yi Gangshun , Li Huanyu , Rangel Victor L. , Mamalis Dimitrios , Aitkenhead Hazel , Davis Benjamin G. , Gilbert Robert J.C. , Duerr Katharina , Gileadi Opher , von Delft Frank
Published: July 2022

The design of proteins that may assemble in a manner that is transferable and modular remains an enduring challenge. In particular, obtaining well-diffracting protein crystals suitable for characterizing ligands or drug candidates and understanding different protein conformations remains a bottleneck for structural studies. Using nanobodies as crystallization chaperones is one strategy to address the problem, but its reliability is uncharacterized and, in this study, we observed it to have a limited success rate. Here we show that by exploring and testing the nanobody-nanobody interfaces predominant in >200 combinations of surface mutations in multiple iterations we can engineer robust crystallization behaviour into the nanobody scaffold. Strikingly, this survey yielded multiple polymorphs, all mediated by the same interface. The resulting ‘Gluebodies’ (Gbs) provide far superior resolution and reliability of diffraction and can be routinely generated for chaperone experiments. We furthermore show that Gbs cannot rescue intrinsically non-crystallizing proteins, but instead are a powerful approach to improve the packing and resolution limit of poorly diffracting crystals. The discovery of an engineered, preferred nanobody interface that arises under kinetic control - trapped here by irreversible crystallization - embodies a protein assembly strategy that could prove even more broadly useful for modular assembly trapped by other irreversible methods.
DREADD activation of the lateral septum alters prosocial and antisocial behaviors, but not partner preferences in male prairie voles
Authors: Sailer Lindsay L. , Park Ashley H. , Galvez Abigail , Ophir Alexander G.
Published: July 2022

Although much has been written on the topic of social behavior, many terms referring to different aspects of social behavior have become inappropriately conflated and the specific mechanisms governing them remains unclear. It is therefore critical that we disentangle the pro- and anti-social elements associated with different forms of social behavior to fully understand the social brain. The lateral septum (LS) mediates social behaviors, emotional processes, and stress responses necessary for individuals to navigate day-to-day social interactions. The LS is particularly important in general and selective prosocial behavior (monogamy) but its role in how these two behavioral domains intersect is unclear. Here, we investigate the effects of chemogenetic-mediated LS activation on social responses in male prairie voles when they are 1) sex-naïve and generally affiliative and 2) after they become pair-bonded and display selective aggression. Amplifying neural activity in the LS augments same-sex social approach behaviors. Despite partner preference formation remaining unaltered, LS activation in pair-bonded males leads to reduced selective aggression while increasing social affiliative behaviors. These results suggest that LS activation alters behavior within certain social contexts, by increasing sex-naïve affiliative behaviors and reducing pair bonding-induced selective aggression with same-sex conspecifics, but not altering bonding with opposite-sex individuals.
Endosome positioning coordinates spatially selective GPCR signaling
Authors: Willette Blair K.A. , Tsvetanova Nikoleta G.
Published: July 2022

G protein-coupled receptors (GPCRs), a class of critical regulators of mammalian physiology, can initiate unique functional responses depending on the subcellular compartment of their activation. Yet, how endosomal receptors transduce location-biased outcomes remains poorly understood. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have largely focused on the biochemical aspect of this regulation through dissection of the relevant factors. Here, we assess the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism coordinating the transduction of spatially biased responses. We focus on the prototypical beta2-adrenergic receptor (β2-AR), which preferentially mediates transcriptional reprogramming via cyclic AMP (cAMP) production from early endosomes. We overcome a technical challenge that has hindered the direct assessment of the role of endosome positioning in this paradigm by devising a strategy to selectively and rapidly redistribute endosomes ‘on command’ in intact cells without perturbing their biochemical composition. Next, we present two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cAMP-dependent transcription with single-cell resolution. We then combine these readouts with rapid endosome relocalization to establish that increasing endosome distance from the nucleus inhibits the initiation of the endosome-dependent response. Lastly, we demonstrate a prominent mechanistic role of phosphodiesterase (PDE)-mediated cAMP hydrolysis in this process. Our study, therefore, illuminates a novel mechanism regulating GPCR function by identifying endosome positioning as a principal mediator of spatially selective receptor signaling.

G protein-coupled receptors (GPCRs) orchestrate essential aspects of mammalian physiology. GPCR function is tightly controlled by endocytic trafficking, where the ligand-activated receptor engages arrestins and clathrin machinery and is subsequently internalized into endosomal compartments1. While the endosome-associated receptor pool was classically presumed to be functionally inactive, it is now clear that receptors can also signal from endosomes2-4. Moreover, endosomal receptors can initiate cellular responses that are distinct from those activated at the plasma membrane. Transcriptional reprogramming was one of the first location-biased GPCR responses to be identified and shown to be stimulated from intracellular receptors5, 6. Since then, compartmentalized signaling has been implicated in the transduction of distinct phosphosignaling7, 8 and in the coordination of unique physiologies and drug actions8-17. Yet, how the endosome selectively facilitates these responses compared to other subcellular compartments remains unclear.
The mammalian forelimb diversity as a morphological gradient of increasing evolutionary versatility
Authors: Rothier Priscila S. , Fabre Anne-Claire , Clavel Julien , Benson Roger , Herrel Anthony
Published: July 2022

Vertebrate limb morphology often reflects the environment, due to variation in locomotor requirements and other ecological traits. However, proximal and distal limb segments may evolve differently to each other, reflecting an anatomical gradient of functional specialization that has been suggested to be impacted by the timing of bone condensation during ontogeny. Here we explore whether the temporal sequence of bone condensation predicts variation in the capacity of evolution to generate morphological diversity between proximal and distal forelimb segments across more than 600 species of mammals. Our findings are consistent with the hypothesis that late developing distal limb elements should display greater morphological variation than more proximal limb elements, which condense earlier during morphogenesis. Distal limb elements, belonging to the autopod, not only exhibit higher diversity of form, but are also more integrated and, on average, show greater evolutionary versatility than intermediate and upper limb segments. Our findings indicate that the macroevolutionary patterns of proximal and distal limb segments are not the same, suggesting that strong functional selection, combined with the higher potential of development to generate variation in more distal limb structures, facilitate the evolution of high autopodial disparity in mammals.
Function and evolution of aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis
Authors: Tsujimoto Hitoshi , Metz Hillery C , Smith Alexis A , Sakamoto Joyce M , Pal Utpal , Rasgon Jason L
Published: July 2022

Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycle, ticks face significant challenges related to water homeostasis. When blood-feeding they must excrete water and ions, but when off-host (for stretches lasting several months) they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. We find that most arachnid AQP-like sequences (including those of I. scapularis) form a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. We next sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. We additionally examined IsAQP1 expression across different life stages and adult female tissues. We found IsAQP1 to be an efficient water channel with salivary gland-specific expression, implying its function may be closely tied to osmoregulation during blood-feeding. Its functional properties were unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance during blood feeding and may hold promise as a target for novel vector control efforts.
Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq ofDrosophilatestes
Authors: Raz Amelie A. , Vida Gabriela S. , Stern Sarah R. , Mahadevaraju Sharvani , Fingerhut Jaclyn M. , Viveiros Jennifer M. , Pal Soumitra , Grey Jasmine R. , Grace Mara R. , Berry Cameron W. , Li Hongjie , Janssens Jasper , Saelens Wouter , Shao Zhantao , Hu Chun , Yamashita Yukiko M. , Przytycka Teresa M. , Oliver Brian , Brill Julie A. , Krause Henry M. , Matunis Erika L. , White-Cooper Helen , DiNardo Stephen , Fuller Margaret T.
Published: July 2022

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis inDrosophilastarting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study (Liet al., 2022). With over 44,000 nuclei and 6,000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers,in situhybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for functionin vivo.
A Model of Hippocampal Replay Driven by Experience and Environmental Structure Facilitates Spatial Learning
Authors: Diekmann Nicolas , Cheng Sen
Published: July 2022

Replay of neuronal sequences in the hippocampus during resting states and sleep play an important role in learning and memory consolidation. Consistent with these functions, replay sequences have been shown to obey current spatial constraints. Nevertheless, replay does not necessarily reflect previous behavior and can construct never-experienced sequences. Here we propose a stochastic replay mechanism that prioritizes experiences based on three variables: 1. Experience strength, 2. experience similarity, and 3. inhibition of return. Using this prioritized replay mechanism to train reinforcement learning agents leads to far better performance than using random replay. Its performance is close to the state-of-the-art, but computationally intensive, algorithm by Mattar & Daw (2018). Importantly, our model reproduces diverse types of replay because of the stochasticity of the replay mechanism and experience-dependent differences between the three variables. In conclusion, a unified replay mechanism generates diverse replay statistics and is efficient in driving spatial learning.
Nicotine-mediated rescue of α-synuclein toxicity requires synaptic vesicle glycoprotein 2
Authors: Olsen Abby L. , Clemens Sabrina G. , Feany Mel B.
Published: July 2022

Background

Parkinson’s disease (PD) is characterized by α-synuclein aggregation and loss of dopamine (DA) neurons in the substantia nigra. Risk of PD arises due to a combination of genetic and environmental factors, which may interact, termed gene-environment (GxE) interactions. An inverse association between smoking and risk of PD is well-established, and a previous genome-wide GxE interaction study identified genetic variation in the synaptic-vesicle glycoprotein 2C (SV2C) locus as an important mediator of the degree to which smoking is inversely associated with PD.

Objective

We sought to determine the mechanism of the smoking-SV2C interaction in a Drosophila model of PD.

Methods

Flies expressing human α-synuclein in all neurons develop the hallmarks of PD, including motor dysfunction, loss of DA neurons, and formation of α-synuclein inclusions. We assessed the effects of increasing doses of nicotine on these parameters of neurodegeneration, in the presence or absence of SV2 knockdown.

Results

α-synuclein-expressing flies treated with nicotine had improvement in locomotion, DA neuron counts, and in α-synuclein aggregation. However, in α-synuclein-expressing flies in which Drosophila orthologs of SV2 were knocked down, nicotine failed to rescue neurodegeneration.

Conclusions

This work confirms a GxE interaction between nicotine and SV2, defines a role for this interaction in α-synuclein proteostasis, and suggests that future clinical trials on nicotine should consider genetic variation in SV2C. Further, this provides proof of concept that our model can be used for mechanistic study of GxE, paving the way for investigation of additional GxE interactions or identification of novel GxE.
Using transfer learning and dimensionality reduction techniques to improve generalisability of machine-learning predictions of mosquito ages from mid-infrared spectra
Authors: Mwanga Emmanuel P. , Siria Doreen J. , Mitton Joshua , Mshani Issa H. , Jimenez Mario Gonzalez , Selvaraj Prashanth , Wynne Klaas , Baldini Francesco , Okumu Fredros O. , Babayan Simon A.
Published: July 2022

Accurate prediction of mosquito population age structures can improve the evaluation of mosquito-targeted interventions since old mosquitoes are more likely to transmit malaria than young ones. Mid-infrared spectroscopy (MIRS) reveals age-associated variation in the biochemical composition of the mosquito cuticle, which can then be used to train machine learning (ML) models to predict mosquito ages. However, these MIRS-ML models are not always generalisable across different mosquito populations. Here, we investigated whether dimensionality reduction applied to the MIRS input data and transfer learning could improve the generalisability of MIRS-ML predictions for mosquito ages. We reared adults of the malaria vector, Anopheles arabiensis, in two insectaries (Ifakara, Tanzania and Glasgow, UK). The heads and thoraces of female mosquitoes of two age classes (1-9 day-olds and 10-17 day-olds) were scanned using an attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrometer (4000 cm-1 to 400 cm-1). The dimensionality of the spectra data was reduced using unsupervised principal component analysis (PCA) or t-distributed stochastic neighbour embedding (t-SNE), and then the spectra were used to train deep learning (DL) and standard machine learning (ML) classifiers. Transfer learning was also evaluated for improving the computational cost of the models when predicting mosquito age classes from new populations. Model accuracies for predicting the age of test mosquitoes from the same insectary as the training samples reached 99% for DL and 92% for ML, but did not generalise to a different insectary, achieving only 46% and 48% for ML for DL, respectively. Dimensionality reduction did not improve the model generalisability between locations but reduced computational time up to 5-fold. However, transfer learning by updating pre-trained models with 2% of mosquitoes from the alternate location brought both DL and standard ML model performance to ~98% accuracy for predicting mosquito age classes in the alternative insectary. Combining dimensionality reduction and transfer learning can reduce computational costs and improve the transferability of both deep learning and standard machine learning models for predicting the age of mosquitoes. Future studies could investigate the optimal quantities and diversity of training data necessary for transfer learning, and implications for broader generalisability to unseen datasets.
Rhizospheric miRNAs affect the plant microbiota
Authors: Middleton Harriet , Dozois Jessica Ann , Monard Cécile , Daburon Virginie , Clostres Emmanuel , Tremblay Julien , Combier Jean-Philippe , Yergeau Étienne , El Amrani Abdelhak
Published: July 2022

Recently, small RNAs have been shown to play important roles in cross-kingdom communication, notably in plant-pathogen relationships. Plant miRNAs were even shown to regulate gene expression in the gut microbiota. But what impact do they have on the plant microbiota? Here we hypothesized that plant miRNAs can be found in the rhizosphere of plants, where they are taken up by rhizosphere bacteria, influencing their gene expression, thereby shaping the rhizosphere bacterial community. We found plant miRNAs in the rhizosphere ofArabidopsis thalianaandBrachypodium distachyon. These plant miRNAs were also found in rhizosphere bacteria, and fluorescent synthetic miRNAs were taken up by cultivated bacteria. A mixture of five plant miRNAs modulated the expression of more than a hundred genes inVariovorax paradoxus, whereas no effect was observed inBacillus mycoides. Similarly, whenV. paradoxuswas grown in the rhizosphere ofArabidopsisthat overexpressed a miRNA, it changed its gene expression profile. The rhizosphere bacterial communities ofArabidopsismutants that were impaired in their miRNA or small RNA pathways differed from wildtype plants. Similarly, bacterial communities ofArabidopsisoverexpressing specific miRNAs diverged from control plants. Finally, the growth and the abundance of specific ASVs of a simplified soil community were affected by exposure to a mixture of synthetic plant miRNAs. Taken together, our results support a paradigm shift in plant-bacteria interactions in the rhizosphere, adding miRNAs to the plant tools shaping microbial assembly.
Dual scRNA-Seq analysis reveals rare and uncommon parasitized cell populations in chronic L. donovani infection
Authors: Karagiannis Konstantinos , Gannavaram Sreenivas , Verma Chaitenya , Bhattacharya Parna , Nakhasi Hira L , Satoskar Abhay
Published: July 2022

Although phagocytic cells are documented targets of Leishmania parasites, it is unclear whether these parasites can infect other cell types. In this study, we describe a computational approach that exploits scRNA-seq to simultaneously analyze the transcriptomic signatures of the host cell and to identify rare and uncommon cells that harbor Leishmania donovani in the spleen and bone marrow. Individual cells were annotated as parasitized based on the presence of L. donovani transcripts that were detected with high accuracy. This unbiased approach allowed identification of heterogenous parasitized cell populations that cannot be detected by conventional methods. Consistent with previous studies, analysis of spleen cells isolated from L. donovani infected mice revealed inflammatory monocytes as the dominant parasitized cells. In addition, megakaryocytes, basophils, and NK cells were found to be the rare cells infected in the spleen. Unexpectedly, hematopoietic stem cells (HSCs), not known to be phagocytic, were the dominant cells parasitized cell in the bone marrow. In addition, eosinophils, megakaryocytes, and basal cells were the rare bone marrow cells found to be infected. scRNA-seq analysis revealed known phagocytic receptors FcγR and CD93 are expressed on HSCs. In vitro studies using purified HSCs showed that these cells can phagocytize L. donovani. Parasitized HSCs were also detectable in the bone marrow of mouse infected with L donovani.. This unbiased dual scRNA-seq approach enables identification of rare and uncommon parasitized cells that could be involved in pathogenesis, persistence, and protective immunity. Further, such approach could be used to study pathogenesis of other infectious agents.
Protein complexes in cells by AI-assisted structural proteomics
Authors: O‘Reilly Francis J. , Graziadei Andrea , Forbrig Christian , Bremenkamp Rica , Charles Kristine , Lenz Swantje , Elfmann Christoph , Fischer Lutz , Stülke Jörg , Rappsilber Juri
Published: July 2022

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enables a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry, cofractionation mass spectrometry (CoFrac-MS) to identify protein-protein interactions in the model Gram-positive bacteriumBacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in theSubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interface, and is applicable to genetically intractable organisms, including pathogenic bacteria.
Dynamic states of cervical epithelia during pregnancy and epithelial barrier disruption
Authors: Cooley Anne , Madhukaran ShanmugaPriyaa , Stroebele Elizabeth , Caraballo Mariano Colon , Wang Lei , Hon Gary C. , Mahendroo Mala
Published: July 2022

The cervical epithelium undergoes continuous changes in proliferation, differentiation, and function that are critical before pregnancy to ensure fertility and during pregnancy to provide a physical and immunoprotective barrier for pregnancy maintenance. Barrier disruption can lead to the ascension of pathogens that elicit inflammatory responses and preterm birth. Here, we identify cervical epithelial subtypes in nonpregnant, pregnant, and in-labor mice using single-cell transcriptome and spatial analysis. We identify heterogeneous subpopulations of epithelia displaying spatial and temporal specificity. Notably, two goblet cell subtypes with distinct transcriptional programs and mucosal networks were dominant in pregnancy. Untimely basal cell proliferation and goblet cells with diminished mucosal integrity characterize barrier dysfunction in mice lacking hyaluronan. These data demonstrate how the cervical epithelium undergoes continuous remodeling to maintain dynamic states of homeostasis in pregnancy and labor, and provide a framework to understand perturbations in epithelial health and host-microbe interactions that increase the risk of premature birth.
Shifted Reverse PAGE: a novel approach based on structure switching for the discovery of riboswitches and aptamers
Authors: Devinck Aurélie , Boutet Emilie , Ouellet Jonathan , Rouag Rihab , Sellamuthu Balasubramanian , Perreault Jonathan
Published: July 2022

Riboswitches are regulatory sequences composed of an aptamer domain capable of binding a ligand and an expression platform that allows the control of the downstream gene expression based on a conformational change. Current bioinformatic methods for their discovery have various limitations. To circumvent this, we developed an experimental technique to discover new riboswitches called SR-PAGE (Shifted Reverse Polyacrylamide Gel Electrophoresis). A ligand-based regulatory molecule is recognized by exploiting the conformational change of the sequence following binding with the ligand within a native polyacrylamide gel. Known riboswitches were tested with their corresponding ligands to validate our method. SR-PAGE was imbricated within an SELEX to enrich switching RNAs from a TPP riboswitch-based degenerate library to change its binding preference from TPP to thiamine. The SR-PAGE technique allows performing a large screening for riboswitches, search in several organisms and test more than one ligand simultaneously.
Maf Family Transcription Factors are Required for Nutrient Uptake in the Neonatal Gut
Authors: Bara Anne M. , Chen Lei , Ma Celina , Underwood Julie , Moreci Rebecca S. , Sumigray Kaelyn , Sun Tongyu , Diao Yarui , Verzi Michael , Lechler Terry
Published: July 2022

There are fundamental differences in the way that neonatal and adult intestines absorb nutrients. In adults, macromolecules are efficiently broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely more on bulk intake of nutrients and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors, MafB and cMaf, as markers of terminally-differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4α/γ, master regulators of the enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of bulk uptake of nutrients. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number in response to loss of Mafs. Finally, we show that deletion of Blimp1, which represses adult enterocyte genes in the neonatal gut, shows highly overlapping changes in gene expression and similar defects in nutrient uptake. This work defines transcriptional regulators that are necessary for bulk uptake in neonatal enterocytes.
Marine viral particles reveal an expansive repertoire of phage-parasitizing mobile elements
Authors: Eppley John M. , Biller Steven J. , Luo Elaine , Burger Andrew , DeLong Edward F.
Published: July 2022

Phage satellites are mobile genetic elements that propagate by parasitizing bacteriophage replication. We report here the discovery of abundant and diverse phage satellites that were packaged as concatemeric repeats within naturally occurring bacteriophage particles in seawater. These same phage-parasitizing mobile elements were found integrated in the genomes of dominant co-occurring bacterioplankton species. Like known phage satellites, many of the marine phage satellites encoded genes for integration, DNA replication, phage interference, and capsid assembly. Many also contained distinctive gene suites indicative of unique virus hijacking, phage interference and mobilization mechanisms. Marine phage satellite sequences were widespread in local and global oceanic virioplankton populations, reflecting their ubiquity, abundance, and temporal persistence in marine planktonic communities worldwide. Their gene content and putative life cycles suggest they may impact host-cell phage immunity and defense, lateral gene transfer, and bacteriophage-induced cell mortality and host and virus productivity. These previously unrecognized marine phage satellites therefore have potential to impact the ecology and evolution of bacteria and their bacteriophages in the ocean, and similar phage parasites likely thrive in many other microbial habitats as well.

Significance statement

Phage satellites are mobile genetic elements that parasitize bacteriophage, thereby exerting profound biological and ecological impacts. To date however, phage satellites have been found primarily in Gram-positive cocci and a few Gram-negative bacteria, many of which are human pathogens. Direct inspection of “wild” marine virus particles however, revealed that phage satellites are widely distributed in the sea, and that their genetic diversity, gene repertoires, and host ranges are much greater than previously supposed. Our analyses provide insight into their parasitic life cycles, potential satellite-helper-phage interactions, and reproductive strategies of these newly recognized phage-parasitizing mobile elements. Their properties, diversity and environmental distributions suggest they exert pervasive influence on marine plankton ecology and bacterial and virus evolution in the sea.
A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals
Authors: Martynowycz Michael W. , Shiriaeva Anna , Clabbers Max T. B. , Nicolas William J. , Weaver Sara J. , Hattne Johan , Gonen Tamir
Published: July 2022

Crystallization of membrane proteins, such as G protein-coupled receptors (GPCRs), is challenging and frequently requires the use of lipidic cubic phase (LCP) crystallization methods. These typically yield crystals that are too small for synchrotron X-ray crystallography, but ideally suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, the viscous nature of LCP makes sample preparation challenging. The LCP layer is often too thick for transmission electron microscopy (TEM), and crystals buried in LCP cannot be identified topologically using a focused ion-beam and scanning electron microscope (FIB/SEM). Therefore, the LCP needs to either be converted to the sponge phase or entirely removed from the path of the ion-beam to allow identification and milling of these crystals. Unfortunately, conversion of the LCP to sponge phase can also deteriorate the sample. Methods that avoid LCP conversion are needed. Here, we employ a novel approach using an integrated fluorescence light microscope (iFLM) inside of a FIB/SEM to identify fluorescently labelled crystals embedded deep in a thick LCP layer. The crystals are then targeted using fluorescence microscopy and unconverted LCP is removed directly using a plasma focused ion beam (pFIB). To assess the optimal ion source to prepare biological lamellae, we first characterized the four available gas sources on standard crystals of the serine protease, proteinase K. However, lamellae prepared using either argon and xenon produced the highest quality data and structures. Fluorescently labelled crystals of the human adenosine receptor embedded in thick LCP were placed directly onto EM grids without conversion to the sponge phase. Buried microcrystals were identified using iFLM, and deep lamellae were created using the xenon beam. Continuous rotation MicroED data were collected from the exposed crystalline lamella and the structure was determined using a single crystal. This study outlines a robust approach to identifying and milling LCP grown membrane protein crystals for MicroED using single microcrystals, and demonstrates plasma ion-beam milling as a powerful tool for preparing biological lamellae.
Investigating Predictive Coding in Younger and Older Children Using MEG and a Multi-Feature Auditory Oddball Paradigm
Authors: Rapaport Hannah , Seymour Robert A. , Benikos Nicholas , He Wei , Pellicano Elizabeth , Sowman Paul F.
Published: July 2022

There is mounting evidence for predictive coding theory from computational, neuroimaging, and psychological research. However there remains a lack of research exploring how predictive brain function develops across childhood. To address this gap, we used paediatric magnetoencephalography (MEG) to record the evoked magnetic fields of 18 younger children (M = 4.1 years) and 19 older children (M = 6.2 years) as they listened to a 12-minute auditory oddball paradigm. For each child, we computed a mismatch field ‘MMF’: an electrophysiological component that is widely interpreted as a neural signature of predictive coding. Consistent with our hypotheses, the older children showed significantly larger MMF amplitudes relative to the younger children. Furthermore, the older children showed a significantly larger MMF amplitude in the right inferior frontal gyrus (IFG; 0.312 to 0.33 s) relative to the younger children, p < .05. These findings support the idea that predictive brain function develops during childhood, with increasing involvement of the frontal cortex in response to prediction errors. These findings contribute to a deeper understanding of the brain function underpinning child cognitive development.

Highlights

This is the first paediatric MEG study to examine the sources underlying the MMF.

Older children showed larger MMF amplitudes in the right inferior frontal gyrus.

Results support the idea that predictive brain function develops during childhood.
Transcriptomic classification of pituitary neuroendocrine tumors causing acromegaly
Authors: Rymuza Julia , Kober Paulina , Rusetska Natalia , Mossakowska Beata J. , Maksymowicz Maria , Nyc Aleksandra , Baluszek Szymon , Zieliński Grzegorz , Kunicki Jacek , Bujko Mateusz
Published: July 2022

Acromegaly results from growth hormone hypersecretion caused by somatotroph pituitary neuroendocrine tumor (PitNET). Our molecular profiling revealed that acromegaly-causing tumors form three distinct transcriptomic subgroups with different histological/clinical features. Transcriptomic subtypes of somatotroph tumors differ in the expression levels of numerous genes including those involved in hormone secretion and genes with known prognostic value. They can be distinguished by determining the expression of marker genes. Transcriptomic group 1 includes ∼20% of acromegaly patients with GNAS mutations-negative, mainly densely granulated tumors with NR5A1 (SF-1) and GIPR co-expression. Group 2 tumors are the most common (46%) and include mainly GNAS-mutated, densely granulated somatotroph and mixed PitNETs. They have significantly smaller size and express favorable prognosis-related genes. Group 3 includes predominantly sparsely granulated somatotroph PitNETs with low GNAS mutations frequency causing ∼35% of acromegaly cases. Ghrelin signaling is implied in their pathogenic mechanism, they have unfavorable gene expression profile, and invasive growth rate. Since a subgroup of somatotroph tumors have high NR5A1 expression, using SF-1 as classification marker specific to gonadotroph PitNETs could be reconsidered.
Bacterial cell widening alters periplasmic size and activates envelope stress responses
Authors: Zietek Matylda , Miguel Amanda , Khusainov Iskander , Shi Handuo , Asmar Abir T , Ram Sri , Wartel Morgane , Sueki Anna , Schorb Martin , Goulian Mark , Collet Jean-François , Beck Martin , Huang Kerwyn Casey , Typas Athanasios
Published: July 2022

The Rcs signal transduction system is a phosphorelay responsible for sensing a wide variety of enterobacterial cell envelope stresses. In Escherichia coli, the Rcs system is required to survive A22 and mecillinam treatment, two drugs that perturb cell size. To test whether cell size changes might be correlated with envelope damage and thereby sensed by the Rcs system, we tuned E. coli cell size via drug inhibition with A22, point mutations to the cell-shape determinant MreB, and mechanically confined growth. In all conditions, cell width was strongly correlated with Rcs activation, with wider cells exhibiting more activation than wild-type. In all conditions, RcsF, the outer membrane-localized upstream component of the Rcs system, was essential for responding to cell width changes. Consistently, several envelope gene deletions known to induce the Rcs system via RcsF resulted in cells that were wider than wild-type. Cryo- electron microscopy revealed that the periplasm of a wide MreB mutant was on average ∼3 nm thinner than wild-type, thereby bringing RcsF closer to the downstream components of the signaling cascade in the inner membrane. Conversely, extending the flexible linker region of RcsF by ∼3 nm increased Rcs activity in wild-type cells. In summary, we propose that the Rcs system responds to changes in cell width because of altered periplasmic thickness.
Modulation of bacterial cell size and growth rate via activation of a cell envelope stress response
Authors: Miguel Amanda , Zietek Matylda , Shi Handuo , Sueki Anna , Maier Lisa , Verheul Jolanda , Blaauwen Tanneke den , Valen David Van , Typas Athanasios , Huang Kerwyn Casey
Published: July 2022

Fluctuating conditions and diverse stresses are typical in natural environments. In response, cells mount complex responses across multiple scales, including adjusting their shape to withstand stress. In enterobacteria, the Rcs phosphorelay is activated by cell envelope damage and by changes to periplasmic dimensions and cell width. Here, we investigated the physiological and morphological consequences of Rcs activation in Escherichia coli in the absence of stresses, using an inducible version of RcsF that mislocalizes to the inner membrane, RcsFIM. Expression of RcsFIM immediately reduced cellular growth rate and the added length per cell cycle in a manner that was directly dependent on induction levels, but independent of Rcs-induced capsule production. At the same time, cells increased intracellular concentration of the cell division protein FtsZ, and decreased the distance between division rings in filamentous cells. Depletion of the Rcs negative regulator IgaA phenocopied RcsFIM induction, indicating that IgaA is essential due to growth inhibition in its absence. However, A22 treatment did not affect growth rate or FtsZ intracellular concentration, despite activating the Rcs system. These findings suggest that the effect of Rcs activation on FtsZ levels is mediated indirectly through growth-rate changes, and highlight feedbacks among the Rcs stress response, growth dynamics, and cell-size control.
Age-related seroprevalence trajectories of seasonal coronaviruses in children
Authors: Luo Yasha , Lv Huibin , Zhao Shilin , Sun Yuanxin , Liu Chengyi , Chen Chunke , Liang Weiwen , Kwok Kin-on , Teo Qi Wen , So Ray TY , Lin Yihan , Deng Yuhong , Li Biyun , Dai Zixi , Zhu Jie , Zhang Dengwei , Fernando Julia , Wu Nicholas C , Tun Hein M. , Bruzzone Roberto , Mok Chris KP , Mu Xiaoping
Published: July 2022

Four seasonal coronaviruses, including HCoV-NL63 and HCoV-229E, HCoV-OC43 and HCoV-HKU1 cause approximately 15–30% of common colds in adults. However, the frequency and timing of early infection with four seasonal coronaviruses in the infant are still not well studied. Here, we evaluated the serological response to four seasonal coronaviruses in 1886 children under 18-year-old to construct the viral infection rates. The antibody levels were also determined from the plasma samples of 485 pairs postpartum women and their newborn babies. This passive immunity waned at one year after birth and the resurgence of the IgGs were found thereafter with the increase of the age. Taken together, our results show the age-related seroprevalence trajectories of seasonal coronaviruses in children and provide useful information for deciding vaccine strategy for coronaviruses in the future.
Proteins with multiple G protein-coupled receptor domains
Authors: Isildayancan Kilic , Kessel Amit , Solan Ron , Kolodny Rachel , Ben-Tal Nir
Published: July 2022

Currently known G protein-coupled receptors (GPCRs) have a single transmembrane domain. Many GPCRs form dimers that have two transmembrane domains (one per protein), and there are indications that this dimeric interaction is functionally meaningful. Here, based on sequence analysis and structure predictions, we report the existence of 57 proteins with two, three, or four GPCR domains within the same protein chain. We analyze the structures of these multi-GPCRs and show that almost all have DRY/NPxxY motifs, a strong indication of signaling activity. By homology, most of the multi-GPCRs that we identified are olfactory-related; a few are chemokine-related. Multi-GPCR candidates are found in various Chordata species including fish, camel, marmite, Chinese hamster, and new world monkeys. The discovery of receptors with multiple transmembrane domains suggests the possibility for signal regulation and amplification within an individual receptor, revealing another step in GPCR evolution and a new layer of complexity in signal transduction.
Honokiol inhibits SARS-CoV-2 replication in cell culture
Authors: Salgado-Benvindo Clarisse , Leijs Anouk A. , Thaler Melissa , Tas Ali , Arbiser Jack L. , Snijder Eric J. , van Hemert Martijn J.
Published: July 2022

SARS-CoV-2 emerged in 2019 and since its global spread has caused the death of over 6 million people. There are currently few antiviral options for treatment of COVID-19. Repurposing of known drugs can be a fast route to obtain molecules that inhibit viral infection and/or modulate pathogenic host responses. Honokiol is a small molecule fromMagnoliatrees, for which several biological effects have been reported,, including anticancer and anti-inflammatory activity. Honokiol has also been shown to inhibit several viruses in cell culture. In this study, we show that honokiol protected Vero E6 cells from SARS-CoV-2-mediated cytopathic effect with an EC50 of 7.8 µM. In viral load reduction assays we observed that honokiol decreased viral RNA copies as well as viral infectious progeny titers. The compound also inhibited SARS-CoV-2 replication in the more relevant A549 cells, expressing ACE2 and TMPRSS2. A time-of-addition assay showed that honokiol inhibited virus replication even when added post infection, suggesting it acts at a post-entry step of the replication cycle. Honokiol was also effective against more recent variants of SARS-CoV-2, including omicron and it inhibited other human coronaviruses as well. Our study suggests that honokiol is an interesting molecule to evaluate in animal studies and clinical trials to investigate its effect on virus replication and pathogenic (inflammatory) host responses.
The Insulin receptor and Insulin like growth factor receptor 5’ UTRs support translation initiation independently of eIF4G1
Authors: Clark Nicholas K. , Harris Meghan T. , Marr Michael T.
Published: July 2022

Protein synthesis is tightly regulated under stress conditions where energy may be scarce. Despite global repression of translation, some transcripts remain actively translated in order for the cell to be able to respond to the insult or prepare to quickly return normal cellular function after the stress ends. For the insulin receptor (Insr) and insulin-like growth factor receptor (Igf1r) transcripts this translation is mediated by an internal ribosome entry site (IRES) in their 5’UTRs that functions independently of eukaryotic initiation factor 4A (eIF4A) and eIF4E. Here we show that these cellular IRESes are also able to promote translation independently of the scaffolding protein eIF4G1 both in vitro and in the cell.

Background

IRES mediated translation initiation requires a different repertoire of factors than canonical cap-dependent translation.

Results

Treatments that inhibit the canonical translation factor eIF4G1 have little or no effect on the ability of the Insr and Igf1r cellular IRESes to promote translation.

Conclusion

Transcripts for two cellular receptors contain RNA elements that facilitate translation initiation without intact eIF4G1

Significance

Cellular IRES mechanisms may resemble viral type III IRESes allowing them to promote translate with a limited number of initiation factors allowing them to work under stress conditions when canonical translation is repressed.
Risk of BA.5 infection in individuals exposed to prior SARS-CoV-2 variants
Authors: Malato João , Ribeiro Ruy M. , Leite Pedro Pinto , Casaca Pedro , Fernandes Eugénia , Antunes Carlos , Fonseca Válter R. , Gomes Manuel Carmo , Graca Luis
Published: July 2022

The SARS-CoV-2 omicron BA.5 subvariant is progressively displacing earlier subvariants, BA.1 and BA.2, in many countries. One possible explanation is the ability of BA.5 to evade immune responses elicited by prior BA.1 and BA.2 infections. The impact of BA.1 infection on the risk of reinfection with BA.5 is a critical issue because adapted vaccines under current clinical development are based on BA.1.

We used the national Portuguese COVID-19 registry to analyze the risk of BA.5 infection in individuals without a documented infection or previously infected during periods of distinct variants’ predominance (Wuhan-Hu-1, alpha, delta, BA.1/BA.2). National predominance periods were established according to the national SARS-CoV-2 genetic surveillance data (when one variant represented >90% of the sample isolates).

We found that prior SARS-CoV-2 infection reduced the risk for BA.5 infection. The protection effectiveness, related to the uninfected group, for a first infection with Wuhan-Hu-1 was 52.9% (95% CI, 51.9 – 53.9%), for Alpha 54.9% (51.2 – 58.3%), for Delta 62.3% (61.4 – 63.3%), and for BA.1/BA.2 80.0% (79.7 – 80.2%).

The results ought to be interpreted in the context of breakthrough infections within a population with a very high vaccine coverage (>98% of the study population completed the primary vaccination series).

In conclusion, infection with BA.1/BA.2 reduces the risk for breakthrough infections with BA.5 in a highly vaccinated population. This finding is critical to appraise the current epidemiological situation and the development of adapted vaccines.