bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimates of persistent inward currents in lower limb muscles are not different between inactive, resistance-trained and endurance-trained young individuals
Authors: Goreau Valentin , Hug François , Jannou Anthony , Dernoncourt François , Crouzier Marion , Cattagni Thomas
Published: July 2023

Persistent inward currents (PICs) increase the intrinsic excitability of α-motoneurons. The main objective of this study was to determine whether estimates of α-motoneuronal PIC magnitude is influenced by chronic endurance and resistance training. We also aimed to investigate whether there is a relationship in the estimates of α-motoneuronal PIC magnitude between muscles. Estimates of PIC magnitude were obtained in three groups of young individuals: resistance-trained (n=12), endurance-trained (n=12), and inactive (n=13). We recorded high-density surface electromyography (HDsEMG) signals from tibialis anterior, gastrocnemius medialis, soleus, vastus medialis, and vastus lateralis. Then, signals were decomposed with convolutive blind source separation to identify motor units spike trains. Participants performed triangular isometric contractions to a peak of 20% of their maximum voluntary contraction. A paired-motor-unit analysis was used to calculate ΔF, which is assumed to be proportional to PIC magnitude. Despite the substantial differences in physical training experience between groups, we found no differences in ΔF, regardless of the muscle. Significant correlations of estimates of PICs magnitude were found between muscles of the same group (VL-VM, SOL-GM). Only one correlation (out of 8) between muscles of different groups was found (GM and TA). Overall, our findings suggest that estimates of PIC magnitude in the lower limb muscles are not influenced by physical training experience in healthy young individuals. They also suggest muscle-specific and muscle group-specific regulations of the diffuse monoamine inputs.
Geographic population structure and distinct population dynamics of globally abundant freshwater bacteria
Authors: Hoetzinger M. , Hahn M.W. , Andersson L.Y. , Buckley N. , Ramsin C. , Buck M. , Nuy J.K. , Garcia S.L. , Puente-Sánchez F. , Bertilsson S.
Published: July 2023

Geographic separation is a principal factor for structuring populations of macroorganisms, with important consequences for evolution, by means of processes such as allopatric speciation. For free-living prokaryotes, implications of geographic separation on their evolution are more unclear. The limited phylogenetic resolution of commonly used markers such as 16S rRNA gene sequences have since long impeded prokaryotic population genetics. However, the vast amount of metagenome sequencing data generated during the last decades from various habitats around the world, now provides an excellent opportunity for such investigations. Here we exploited publicly available and new freshwater metagenomes in combination with genomes of abundant freshwater bacteria to study the impact of geographic separation on population structure. We focused on species that were detected across broad geographic ranges at high enough sequence coverage for meaningful population genomic analyses, i.e. members of the predominant freshwater taxa acI, LD12,PolynucleobacterandCa. Methylopumilus. Population differentiation increased significantly with spatial distance in all species, but notable dispersal barriers (e.g. oceanic) were not apparent. Yet, the different species showed contrasting rates of geographic divergence and strikingly different population dynamics in time series within individual lakes. While certain populations hardly diverged over several years, others displayed high divergence after merely a few months, similar in scale to populations separated by thousands of kilometers. We speculate that populations with higher strain diversity evolve more monotonously, while low strain diversity enables more drastic clonal expansion of genotypes which will be reflected in strong but transient differentiation between temporally or spatially adjacent populations.
Bidirectional Regulation of Motor Circuits Using Magnetogenetic Gene Therapy
Authors: Unda Santiago R. , Pomeranz Lisa E. , Marongiu Roberta , Yu Xiaofei , Kelly Leah , Hassanzadeh Gholamreza , Molina Henrik , Vaisey George , Wang Putianqi , Dyke Jonathan P. , Fung Edward K. , Grosenick Logan , Zirkel Rick , Antoniazzi Aldana M. , Norman Sofya , Liston Conor M. , Schaffer Chris , Nishimura Nozomi , Stanley Sarah A. , Friedman Jeffrey M. , Kaplitt Michael G.
Published: July 2023

Here we report a novel suite of magnetogenetic tools, based on a single anti-ferritin nanobody-TRPV1 receptor fusion protein, which regulated neuronal activity when exposed to magnetic fields. AAV-mediated delivery of a floxed nanobody-TRPV1 into the striatum of adenosine 2a receptor-cre driver mice resulted in motor freezing when placed in an MRI or adjacent to a transcranial magnetic stimulation (TMS) device. Functional imaging and fiber photometry both confirmed activation of the target region in response to the magnetic fields. Expression of the same construct in the striatum of wild-type mice along with a second injection of an AAVretro expressing cre into the globus pallidus led to similar circuit specificity and motor responses. Finally, a mutation was generated to gate chloride and inhibit neuronal activity. Expression of this variant in subthalamic nucleus in PitX2-cre parkinsonian mice resulted in reduced local c-fos expression and motor rotational behavior. These data demonstrate that magnetogenetic constructs can bidirectionally regulate activity of specific neuronal circuits non-invasivelyin-vivousing clinically available devices.

Teaser

A novel magnetogenetics toolbox to regulate neural circuitsin-vivo.
Genome size and repeat content contribute to a complex architecture of flowering time inAmaranthus tuberculatus
Authors: Kreiner Julia M. , Hnatovska Solomiya , Stinchcombe John R. , Wright Stephen I.
Published: July 2023

Genome size variation, largely driven by repeat content, is poorly understood within and among populations, limiting our understanding of its significance for adaptation. Here we characterize intraspecific variation in genome size and repeat content across 186 individuals ofAmaranthus tuberculatus, a ubiquitous native weed that shows flowering time adaptation to climate across its range and in response to agriculture. K-mer based genome size estimates vary by up to 20% across individuals, with transposable elements, unknown repeats, and rDNAs being the primary contributors to this variability. The additive effect of this variation has important phenotypic consequences—individuals with more repeats, and thus larger genomes, show slower flowering times and growth rates. Compared to newly-characterized gene copy number and polygenic nucleotide changes underlying variation in flowering time, we show that genome size remains a modest but significant contributor to the genetic basis of flowering time. Differences in flowering time across sexes and habitats are not mirrored by genome size variation, but rather polygenic variation and a gene copy number variant within the ATP synthesis pathway. Repeat content nonetheless shows non-neutral distributions across the genome, and across latitudinal and environmental gradients, reflecting numerous governing processes that in turn influence quantitative genetic variation for phenotypes key to plant adaptation.

Author Summary

The remarkable and seemingly inconsequential variation in genome size across species has long been an enigma in evolutionary biology. Calling this viewpoint into question, correlations between genome size variation and traits linked to fitness are increasingly uncovered. While this suggests that DNA content itself may be a source of adaptive genetic variation, repeat elements that propagate at the cost of the host are known to largely mediate this variation and may thus limit adaptive potential. Here we look to disentangle these multi-level dynamics, characterizing repeat dynamics across the genome and among individuals across diverse collections of a widespread agricultural weed, linking repeat content to genome size variation, and characterizing the relative importance of its phenotypic consequences. InAmaranthus tuberculatus, we find non-neutral repeat distributions across individuals across the range, and while this repeat variation underlies both variation in genome size and flowering time, we show that it makes a relatively minor contribution to variation in a fitness-related trait across the landscape relative to monogenic and polygenic features. Together, this work broadens our perspective on the complex selective dynamics that govern intraspecific variation in genome size and traits key to fitness in plants.
Interchromosomal contacts between regulatory regions trigger stable transgenerational epigenetic inheritance in Drosophila
Authors: Fitz-James Maximilian H. , Sabarís Gonzalo , Sarkies Peter , Bantignies Frédéric , Cavalli Giacomo
Published: July 2023

Non-genetic information can be inherited across generations by the process of Transgenerational Epigenetic Inheritance (TEI). TEI can be established by various triggering events, including transient genetic perturbations. InDrosophila, hemizygosity of theFab-7regulatory element triggers inheritance of the histone mark H3K27me3 at a homologous locus on another chromosome, resulting in heritable epigenetic differences in eye colour. By mutating transcription factor binding sites within theFab-7element, we demonstrate the importance of two proteins in the establishment and maintenance of TEI: GAGA-factor and Pleiohomeotic. We show that these proteins function by recruiting the Polycomb Repressive Complex 2 and by mediating interchromosomal chromatin contacts betweenFab-7and its homologous locus. Finally, using anin vivosynthetic biology system to induce them, we show that chromatin contacts alone can establish TEI, providing a mechanism by which hemizygosity of one locus can establish epigenetic memory at anotherin transthrough long-distance chromatin contacts.
Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism
Authors: Lund Philip M. , Kristensen Kasper , Larsen Nanna W. , Knuhtsen Astrid , Hansen Morten B. , Hjørringgaard Claudia U. , Eriksen Anne Z. , Urquhart Andrew J. , Mortensen Kim I. , Simonsen Jens B. , Andresen Thomas L. , Larsen Jannik B.
Published: July 2023

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Effortsin vitroandin vivoinvestigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it’s membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.

Graphical Abstract

Highlights

- Lipidating the therapeutic peptide salmon calcitonin alters its biophysical characteristics, including oligomer size, hydrophobicity and membrane activity.

- Fluorescent microscopy of single GUVs enables the determination of peptide mediated reporter dye influx behavior as either graded or All-or-None, which is coupled to either smaller membrane perturbations or peptide pore formation.

- Modulating the number of hydrocarbons constituting the lipidation moieties determines the membrane permeation mechanism.

- By increasing the lipid chain length lipidated of salmon calcitonin goes from displaying smaller membrane perturbations to a peptide pore formation mechanism.

- Effective membrane translocation of lipidated salmon calcitonin requires a peptide mediated pore forming mechanism.
Regression and Alignment for Functional Data and Network Topology
Authors: Tu Danni , Wrobel Julia , Satterthwaite Theodore D , Goldsmith Jeff , Gur Ruben C , Gur Raquel E , Gertheiss Jan , Bassett Dani S , Shinohara Russell T
Published: July 2023

In the brain, functional connections form a network whose topological organization can be described by graph-theoretic network diagnostics. These include characterizations of the community structure, such as modularity and participation coefficient, which have been shown to change over the course of childhood and adolescence. To investigate if such changes in the functional network are associated with changes in cognitive performance during development, network studies often rely on an arbitrary choice of pre-processing parameters, in particular the proportional threshold of network edges. Because the choice of parameter can impact the value of the network diagnostic, and therefore downstream conclusions, we propose to circumvent that choice by conceptualizing the network diagnostic as a function of the parameter. As opposed to a single value, a network diagnostic curve describes the connectome topology at multiple scales--from the sparsest group of the strongest edges to the entire edge set. To relate these curves to executive function and other covariates, we use scalar-on-function regression, which is more flexible than previous functional data-based models used in network neuroscience. We then consider how systematic differences between networks can manifest in misalignment of diagnostic curves, and consequently propose a supervised curve alignment method that incorporates auxiliary information from other variables. Our algorithm performs both functional regression and alignment via an iterative, penalized, and nonlinear likelihood optimization. The illustrated method has the potential to improve the interpretability and generalizability of neuroscience studies where the goal is to study heterogeneity among a mixture of function- and scalar-valued measures.
A Generalized Framework for the Calcium Control Hypothesis Describes Weight-Dependent Synaptic Plasticity
Authors: Moldwin Toviah , Azran Li Shay , Segev Idan
Published: July 2023

The brain modifies synaptic strengths to store new information via long-term potentiation (LTP) and long-term depression (LTD). Evidence has mounted that long-term plasticity is controlled via concentrations of calcium ([Ca2+]) in postsynaptic spines. Several mathematical models describe this phenomenon, including those of Shouval, Bear, and Cooper (SBC) (Shouval et al., 2002, 2010) and Graupner and Brunel (GB)(Graupner & Brunel, 2012). Here we suggest a generalized version of the SBC and GB models, based on a fixed point – learning rate (FPLR) framework, where the synaptic [Ca2+] specifies a fixed point toward which the synaptic weight approaches asymptotically at a [Ca2+]-dependent rate. The FPLR framework offers a straightforward phenomenological interpretation of calcium-based plasticity:the calcium concentration tells the synaptic weight where it is going and how fast it goes there. The FPLR framework can flexibly incorporate various experimental findings, including the existence of multiple regions of [Ca2+] where no plasticity occurs, or plasticity in cerebellar Purkinje cells, where the directionality of calcium-based synaptic changes is thought to be reversed relative to cortical and hippocampal neurons. We also suggest a modeling approach that captures the dependency of late-phase plasticity stabilization on protein synthesis. We demonstrate that due to the asymptotic, saturating nature of synaptic changes in the FPLR rule, the result of frequency- and spike-timing-dependent plasticity protocols are weight-dependent. Finally, we show how the FPLR framework can explain plateau potential-induced place field formation in hippocampal CA1 neurons, also known as behavioral time scale plasticity (BTSP).
EASYSTIFF®, a portable and innovative device able to separately analyze each skin compartment for the evaluation of mechanical properties
Authors: Runel Gael , Lapart Jean-André , Chlasta Julien
Published: July 2023

Human skin is submitted to various factors leading to either extrinsic or intrinsic aging. In addition to inducing molecular and morphological changes, skin aging is characterized by modifications in skin biomechanical properties modifications. These modifications can be used as biomarkers to both evaluate the degree of advancement in skin aging but also to detect potential pathologies. Hence, it is from critical interest to be able to evaluate skin biomechanical properties to fully detect and monitor these alterations. Here we introduce a portable and innovative®device, EASYSTIFF®, which is thought toin vivomeasure stiffness properties of skin. Based on indentation principle, EASYSTIFF allows to identify both global stiffness of skin (all confounded cutaneous compartment) or compartmentalized stiffness, in which stiffness values of each compartment, i.e., the stratum corneum, the epidermis, the dermis and the hypodermis, are separately analyzed. Thus, EASYSTIFF is a complementary tool to 2D and 3Din/ex vivomodel and an alternative to existing devices that measure skin mechanical properties, for assessing the effectiveness of cosmetic products, monitoring skin stiffness over time and to provide better understanding in the evolution of skin mechanical properties.
Interoceptive signals shape the earliest markers and neural pathway to awareness at the visual threshold
Authors: Leupin Viviana , Britz Juliane
Published: July 2023

Variations in interoceptive signals from the baroreceptors (BRs) across the cardiac and respiratory cycle can modulate cortical excitability and so affect awareness. It remains debated at what stages of processing they affect awareness-related event-related potentials (ERPs) in different sensory modalities. We investigated the influence of the cardiac (systole/diastole) and the respiratory (inhalation/exhalation) phase on awareness-related ERPs. Subjects discriminated visual threshold stimuli while their electroencephalogram (EEG), electrocardiogram (ECG) and respiration were simultaneously recorded. We compared ERPs and their intracranial generators for stimuli classified correctly with and without awareness as a function of the cardiac and respiratory phase. Cyclic variations of interoceptive signals from the baroreceptors (BRs) modulated both the earliest electrophysiological markers and the trajectory of brain activity when subjects became aware of the stimuli: an early sensory component (P1) was the earliest marker of awareness for low (diastole/inhalation) and a perceptual component (visual awareness negativity, VAN) for high (systole/exhalation) BR activity, indicating that BR signals interfere with the sensory processing of the visual input. Likewise, activity spread from the primary visceral cortex (posterior insula) to posterior parietal cortices during high and from associative interoceptive centers (anterior insula) to prefrontal cortex during low BR activity. Consciousness is thereby resolved in cognitive/associative regions when BR is low and in perceptual centers when it is high. Our results suggest that cyclic fluctuations of BR signaling affect both the earliest markers of awareness and the brain processes underlying conscious awareness.

Significance Statement

The brain continuously processes stimuli from inside and outside the body, and interoceptive stimuli can modulate the perception of external stimuli. Cardiac and respiratory rhythms are important pacemakers of the organism, and we show how they shape awareness-related brain activity for visual threshold stimuli in two ways. Variations of baroreceptor (BR) activity across the cardiac and respiratory cycle affect 1) the earliest electrophysiological marker (P1 for low (diastole/inhalation), VAN for high (systole/exhalation) BR activity) and 2) the brain areas activated (frontal cortex for low and parietal cortex for high BR activity) when subjects become aware of a stimulus. Cyclic variations of bodily signal can modulate cortical excitability and so shape the pathway to awareness and we propose to consider them as functionally relevant signals rather dismissing them as noise.
Differential Adaptation and Remodeling of the Tumor Microenvironment in Breast Cancer Cells During the Stages of Brain Metastasis
Authors: Wu Xinquan , Zhang Yan , Huang Zhenyong , Li Nan
Published: July 2023

Objective

To integrate macro-transcriptomics and single-cell sequencing to analyze the differential gene expression between primary and brain metastatic breast cancer cells, as well as between active and dormant cancer cells within brain metastases, exploring their adaptation and remodeling abilities at different stages of brain metastasis.

Methods

Four public datasets were used: three mRNA microarray datasets from breast cancer and brain metastasis tissues, and one single-cell RNA sequencing (scRNA-seq) dataset from active and dormant brain metastatic cells. Gene differential expression, pathway enrichment, and cell clustering analyses were performed to compare primary and metastatic breast cancer cells, as well as active and dormant cells in brain metastases, focusing on gene expression, metabolic pathways, and functional pathways.

Results

Metastatic breast cancer cells showed weakened pathways related to the extracellular matrix and protein digestion. Active cells exhibited enhanced cell cycle regulation, tumor proliferation, and hypoxia resistance pathways compared to dormant cells. Clustering analysis revealed that cluster 6, unique to dormant cells, had enhanced functions in epithelial-mesenchymal transition (EMT), extracellular matrix (ECM), collagen formation, tumor inflammation siganure (TIS), and IL-10 signaling. Cluster 3 and 4 in active cells had enhanced DNA replication and tumor proliferation pathways, respectively.

Conclusion

This study highlights the role of cancer cell characteristics and heterogeneity in the invasiveness of brain metastasis. Understanding these mechanisms can guide the development of more effective treatment strategies for breast cancer brain metastasis (BCBM) patients.
From goal to outcome: analyzing the progression of biomedical sciences PhD careers in a longitudinal study using an expanded taxonomy
Authors: Brown Abigail M. , Meyers Lindsay C. , Varadarajan Janani , Ward Nicholas J. , Cartailler Jean-Philippe , Chalkley Roger G. , Gould Kathleen L. , Petrie Kimberly A.
Published: July 2023

Biomedical sciences PhDs pursue a wide range of careers inside and outside academia. However, there is little data regarding how career interests of PhD students relate to the decision to pursue postdoctoral training or to their eventual career outcomes. Here, we present the career goals and career outcomes of 1,452 biomedical sciences PhDs who graduated from Vanderbilt University between 1997-2021. We categorized careers using an expanded three-tiered taxonomy and flags that delineate key career milestones. We also analyzed career goal changes between matriculation and defense, and the reasons why students became more- or less-interested in research-intensive faculty careers. We linked students’ career goal at defense to whether they did a postdoc, the duration of time between defense and the first non-training position, the career area of the first non-training position, and the career area of the job at ten years after graduation. Finally, we followed individual careers for ten years after graduation to characterize movement between different career areas over time. We found that most students changed their career goal during graduate school, declining numbers of alumni pursued postdoctoral training, many alumni entered first non-training positions in a different career area than their goal at defense, and the career area of the first non-training position was a good indicator of the job that alumni held 10 years after graduation. Our findings emphasize that students need a wide range of career development opportunities and career mentoring during graduate school to prepare them for futures in research and research-related professions.
Differences in cortical surface area in developmental language disorder
Authors: Bahar Nilgoun , Cler Gabriel J , Krishnan Saloni , Asaridou Salomi S , Smith Harriet J , Willis Hanna E , Healy Máiréad P , Watkins Kate E
Published: July 2023

Approximately seven per cent of children have developmental language disorder (DLD), a neurodevelopmental condition associated with persistent language learning difficulties without a known cause. Our understanding of the neurobiological basis of DLD is limited. Here, we used FreeSurfer to investigate cortical surface area and thickness in 54 children and adolescents with DLD and 74 age-matched controls aged 10-16 years. We also examined cortical asymmetries in DLD using an automated surface-based technique. Those with DLD showed smaller surface area bilaterally in the inferior frontal gyrus extending to the anterior insula, in the posterior temporal and ventral occipito-temporal cortex, and in portions of the anterior cingulate and superior frontal cortex. There were no differences in cortical thickness, nor in asymmetry of these cortical metrics. Post-hoc exploratory analyses revealed that surface area in the left fusiform and inferior frontal cortex related to children’s reading and non-word repetition scores, respectively. This study highlights the importance of distinguishing between surface area and cortical thickness in investigating the brain basis of neurodevelopmental disorders and suggests the development of cortical surface area to be of importance to DLD. Future longitudinal studies are required to understand the developmental trajectory of these cortical differences in DLD and how they relate to language maturation.
Multivalent Exosome based protein vaccine: a “mix and match” approach to epidemic viruses’ challenges
Authors: Cacciottolo Mafalda , Hsieh Li-En , Li Yujia , LeClaire Michael J. , Nice Justin B , Twaddle Ryan , Mora Ciana , Young Meredith , Angeles Jenna , Lau Christy , Jin Cindy , Elliott Kristi , Sun Minghao
Published: July 2023

Endemic viruses are becoming increasingly the norm, and the development of a rapid and effective vaccine is emergent. Here, we used our StealthX ™ exosome platform to express either Influenza H3 (Stealth™ X-Hemagglutinin, STX-H3) or SARS-CoV-2 Delta spike (Stealth™ X-Spike, STX-S) protein on the surface and facilitate their trafficking to the exosomes. When administered as single product, both STX-H3 and STX-S induced a strong immunization with the production of a potent humoral and cellular immune response in mice. Interestingly, these effects were obtained with administration of nanograms of protein and without adjuvant. Therefore, we tested the possibility of a multivalent vaccine: STX-H3 and STX-S exosomes were formulated together in a “mix and match” approach and the immune response was further evaluated. We showed that our STX-H3+S cocktail vaccine is as effective as the single components administered separately, resulting in a strong antibody and T-cell response. Our data show that our exosome platform has an enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation, and for therapeutics by enabling cell and tissue specific targeting.
Neural representation of spatial and non-spatial auditory attention in EEG signals
Authors: An Winko W. , Noyce Abigail , Pei Alexander , Shinn-Cunningham Barbara
Published: July 2023

Neural representation, capturing the content and format of encoded information, provides insight into the internal states of neural units. Studies of neural representation contrast with studies of neural processes, which focus on how one neural unit influences another. Representational similarity analysis (RSA), a multivariate analysis approach, has been used in previous studies to explore the neural representation of object categories in various neuroimaging modalities. In this study, we employed RSA to examine the neural representation of executive function. We designed an experiment involving a rich set of conditions where participants engaged in an auditory task requiring either spatial or non-spatial attention. We extracted representational features from their electroencephalography (EEG) scalp voltage and alpha power and compared these features with ideal conceptual models representing perfect categorization of different attentional states. The results demonstrate the feasibility of investigating internal cognitive states using RSA. Specifically, we identified time intervals during which attentional state contrasts, such as differences between attention types or locations, manifested in the measured neural responses from scalp voltage and alpha power distributions.
Amplicon-guided isolation and cultivation of previously uncultured microbial species from activated sludge
Authors: Verhoeven Maarten D. , Nielsen Per H. , Dueholm Morten K. D.
Published: July 2023

Microbes are fundamental for biological wastewater treatment. However, most microbial species found in activated sludge (AS) from wastewater treatment plants (WWTPs) have never been isolated and grown as pure cultures, thus limiting our understanding of the underlying biological processes. To change this, we here introduce an experimental setup where the plating of dispersed AS bacteria are combined with 16S rRNA gene amplicon sequencing of total plate biomass for rapid identification of growth conditions that allow for the isolation of key microbial species in AS. We show that agarose plates composed of AS fluid supplemented with various carbon sources support the growth of many previously uncultivated AS bacteria. To confirm that the approach can also be used to isolate previously uncultured species, we picked 200 colonies from the plates for growth in liquid medium. This resulted in 185 growing cultures representing 102 strains based on unique 16S rRNA gene V1-V3 amplicon sequence variants (ASVs). Classification of the ASVs with the MiDAS 4 database revealed 48 distinct genera, including the previously uncultured AAP99,Ca. Propionivibrio, Ellin6067, midas_g_12, andCa. Brachybacter. Among the ASVs that obtained species-level classification, we observed 43 unique species of which 29 were only classified based on the MiDAS placeholder taxonomy highlighting the potential for culturing many novel taxa. Preparation of glycerol stocks and subsequent validation by restreaking on plates resulted in 10 pure cultures of which six represent core or conditional rare or abundant (CRAT) species observed within the MiDAS global survey of WWTPs.

Importance

Biological wastewater treatment relies on complex microbial communities that assimilate nutrients and break down pollutants in the wastewater. Knowledge about the physiology and metabolism of bacteria in wastewater treatment plants (WWTPs) may therefore be used to improve the efficacy and economy of wastewater treatment. Our current knowledge is largely based on 16S rRNA gene amplicon profiling, fluorescence in situ hybridization studies, and predictions based on metagenome-assembled genomes. Bacterial isolates are often required to validate genome-based predictions as they allow researchers to analyze a specific species without interference from other bacteria and with simple bulk measurements. Unfortunately, there are currently very few pure cultures of microbes commonly found in WWTPs. To address this, we introduce an isolation strategy that takes advantage of state-of-the-art microbial profiling techniques to uncover suitable growth conditions for key WWTP microbes. We furthermore demonstrate that this information can be used to isolate key organisms representing global WWTPs.
CRIMP: A CRISPR/Cas9 Insertional Mutagenesis Protocol and the CRIMP Toolkit
Authors: Miles Lee B , Calcinotto Vanessa , Sonntag Carmen , Lee Clara , Bryson-Richardson Robert J
Published: July 2023

We developed a highly efficient targeted insertional mutagenesis system, CRIMP, and an associated plasmid toolkit, CRIMPkit, that disrupts native gene expression by inducing complete transcriptional termination to produce null mutant alleles without inducing genetic compensation. The CRIMPkit contains over 30 ready-to-use plasmid vectors allowing easy and complete mutagenesis of any gene in any reading frame without requiring custom sequences, modification, or subcloning, and also provides a fluorescent readout of successfully mutagenised fish.
Amyloid β accelerates age-related proteome-wide protein insolubility
Authors: Anderton Edward , Chamoli Manish , Bhaumik Dipa , King Christina D. , Xie Xueshu , Foulger Anna , Andersen Julie K. , Schilling Birgit , Lithgow Gordon J.
Published: July 2023

Loss of proteostasis is a highly conserved feature of aging across model organisms and typically results in the accumulation of insoluble protein aggregates. Protein insolubility is a central feature of major age-related neurodegenerative diseases, including Alzheimer’s Disease (AD), where hundreds of insoluble proteins associate with aggregated amyloid beta (Aβ) in senile plaques. Moreover, proteins that become insoluble during aging in model organisms are capable of accelerating Aβ aggregation in vitro. Despite the connection between aging and AD risk, therapeutic approaches to date have overlooked aging-driven protein insolubility as a contributory factor. Here, using an unbiased proteomics approach, we questioned the relationship between Aβ and age-related protein insolubility. We demonstrate that Aβ expression drives proteome-wide protein insolubility in C. elegans and this insoluble proteome closely resembles the insoluble proteome driven by normal aging, suggesting the possibility of a vicious feedforward cycle of aggregation in the context of AD. Importantly, using human genome-wide association studies (GWAS), we show that the CIP is replete with biological processes implicated not only in neurodegenerative diseases but also across a broad array of chronic, age-related diseases (CARDs). This provides suggestive evidence that age-related loss of proteostasis could play a role in general CARD risk. Finally, we show that the CIP is enriched with proteins that modulate the toxic effects of Aβ and that the gut-derived metabolite, Urolithin A, relieves Aβ toxicity, supporting its use in clinical trials for dementia and other age-related diseases.
Mechanism of target site selection by type V-K CRISPR-associated transposases
Authors: George Jerrin Thomas , Acree Christopher , Park Jung-Un , Kong Muwen , Wiegand Tanner , Pignot Yanis Luca , Kellogg Elizabeth H. , Greene Eric C. , Sternberg Samuel H.
Published: July 2023

Unlike canonical CRISPR-Cas systems that rely on RNA-guided nucleases for target cleavage, CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to facilitate RNA-guided transposition of large genetic payloads. Type V-K CASTs offer several potential upsides for genome engineering, due to their compact size, easy programmability, and unidirectional integration. However, these systems are substantially less accurate than type I-F CASTs, and the molecular basis for this difference has remained elusive. Here we reveal that type V-K CASTs undergo two distinct mobilization pathways with remarkably different specificities: RNA-dependent and RNA-independent transposition. Whereas RNA-dependent transposition relies on Cas12k for accurate target selection, RNA-independent integration events are untargeted and primarily driven by the local availability of TnsC filaments. The cryo-EM structure of the untargeted complex reveals a TnsB-TnsC-TniQ transpososome that encompasses two turns of a TnsC filament and otherwise resembles major architectural aspects of the Cas12k-containing transpososome. Using single-molecule experiments and genome-wide meta-analyses, we found that AT-rich sites are preferred substrates for untargeted transposition and that the TnsB transposase also imparts local specificity, which collectively determine the precise insertion site. Knowledge of these motifs allowed us to direct untargeted transposition events to specific hotspot regions of a plasmid. Finally, by exploiting TnsB’s preference for on-target integration and modulating the availability of TnsC, we suppressed RNA-independent transposition events and increased type V-K CAST specificity up to 98.1%, without compromising the efficiency of on-target integration. Collectively, our results reveal the importance of dissecting target site selection mechanisms and highlight new opportunities to leverage CAST systems for accurate, kilobase-scale genome engineering applications.
In vivo imaging in transgenic songbirds reveals superdiffusive neuron migration in the adult brain
Authors: Shvedov Naomi R. , Analoui Sina , Dafalias Theresia , Bedell Brooke L. , Gardner Timothy J. , Scott Benjamin B.
Published: July 2023

Neuron migration is a key phase of neurogenesis, critical for the assembly and function of neuronal circuits. In songbirds, this process continues throughout life, but how these newborn neurons disperse through the adult brain is unclear. We addressed this question usingin vivotwo-photon imaging in transgenic songbirds that express GFP in young neurons. In juvenile and adult birds, migratory cells were present at a high density, traveled in all directions, and made frequent course changes. Notably, these dynamic migration patterns were well fit by a superdiffusive model. Simulations revealed that these diffusion-like dynamics were sufficient to disperse new neurons throughout the song nucleus HVC. These results suggest that diffusion-like migration may underlie the formation and maintenance of nuclear brain structures in the postnatal brain and indicate that transgenic songbirds are a useful resource for future studies into the mechanisms of adult neurogenesis.

Highlights

Transgenic songbirds express GFP in a neurogenic lineage

GFP expression is strong and sparse enough to track single cellsin vivo

Adult neuron migration is well fit by a superdiffusive model

Superdiffusive migration is sufficient to populate HVC in simulation
Find, Label, Annotate Genomes: FLAG, a fully automated tool for structural and functional gene annotation
Authors: Troy William , Damas Joana , Titus Alexander J. , Cantarel Brandi L.
Published: July 2023

Recent advances in long-read sequencing technologies and the efforts of projects aimed at increasing the universe of sequenced reference genomes have led to a growth in the number of whole genomes sequenced for non-model organisms. Still, 81% of the over 36,000 unique publicly available eukaryotic genomes in the NCBI database lack gene structure annotations (1). While there are many open-source tools available for each step in the annotation process, many of these tools are designed for chromosomal assemblies with available transcript data from the same or very closely related organisms. Here we present “Find, Label, Annotate Genomes” (FLAG), a fully automated genome annotation workflow. FLAG (i) works on any computing environment, (ii) runs automatically without initial training data, (iii) generates structural and functional annotations, (iv) performs accurately with fragmented genomes, (v) does not require species-specific extrinsic evidence (transcript sequences) and (vi) includes quality control steps to evaluate annotation completeness. We compared the gene annotations generated by FLAG and publicly available annotations from 12 eukaryotic organisms, including 1 plant, 8 living animals, and 1 extinct animal. In fragmented genomes, FLAG annotations provided an average of 18% increase in complete BUSCO scores and 15x lower error rate for the predicted number of protein-coding genes when compared to published BRAKER2 annotations. With FLAG-Refiner further improved results by decreasing the error rate to 17x lower when compared to published BRAKER2 annotations. In high-quality model organisms, FLAG demonstrates comparable results to those of the NCBI EGAP pipeline, underscoring its robustness and accuracy in gene prediction across diverse taxa and assembly qualities.
Evolving together: Cassandra retrotransposons gradually mirror promoter mutations of the 5S rRNA genes
Authors: Maiwald Sophie , Mann Ludwig , Garcia Sònia , Heitkam Tony
Published: July 2023

The 5S rRNA genes are among the most conserved nucleotide sequences across all species. Similar to the 5S preservation we observe the occurrence of 5S-related non-autonomous retrotransposons, so-called Cassandra. Cassandras harbor highly conserved 5S rDNA-related sequences within their long terminal repeats (LTRs), advantageously providing them with the 5S internal promoter. However, the dynamics of Cassandra retrotransposon evolution in the context of 5S rRNA gene sequence information and structural arrangement are still unclear, especially: 1) do we observe repeated or gradual domestication of the highly conserved 5S promoter by Cassandras and 2) do changes in 5S organization such as in the linked 35S-5S rDNA arrangements impact Cassandra evolution? Here, we show evidence for gradual co-evolution of Cassandra sequences with their corresponding 5S rDNAs. To follow the impact of 5S rDNA variability on Cassandra TEs, we investigate the Asteraceae family where highly variable 5S rDNAs, including 5S promoter shifts and both linked and separated 35S-5S rDNA arrangements have been reported. Cassandras within the Asteraceae mirror 5S rDNA promoter mutations of their host genome, likely as an adaptation to the host’s specific 5S transcription factors and hence compensating for evolutionary changes in the 5S rDNA sequence. Changes in the 5S rDNA sequence and in Cassandras seem uncorrelated with linked/separated rDNA arrangements. We place all these observations into the context of angiosperm 5S rDNA-Cassandra evolution, discuss Cassandra’s origin hypotheses (single or multiple) and Cassandra’s possible impact on rDNA and plant genome organization, giving new insights into the interplay of ribosomal genes and transposable elements.
SQuHIVLa: A novel assay for Specific Quantification of inducible HIV-1 reservoir by LAMP
Authors: Hossain Tanvir , Lungu Cynthia , de Schrijver Sten , Kuali Mamokoena , Rao Shringar , Ngubane Ayanda , Wai Kan Tsung , Palstra Robert-Jan , Madlala Paradise , Ndung’u Thumbi , Mahmoudi Tokameh
Published: July 2023

Strategies toward HIV-1 cure aim to clear, inactivate, reduce or immunologically control the virus from a pool of latently infected cells such that combination antiretroviral therapy (cART) can be safely interrupted. In order to assess the impact of any putative curative interventions on the size and inducibility of the latent HIV-1 reservoir, robust and scalable assays are needed to precisely quantify the frequency of infected cells containing inducible replication competent HIV-1. Here, we present Specific Quantification of Inducible HIV-1 by LAMP (SQuHIVLa), a novel assay that leverages the high sensitivity and specificity of RT-LAMP, performed in a single reaction, to detect and quantify cells expressing Tat/Rev msRNA upon activation. Our LAMP primer/probe design exclusively detects subtype-specific HIV-1 Tat/Rev msRNA and exhibits high sensitivity, specificity, and reproducibility. Using SQuHIVLa we quantified the inducible viral reservoir in CD4+ T cells from a diverse group of people living with HIV-1 subtypes B and C on cART. SQuHIVLa presents a high throughput, scalable and specific HIV-1 reservoir quantification tool that is amenable to resource limited settings.
Deep brain stimulation at the basolateral amygdala alters whole-brain connectivity and synchronization and social behaviors ofTbr1deficient mice
Authors: Hsu Tsan-Ting , Huang Tzyy-Nan , Wang Chien-Yao , Hueh Yi-Ping
Published: July 2023

Autism spectrum disorders (ASD) are considered neural dysconnectivity syndromes. To better understand ASD and uncover potential treatments, it is imperative to know and dissect the connectivity deficits under conditions of autism. Here, we apply a whole-brain immunostaining and quantification platform to demonstrate impaired structural and functional connectivity and aberrant whole-brain synchronization in aTbr1+/–autism mouse model. We express a channelrhodopsin variant oChIEF fused with Citrine at the basolateral amygdala (BLA) to outline the axonal projections of BLA neurons. By activating the BLA under blue light theta-burst stimulation (TBS), we then evaluate the effect of BLA activation on C-FOS expression at a whole brain level to represent neural activity. We show thatTbr1haploinsufficiency almost completely disrupts contralateral BLA axonal projections and results in mistargeting in both ipsilateral and contralateral hemispheres, thereby globally altering BLA functional connectivity. Based on correlated C-FOS expression among brain regions, we further show thatTbr1deficiency severely disrupts whole-brain synchronization in the absence of salient stimulation.Tbr1+/–and wild-type (WT) mice exhibit opposing responses to TBS-induced amygdalar activation, reducing synchronization in WT mice but enhancing it inTbr1+/–mice. Whole-brain modular organization and inter-module connectivity are also affected by Tbr1 deficiency and amygdalar activation. Following BLA activation by TBS, the synchronizations of the whole brain and the default mode network, a specific subnetwork highly relevant to ASD, are enhanced inTbr1+/–mice, implying a potential ameliorating effect of amygdalar stimulation on brain function. Indeed, TBS-mediated BLA activation increases nose-to-nose social interactions ofTbr1+/–mice, strengthening evidence for the role of amygdalar connectivity in social behaviors. Our high-resolution analytical platform reveals the inter- and intra-hemispheric connectopathies arising from ASD. Our study emphasizes the defective synchronization at a whole-brain scale caused byTbr1deficiency and implies a potential beneficial effect of deep brain stimulation at the amygdala for TBR1-linked autism.
Structural reconstruction of individual filaments in Aβ42 fibril populations assembledin vitroreveal rare species that resembleex vivoamyloid polymorphs from human brains
Authors: Aubrey Liam D. , Lutter Liisa , Fennell Kate , Purton Tracey J. , Ward Natasha , Serpell Louise C. , Xue Wei-Feng
Published: July 2023

Structural polymorphism has been demonstrated for bothin vitroandex vivoamyloid fibrils associated with disease. The manner in which different filament structures are assembled from common building blocks remains unclear but the assembly environment is likely to be a key determinant. To address this, three-dimensional reconstruction of individual filament structures was conducted from atomic force microscopy images to map the structural polymorphism landscape of Aβ42amyloid fibril populations formedin vitrounder most frequently used buffer conditions. The data show sensitivity of Aβ42fibril polymorphism to the assembly environment in both the magnitude of heterogeneity and the types of filament species formed. However, some conserved fibril polymorphs were observed across the experimental conditions. Excitingly, by matching individual filament structures to cryo-electron microscopy derived structural data, rare species in these heterogeneous population clouds that show remarkable similarity to Aβ42amyloid polymorphs purified from human patient brains were discovered. These results linkin vitroexperimental approaches with structures formedin vivo, and highlight the polymorph distribution, and the type and magnitude of structural variations within these heterogeneous molecular distributions as important factors in amyloid biology.