bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication protein Rep provides selective advantage to viruses in the presence of CRISPR-Cas immunity
Authors: Zhang Weijia , Bhoobalan-Chitty Yuvaraj , Zhai Xichuan , Hui Yan , Hansen Lars Hestbjerg , Deng Ling , Peng Xu
Published: November 2021

Prokaryotic viruses express anti-CRISPR (Acr) proteins to inhibit the host adaptive immune system, CRISPR-Cas. While the virus infection biology was shown to be strongly dependent on the relative strengths of the host CRISPR-Cas and viral Acrs, little is known about the role of the core processes of viral life cycle (replication, packaging etc) in defence/anti-defence arms race. Here, we demonstrate the selective advantage provided by a replication initiator, Rep, in the context of CRISPR-Acr interactions. First, we developed a two-host based CRISPR-Cas genome editing tool for the deletion of highly conserved and thus potentially important viral genes. Using this strategy, we deleted a highly conserved Rep-coding gene, gp16, from the genome of Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The knockout mutant (Δgp16) produced around 4 fold less virus in a CRISPR-null host, suggesting that Rep is the major contributor to replication initiation in Rudiviridae. Indeed, DNA sequencing revealed Rep-dependent replication initiation from the viral genome termini, in addition to Rep-independent replication initiation from non-terminal sites. Intriguingly, the lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the Δgp16 mutant much more quickly in CRISPR-containing host than in CRISPR-null host, demonstrating a selective advantage provided by Rep in the presence of host CRISPR-Cas immunity. Despite the non-essentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.

Importance

CRISPR-Cas and anti-CRISPR proteins are accessary to prokaryotes and their viruses respectively. To date, research has been focused on their diversity, molecular mechanisms and application in genome editing. How CRISPR-Acr arms race influence the evolution of viral core genes involved in the basic virus life cycle remained a gap of knowledge so far. This study provides the first evidence that CRISPR-Acr arms race poses a selection pressure on the efficiency of viral genome replication, forcing viruses to evolve highly productive replication machineries..
Genetic regulation of serum IgA levels and susceptibility to common immune, infectious, kidney, and cardio-metabolic traits
Authors: Liu Lili , Khan Atlas , Sanchez-Rodriguez Elena , Zanoni Francesca , Li Yifu , Steers Nicholas , Balderes Olivia , Zhang Junying , Krithivasan Priya , LeDesma Robert A. , Fischman Clara , Hebbring Scott J. , Harley John B. , Moncrieffe Halima , Kottyan Leah C. , Namjou-Khales Bahram , Walunas Theresa L. , Knevel Rachel , Raychaudhuri Soumya , Karlson Elizabeth W. , Denny Joshua C. , Stanaway Ian B. , Crosslin David , Rauen Thomas , Floege Jürgen , Eitner Frank , Moldoveanu Zina , Reily Colin , Knoppova Barbora , Hall Stacy , Sheff Justin T. , Julian Bruce A. , Wyatt Robert J. , Suzuki Hitoshi , Xie Jingyuan , Chen Nan , Zhou Xujie , Zhang Hong , Hammarström Lennart , Viktorin Alexander , Magnusson Patrik K. E. , Shang Ning , Hripcsak George , Weng Chunhua , Rundek Tatjana , Elkind Mitchell S. V. , Oelsner Elizabeth C. , Barr R. Graham , Ionita-Laza Iuliana , Novak Jan , Gharavi Ali G. , Kiryluk Krzysztof
Published: November 2021

Immunoglobulin A (IgA) mediates mucosal responses to food antigens and the intestinal microbiome and has a known role in susceptibility to mucosal pathogens, celiac disease, inflammatory bowel disease, and IgA nephropathy. We performed genetic analyses of serum IgA levels in 41,263 individuals of diverse ancestries. We observed unexpected variability in IgA levels across major ancestral populations, with African ancestry being reproducibly associated with higher serum IgA levels compared to other ancestries. The trans-ethnic GWAS analysis identified 20 genome-wide significant loci associated with serum IgA levels, including nine known and 11 novel loci. Systematic co-localization analysis with blood and primary immune cell expression QTLs prioritized candidate genes for 14 of 20 loci. Most GWAS loci encoded genes that produce immune defects and IgA abnormalities when genetically manipulated in mice. We uncovered positive genetic correlations of serum IgA levels with IgA nephropathy, type 2 diabetes and body mass index, as well as negative genetic correlations with celiac disease, inflammatory bowel disease, several infections, and intestinal microbiome diversity. Our findings provide novel insights into the genetic regulation of IgA production and its potential role in human disease.
Humoral immune response after COVID-19 infection or BNT162b2 vaccine among older adults: evolution over time and protective thresholds
Authors: Meyer Maxence , Constancias Florentin , Worth Claudia , Meyer Anita , Muller Marion , Boussuge Alexandre , Kaltenbach Georges , Schmitt Elise , Chayer Saïd , Velay Aurélie , Vogel Thomas , Fafi-Kremer Samira , Karcher Patrick
Published: November 2021

INTRODUCTION

The objectives of this study were to assess the dynamics of the SARS-CoV-2 anti-RBD IgG response over time among older people after COVID-19 infection or vaccination and its comparison with speculative levels of protection assumed by current data.

METHODS

From November 2020 to October 2021, we included geriatric patients with serological test results for COVID-19.

We considered antibody titre thresholds thought to be high enough to protect against SARS-CoV-2 infection: 141 BAU/ml for protection/vaccine efficacy > 89.3%.

Three cohorts are presented. A vaccine group (n=34) that received two BNT162b2/Comirnaty injections 21 days apart, a group of natural COVID-19 infection (n=32) and a third group who contracted COVID-19 less than 15 days after the first BNT162b2/Comirnaty injection (n=17).

RESULTS

83 patients were included, the median age was 87 (81-91) years.

In the vaccine group at 1 month since the first vaccination, the median BAU/ml with IQR was 620 (217-1874) with 87% of patients above the threshold of 141 BAU/ml. Seven months after the first vaccination the BAU/ml was 30 (19-58) with 9.5% of patients above the threshold of 141 BAU/ml.

In the natural COVID-19 infection group, at 1 month since the date of first symptom onset, the median BAU/ml was 798 (325-1320) with 86.7% of patients above the threshold of 141 BAU/ml and fell to 88 (37-385) with 42.9% of patients above the threshold of 141 BAU/ml at 2 months. The natural infection group was vaccinated three months after the infection. Five months after the end of the vaccination cycle the BAU/ml was 2048 (471-4386) with 83.3% of patients above the threshold of 141 BAU/ml.

DISCUSSION

On the humoral level, this supports the clinical results describing the decrease in vaccine protection over time.
Low neutralizing antibody titers against the Mu variant of SARS-CoV-2 in BNT162b2 vaccinated individuals
Authors: Álvarez-Díaz Diego A. , Muñoz Ana Luisa , Tavera-Rodríguez Pilar , Herrera-Sepúlveda María T. , Ruiz-Moreno Hector Alejandro , Laiton-Donato Katherine , Franco-Muñoz Carlos , Pelaez-Carvajal Dioselina , Cuellar Diego , Muñoz-Ramirez Alejandra M. , Galindo Marisol , Arias-Ramirez Edgar J. , Mercado-Reyes Marcela
Published: November 2021

Background

Global surveillance programs for the virus that causes COVID-19 are showing the emergence of variants with mutations in the Spike protein, including the Mu variant, recently declared a Variant of Interest (VOI) by the World Health Organization. Genomic and laboratory surveillance is important in these types of variants because they may be more infectious or less susceptible to antiviral treatments and vaccine-induced antibodies.

Objectives

To evaluate the sensitivity of the Mu variant (B.1.621) to neutralizing antibodies induced by the BNT162b2 vaccine.

Study design

Three of the most predominant SARS-CoV-2 variants in Colombia during the epidemiological peaks of 2021 were isolated. Microneutralization assays were performed by incubating 120 TCDI50 of each SARS-CoV-2 isolate with five 2-fold serial dilutions of sera from 14 BNT162b2 vaccinated volunteers. The MN50 titer was calculated by the Reed-Muench formula

Results

The three isolated variants were Mu, a Variant of Interest (VOI), Gamma, a variant of concern (VOC), and B.1.111 that lacks genetic markers associated with greater virulence. At the end of August, the Mu and Gamma variants were widely distributed in Colombia. Mu was predominant (49%), followed by Gamma (25%). In contrast, B.1.111 became almost undetectable. The evaluation of neutralizing antibodies suggests that patients vaccinated with BNT162-2 generate neutralizing antibody titers against the Mu variant at significantly lower concentrations relative to B.1.111 and Gamma.

Conclusions

This study shows the importance of continuing with surveillance programs of emerging variants as well as the need to evaluate the neutralizing antibody response induced by other vaccines circulating in the country against Mu and other variants with high epidemiological impact.

Highlights

Mu and Gamma variants represented 49% and 25% of cases in Colombia by August 2021.

Increased proportion of SARS-COV-2 cases were mostly associated with Mu variant, despite being detected simultaneously with the VOC Gamma

The Mu variant remarkably escapes from neutralizing antibodies elicited by the BNT162b2-vaccine

Laboratory studies of neutralizing antibodies are useful to determine the efficacy of SARS-CoV-2 vaccines against VOC and VOI.
Smart soils to observe hidden rhizosphere processes
Authors: Patko Daniel , Yang Qizhi , Liu Yangminghao , Falireas Panagiotis , Briou Benoit , Tawade Bhausaheb V , George Timothy S , Daniell Tim J , MacDonald Michael P , Ladmiral Vincent , Ameduri Bruno , Dupuy Lionel X
Published: November 2021

Agriculture must reduce green-house gas emission and pollution, produce safer and healthier food, closer to home, reducing waste whilst delivering more diverse diets to a growing world population. Soils could enable this transformation, but unfortunately, they have a hugely complex and opaque structure and studies of its myriad of mechanisms are difficult. Here, the fabrication of smart soils for the screening of below-ground bio-processes is demonstrated. Particles were generated from fluoropolymer waste with functionalisation converting them into sensors able to report on key chemical dynamics. Tailored functionalization was obtained by radical terpolymerisation to improve growth conditions and sensing capabilities. The study demonstrates the potential for the development of accelerated genetic or agrochemical screens and could pave the way for improved models for rhizosphere dynamics.
Glyphosate and its formulations Roundup Bioflow and RangerPro alter bacterial and fungal community composition in the rat caecum microbiome
Authors: Mesnage Robin , Panzacchi Simona , Bourne Emma , Mein Charles A , Perry Melissa J , Hu Jianzhong , Chen Jia , Mandrioli Daniele , Belpoggi Fiorella , Antoniou Michael N
Published: November 2021

The potential health consequences of glyphosate-induced gut microbiome alterations have become a matter of intense debate. As part of a multifaceted study investigating toxicity, carcinogenicity and multigenerational effects of glyphosate and its commercial herbicide formulations, we assessed changes in bacterial and fungal populations in the caecum microbiota of rats exposed prenatally until adulthood (13 weeks after weaning) to three doses of glyphosate (0.5, 5, 50 mg/kg body weight/day), or to the formulated herbicide products Roundup Bioflow and RangerPro at the same glyphosate-equivalent doses. Caecum bacterial microbiota were evaluated by 16S rRNA sequencing whilst the fungal population was determined by ITS2 amplicon sequencing. Results showed that both fungal and bacterial diversity were affected by the Roundup formulations in a dose-dependent manner, whilst glyphosate alone significantly altered only bacterial diversity. At taxa level, a reduction in Bacteroidota abundance, marked by alterations in the levels of Alloprevotella, Prevotella and Prevotellaceae UCG-003, was concomitant to increased levels of Firmicutes (e.g., Romboutsia, Dubosiella, Eubacterium brachy group or Christensenellaceae) and Actinobacteria (e.g., Enterorhabdus, Adlercreutzia, or Asaccharobacter). Treponema and Mycoplasma also had their levels reduced by the pesticide treatments. Analysis of fungal composition indicated that the abundance of the rat gut commensal Ascomycota Kazachstania was reduced while the abundance of Gibberella, Penicillium, Claviceps, Cornuvesica, Candida, Trichoderma and Sarocladium were increased by exposure to the Roundup formulations, but not to glyphosate. Altogether, our data suggest that glyphosate and its Roundup RangerPro and Bioflow caused profound changes in caecum microbiome composition by affecting the fitness of major commensals, which in turn reduced competition and allowed opportunistic fungi to grow in the gut, in particular in animals exposed to the herbicide formulations. This further indicates that changes in gut microbiome composition might influence the long-term toxicity, carcinogenicity and multigenerational effects of glyphosate-based herbicides.
Swarm learning for decentralized artificial intelligence in cancer histopathology
Authors: Saldanha Oliver Lester , Quirke Philip , West Nicholas P. , James Jacqueline A. , Loughrey Maurice B. , Grabsch Heike I. , Salto-Tellez Manuel , Alwers Elizabeth , Cifci Didem , Laleh Narmin Ghaffari , Seibel Tobias , Gray Richard , Hutchins Gordon G. A. , Brenner Hermann , Yuan Tanwei , Brinker Titus J. , Chang-Claude Jenny , Khader Firas , Schuppert Andreas , Luedde Tom , Foersch Sebastian , Muti Hannah Sophie , Trautwein Christian , Hoffmeister Michael , Truhn Daniel , Kather Jakob Nikolas
Published: November 2021

Artificial Intelligence (AI) can extract clinically actionable information from medical image data. In cancer histopathology, AI can be used to predict the presence of molecular alterations directly from routine histopathology slides. However, training robust AI systems requires large datasets whose collection faces practical, ethical and legal obstacles. These obstacles could be overcome with swarm learning (SL) where partners jointly train AI models, while avoiding data transfer and monopolistic data governance. Here, for the first time, we demonstrate the successful use of SL in large, multicentric datasets of gigapixel histopathology images comprising over 5000 patients. We show that AI models trained using Swarm Learning can predict BRAF mutational status and microsatellite instability (MSI) directly from hematoxylin and eosin (H&E)-stained pathology slides of colorectal cancer (CRC). We trained AI models on three patient cohorts from Northern Ireland, Germany and the United States of America and validated the prediction performance in two independent datasets from the United Kingdom using SL-based AI models. Our data show that SL enables us to train AI models which outperform most locally trained models and perform on par with models which are centrally trained on the merged datasets. In addition, we show that SL-based AI models are data efficient and maintain a robust performance even if only subsets of local datasets are used for training. In the future, SL can be used to train distributed AI models for any histopathology image analysis tasks, overcoming the need for data transfer and without requiring institutions to give up control of the final AI model.
TK216 targets microtubules in Ewing sarcoma cells
Authors: Povedano Juan Manuel , Li Vicky , Lake Katherine E. , Bai Xin , Rallabandi Rameshu , Kim Jiwoong , Xie Yang , De Brabander Jef K. , McFadden David G.
Published: November 2021

Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11;22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for EWS patients. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding α-tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.
A Time-calibrated Firefly (Coleoptera: Lampyridae) Phylogeny: Using Genomic Data for Divergence Time Estimation
Authors: Höhna Sebastian , Lower Sarah E. , Duchen Pablo , Catalán Ana
Published: November 2021

Fireflies (Coleoptera: Lampyridae) consist of over 2,000 described extant species. A well-resolved phylogeny of fireflies is important for the study of their population genetics, bioluminescence, evolution, and conservation. We used a recently published anchored hybrid enrichment dataset (AHE; 436 loci for 88 Lampyridae species and 10 outgroup species) and state-of-the-art statistical methods (the fossilized birth-death-range process implemented in a Bayesian framework) to estimate a time-calibrated phylogeny of Lampyridae. Unfortunately, estimating calibrated phylogenies using AHE and the latest and most robust time-calibration strategies is not possible because of computational constraints. As a solution, we subset the full dataset by applying three different strategies: (i) using the most complete loci, (ii) using the most homogeneous loci, and (iii) using the loci with the highest accuracy to infer the well established Photinus clade. The estimated topology using the three data subsets agreed on almost all major clades and only showed minor discordance within less supported nodes. The estimated divergence times overlapped for all nodes that are shared between the topologies. Thus, divergence time estimation is robust as long as the topology inference is robust and any well selected data subset suffices. Additionally, we observed an un-expected amount of gene tree discordance between the 436 AHE loci. Our assessment of model adequacy showed that standard phylogenetic substitution models are not adequate for any of the 436 AHE loci which is likely to bias phylogenetic inferences. We performed a simulation study to explore the impact of (a) incomplete lineage sorting, (b) uniformly distributed and systematic missing data, and (c) systematic bias in the position of highly variable and conserved sites. For our simulated data, we observed less gene tree variation which shows that the empirically observed amount of gene tree discordance for the AHE dataset is unexpected and needs further investigation.
DomBpred: protein domain boundary predictor using inter-residue distance and domain-residue level clustering
Authors: Yu Zhongze , Peng Chunxiang , Liu Jun , Zhang Biao , Zhou Xiaogen , Zhang Guijun
Published: November 2021

Domain boundary prediction is one of the most important problems in the study of protein structure and function, especially for large proteins. At present, most domain boundary prediction methods have low accuracy and limitations in dealing with multi-domain proteins. In this study, we develop a sequence-based protein domain boundary predictor, named DomBpred. In DomBpred, the input sequence is firstly classified as either a single-domain protein or a multi-domain protein through a designed effective sequence metric based on a constructed single-domain sequence library. For the multi-domain protein, a domain-residue level clustering algorithm inspired by Ising model is proposed to cluster the spatially close residues according inter-residue distance. The unclassified residues and the residues at the edge of the cluster are then tuned by the secondary structure to form potential cut points. Finally, a domain boundary scoring function is proposed to recursively evaluate the potential cut points to generate the domain boundary. DomBpred is tested on a large-scale test set of FUpred comprising 2549 proteins. Experimental results show that DomBpred better performs than the state-of-the-art methods in classifying whether protein sequences are composed by single or multiple domains, and the Matthew’s correlation coefficient is 0.882. Moreover, on 849 multi-domain proteins, the domain boundary distance and normalised domain overlap scores of DomBpred are 0.523 and 0.824, respectively, which are 5.0% and 4.2% higher than those of the best comparison method, respectively. Comparison with other methods on the given test set shows that DomBpred outperforms most state-of-the-art sequence-based methods and even achieves better results than the top-level template-based method.
The effect of external stimulation on functional networks in the aging healthy human brain
Authors: Escrichs Anira , Perl Yonatan Sanz , Martínez-Molina Noelia , Biarnes Carles , Garre-Olmo Josep , Fernández-Real José Manuel , Ramos Rafel , Martí Ruth , Pamplona Reinald , Brugada Ramon , Serena Joaquin , Ramió-Torrentà Lluís , Coll-De-Tuero Gabriel , Gallart Luís , Barretina Jordi , Vilanova Joan C. , Mayneris-Perxachs Jordi , Saba Luca , Pedraza Salvador , Kringelbach Morten L. , Puig Josep , Deco Gustavo
Published: November 2021

Understanding the brain changes occurring during aging can provide new insights for developing treatments that alleviate or reverse cognitive decline. Neurostimulation techniques have emerged as potential treatments for brain disorders and to improve cognitive functions. Nevertheless, given the ethical restrictions of neurostimulation approaches, in silico perturbation protocols based on causal whole-brain models are fundamental to gaining a mechanistic understanding of brain dynamics. Furthermore, this strategy could serve as a more specific biomarker relating local activity with global brain dynamics. Here, we used a large resting-state fMRI dataset divided into middle-aged (N=310, aged < 65 years) and older adults (N=310, aged ≥ 65) to characterize brain states in each group as a probabilistic metastable substate (PMS) space, each with a probabilistic occurrence and frequency. Then, we fitted the PMS to a whole-brain model and applied in silico stimulations with different intensities in each node to force transitions from the brain states of the older group to the middle-age group. We found that the precuneus, a brain area belonging to the default mode network and the rich club, was the best stimulation target. These findings might have important implications for designing neurostimulation interventions to revert the effects of aging on whole-brain dynamics.
Model-checking ecological state-transition graphs
Authors: Thomas Colin , Cosme Maximilien , Gaucherel Cédric , Pommereau Franck
Published: November 2021

Model-checking is a methodology developed in computer science to automatically assess the dynamics of discrete systems, by checking if a system modelled as a state-transition graph satisfies a dynamical property written as a temporal logic formula. The dynamics of ecosystems have been drawn as state-transition graphs for more than a century, from state-and-transition models to assembly graphs. Thus, model-checking can provide insights into both empirical data and theoretical models, as long as they sum up into state-transition graphs. While model-checking proved to be a valuable tool in systems biology, it remains largely underused in ecology. Here we promote the adoption of the model-checking toolbox in ecology through its application to an illustrative example. We assessed the dynamics of a vegetation model inspired from state-and-transition models by model-checking Computation Tree Logic formulas built from a proposed catalogue of patterns. Model-checking encompasses a wide range of concepts and available software, mentioned in discussion, thus its implementation can be fitted to the specific features of the described system. In addition to the automated analysis of ecological state-transition graphs, we believe that defining ecological concepts with temporal logics could help clarifying and comparing them.

Author summary

Ecologists have drawn state-transition graphs representing the dynamics of ecosystems for more than a century. Model-checking is an automated method for the analysis of such graphs developed in computer science and acknowledged by a Turing award in 2007. Ecologists appear to be mostly unaware of model-checking despite its successes in systems biology to assess the dynamics of biological networks.

We promote model-checking of ecological state-transition graphs through its application to an illustrative vegetation model. We exemplify the insights provided by model-checking by assessing management policies aiming to tackle savanna encroachment. We also provide a catalogue of patterns to help ecologists with the difficulty of formally expressing dynamical properties. We also discuss the wide range of model-checking concepts and available software, enabling to fit the specific features of the studied system, such as durations or probabilities.

Model-checking can be applied to both empirical data and theoretical models, as long as they sum up into state-transition graphs. It provides automated and accurate answers to complex questions that could barely be analysed through human examination, if not impossible to answer this way. In addition to the automated analysis of ecological state-transition graphs, we believe that formally defining ecological concepts within the model-checking framework could help in clarifying and comparing them.
DETIRE: A Hybrid Deep Learning Model for identifying Viral Sequences from Metagenomes
Authors: Miao Yan , Liu Fu , Hou Tao , Liu Qiaoliang , Dong Tian , Liu Yun
Published: November 2021

A metagenome contains all DNA sequences from an environmental sample, including viruses, bacteria, fungi, actinomycetes and so on. Since viruses are of huge abundance and have caused vast mortality and morbidity to human society in history as a kind of major pathogens, detecting viruses from metagenomes plays a crucial role in analysing the viral component of samples and is the very first step for clinical diagnosis. However, detecting viral fragments directly from the metagenomes is still a tough issue because of the existence of huge number of short sequences. In this paper, a hybrid Deep lEarning model for idenTifying vIral sequences fRom mEtagenomes (DETIRE), is proposed to solve the problem. Firstly, the graph-based nucleotide sequence embedding strategy is utilized to enrich the expression of DNA sequences by training an embedding matrix. Then the spatial and sequential features are extracted by trained CNN and BiLSTM networks respectively to improve the feature expression of short sequences. Finally, the two set of features are weighted combined for the final decision. Trained by 220,000 sequences of 500bp subsampled from the Virus and Host RefSeq genomes, DETIRE identifies more short viral sequences (<1,000bp) than three latest methods, DeepVirFinder, PPR-Meta and CHEER. DETIRE is freely available athttps://github.com/crazyinter/DETIRE.
Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing
Authors: Gimber Niclas , Strauss Sebastian , Jungmann Ralf , Schmoranzer Jan
Published: November 2021

Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach ‘DNA-PAINT’ robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by ‘Exchange-PAINT’ that requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3 – 6 nm) and multicolor registration of dual and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.
Oscillatory population-level activity of dorsal raphe serotonergic neurons sculpts sleep structure
Authors: Kato Tomonobu , Mitsukura Yasue , Yoshida Keitaro , Mimura Masaru , Takata Norio , Tanaka Kenji F.
Published: November 2021

Dorsal raphe (DR) 5-HT neurons are involved in regulating sleep-wake transitions. Previous studies demonstrated that single-unit activity of DR 5-HT neurons is high during wakefulness, decreases during non–rapid eye movement (NREM) sleep, and ceases during rapid eye movement (REM) sleep. However, characteristics of the population-level activity of DR 5-HT neurons, which can influence the entire brain, are largely unknown. Here we measured population activities of 5-HT neurons in male and female mouse DR across the sleep-wake cycle by a ratiometric fiber photometry system. We found a slow oscillatory activity of compound intracellular Ca2+ signals during NREM sleep. The trough of concave 5-HT activity increased along with sleep progression, but the 5-HT activity level always returned to that seen in wake periods. When the trough reached the minimum level and remained there, REM sleep initiated. We also found a unique coupling of the oscillatory 5-HT activity and EEG power fluctuation, suggesting that EEG fluctuation is a proxy for 5-HT activity. Optogenetic activation of 5-HT neurons during NREM sleep triggered a high EMG power and induced wakefulness. Optogenetic inhibition induced REM sleep or sustained NREM with an EEG power increase and EEG fluctuation. These manipulations demonstrated a causal role of DR 5-HT neurons in sculpting sleep-wake structure. We also observed EEG fluctuations in human males during NREM sleep, implicating the existence of 5- HT oscillatory activity in humans. We propose that NREM sleep is not a monotonous state, but that it is dynamically regulated by the oscillatory population activity of DR 5- HT neurons.

Significant statement

Previous studies have demonstrated single-cell 5-HT neuronal activity across sleep- wake conditions; however, population-level activities of these neurons are largely unknown. We monitored dorsal raphe (DR) 5-HT population activity using a fiber photometry system in mice and demonstrated that activity was highest during wakefulness, and lowest during rapid eye movement (REM) sleep. Surprisingly, during non-REM (NREM) sleep, the 5-HT population activity decreased with an oscillatory pattern, coinciding with EEG fluctuations. We examined the causal role of these 5-HT neuron activities by optogenetics and found that DR 5-HT neurons sculpted sleep-wake conditions by influencing EEG and EMG patterns. We found similar EEG fluctuations in a human sleep EEG study, suggesting the presence of oscillatory 5-HT neuron activity during NREM across species.
A scalable analytical approach from bacterial genomes to epidemiology
Authors: Didelot Xavier , Parkhill Julian
Published: November 2021

Recent years have seen a remarkable increase in the practicality of sequencing whole genomes from large numbers of bacterial isolates. The availability of this data has huge potential to deliver new insights into the evolution and epidemiology of bacterial pathogens, but the scalability of the analytical methodology has been lagging behind that of the sequencing technology. Here we present a step-by-step approach for such large-scale genomic epidemiology analyses, from bacterial genomes to epidemiological interpretations. A central component of this approach is the dated phylogeny, which is a phylogenetic tree with branch lengths measured in units of time. The construction of dated phylogenies from bacterial genomic data needs to account for the disruptive effect of recombination on phylogenetic relationships, and we describe how this can be achieved. Dated phylogenies can then be used to perform fine-scale or large-scale epidemiological analyses, depending on the proportion of cases for which genomes are available. A key feature of this approach is computational scalability, and in particular the ability to process hundreds or thousands of genomes within a matter of hours. This is a clear advantage of the step-by-step approach described here. We discuss other advantages and disadvantages of the approach, as well as potential improvements and avenues for future research.
A 1-bp deletion in bovineQRICH2causes low sperm count and immotile sperm with multiple morphological abnormalities
Authors: Hiltpold Maya , Janett Fredi , Mapel Xena Marie , Kadri Naveen Kumar , Fang Zih-Hua , Schwarzenbacher Hermann , Seefried Franz R , Spengeler Mirjam , Witschi Ulrich , Pausch Hubert
Published: November 2021

Background

Semen quality and male fertility are monitored in artificial insemination bulls to ensure high insemination success rates. Only ejaculates that fulfill minimum quality requirements are processed and eventually used for artificial inseminations. We examined 70,990 ejaculates from 1343 Brown Swiss bulls to identify bulls from which all ejaculates were rejected due to low semen quality. This procedure identified a bull that produced twelve ejaculates with an aberrantly low number of sperm (0.2±0.2 × 109sperm per ml) which were mostly immotile due to multiple morphological abnormalities.

Results

The genome of the bull was sequenced at 12-fold coverage to investigate a suspected genetic cause. Comparing the sequence variant genotypes of the bull with those from 397 fertile bulls revealed a 1-bp deletion in the coding sequence ofQRICH2encoding glutamine rich 2 as a compelling candidate causal variant. The 1-bp deletion causes a frameshift in translation and induces a premature termination codon (ENSBTAP00000018337.1:p.Cys1644AlafsTer52). The analysis of testis transcriptomes from 76 bulls showed that the transcript with the premature termination codon is subjected to nonsense-mediated mRNA decay. The 1-bp deletion resides on a 675 kb haplotype spanning 181 SNPs from the Illumina BovineHD Bead chip. The haplotype segregates at a frequency of 5% in the Brown Swiss cattle population. This analysis also identified another bull that carried the 1-bp deletion in the homozygous state. Semen analyses from the second bull confirmed low sperm concentration and immotile sperm with multiple morphological abnormalities primarily affecting the sperm flagellum and, to a lesser extent, the sperm head.

Conclusions

A recessive loss-of-function allele of bovineQRICH2likely causes low sperm concentration and immotile sperm with multiple morphological abnormalities. Routine sperm analyses unambiguously identify homozygous bulls. A direct gene test can be implemented to monitor the frequency of the undesired allele in cattle populations.
Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity
Authors: Ziv Omer , Farberov Svetlana , Lau Jian You , Miska Eric , Kudla Grzegorz , Ulitsky Igor
Published: November 2021

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.
Human macrophage polarization determines bacterial persistence ofStaphylococcus aureusin a liver-on-chip-based infection model
Authors: Siwczak Fatina , Cseresnyes Zoltan , Carlstedt Swen , Sigmund Anke , Gröger Marko , Surewaard Bas G.J. , Werz Oliver , Figge Marc Thilo , Tuchscherr Lorena , Löffler Bettina , Mosig Alexander S.
Published: November 2021

Infections withStaphylococcus aureus(S. aureus) have been reported from various organs ranging from asymptomatic colonization to severe infections and sepsis associated with multiple organ dysfunction. Although considered an extracellular pathogen,S. aureuscan invade and persist in professional phagocytes such as monocytes and macrophages. Its capability to persist and manipulate phagocytes is considered a critical step to evade host antimicrobial reactions. For the first time we leveraged a human liver-on-chip model and tailored image analysis algorithms to demonstrate thatS. aureus(USA300) specifically targets macrophages in the liver models as essential niche facilitating bacterial persistence and phenotype switching to small colony variants (SCVs).In vitroM2 polarization was found to favor SCV-formation and was associated with increased intracellular bacterial loads in macrophages, increased cell death, and impaired recruitment of circulating monocytes to sites of infection. These findings expand the knowledge about the role of liver macrophages in the course of systemic infection. Further, the results might be relevant for understanding infection mechanisms in patients with chronic liver disease such as fibrosis that display increased frequencies of M2 polarized liver macrophages and have a higher risk for developing chronic infections and relapsing bacteremia.
Exploring genome gene content and morphological analysis to test recalcitrant nodes in the animal phylogeny
Authors: Juravel Ksenia , Porras Luis , Höhna Sebastian , Pisani Davide , Wörheide Gert
Published: November 2021

An accurate phylogeny of animals is needed to clarify their evolution, ecology, and impact on shaping the biosphere. Although datasets of several hundred thousand amino acids are nowadays routinely used to test phylogenetic hypotheses, key deep nodes in the metazoan tree remain unresolved: the root of animals, the root of Bilateria, and the monophyly of Deuterostomia. Instead of using the standard approach of amino acid datasets, we performed analyses of newly assembled genome gene content and morphological datasets to investigate these recalcitrant nodes in the phylogeny of animals. We explored extensively the choices for assembling the genome gene content dataset and model choices of morphological analyses. Our results are robust to these choices and provide additional insights into the early evolution of animals, they are consistent with sponges as the sister group of all the other animals, the worm-like bilaterian lineage Xenacoelomorpha as the sister group of the other Bilateria, and tentatively support monophyletic Deuterostomia.
STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia
Authors: Dinarello Alberto , Betto Riccardo Massimiliano , Diamante Linda , Cioccarelli Chiara , Meneghetti Giacomo , Peron Margherita , Tesoriere Annachiara , Laquatra Claudio , Tiso Natascia , Martello Graziano , Argenton Francesco
Published: November 2021

STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. Here we report that STAT3 is required for the HIF1α-dependent response to hypoxia. In Stat3 knock-out pluripotent embryonic stem cells (ESCs), a large fraction of HIF1α target genes is not induced by hypoxia. Mechanistically, STAT3 does not regulate neither HIF1α expression nor stability, rather, it physically interacts with it in the nucleus. In vivo, we observed that both genetic and chemical inactivation of Stat3 blunted physiological responses to hypoxia, such as angiogenesis, erythropoiesis, and immune cell mobilization. Such defects were accompanied with faulty transcriptional activity of HIF1α. In sum, our data reveal that STAT3 and HIF1α cooperatively mediate the physiological response to hypoxia.
A chemerin peptide analog stimulates tumor growth in two xenograft mouse models of human colorectal carcinoma
Authors: Friebus-Kardash Justa , Schulz Petra , Reinicke Sandy , Karthaus Cordula , Schefer Quirino , Bandholtz Sebastian , Grötzinger Carsten
Published: November 2021

Background

Chemerin plasma concentration has been reported to be positively correlated with the risk of colorectal cancer. However, the potential regulation of CRC tumorigenesis and progression has not yet been investigated in an experimental setting. This study addresses this hypothesis by investigating proliferation, colony formation and migration of CRC cell lines in vitro as well as in animal models.

Methods

In vitro, microscopic assays to study proliferation as well as a scratch-wound assay for migration monitoring were applied using the human CRC cell lines HCT116, HT29 and SW620 under the influence of the chemerin analog CG34. The animal study investigated HCT116-luc and HT29-luc subcutaneous tumor size and bioluminescence during treatment with CG34 versus control, followed by ex-vivo analysis of vessel density and mitotic activity.

Results

While proliferation of the three CRC cell lines in monolayers was not clearly stimulated by CG34, the chemerin analog promoted colony formation in three-dimensional aggregates. An effect on cell migration was not observed. In the treatment study, CG34 significantly stimulated both growth and bioluminescence signal of HCT116-luc and HT29-luc xenografts.

Conclusions

The results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC.
Partial prevention of glucocorticoid-induced osteocyte deterioration with osteocrin gene therapy
Authors: Mazur Courtney M. , Castro Andrade Christian D. , Sato Tadatoshi , Bruce Michael , Bouxsein Mary L. , Wang Jialiang S. , Wein Marc N.
Published: November 2021

Glucocorticoid (GC)-induced osteoporosis and subsequent bone fragility are preceded by death and dysfunction at the cellular level. In particular, short-term glucocorticoid excess suppresses osteocyte remodeling of the surrounding bone mineral, causes apoptosis of osteoblasts and osteocytes, and disrupts homeostatic bone remodeling. Preventing apoptosis and preserving osteocyte morphology and function could be effective means of preventing bone loss during glucocorticoid excess. We hypothesized that osteocrin, which preserves osteocyte viability and morphology in other models where osteocyte defects exist, could prevent osteocyte death and dysfunction in a GC excess model. We used a liver-targeted adeno-associated virus (AAV8) to induce osteocrin overexpression in mice one week prior to implantation with prednisolone or placebo pellets. After 28 days, tissues were collected for micro-CT and histological analysis. GC excess caused the expected reduction in cortical bone thickness and osteocyte canalicular length in control AAV8-treated mice, and these effects were blunted in mice overexpressing osteocrin. However, GC-induced changes in cortical porosity, trabecular bone mass, and gene expression were not prevented by osteocrin. While the mechanism of osteocrin’s effects on osteocyte morphology warrants further investigation, this study does not support a role for this model of osteocrin supplementation to combat the full skeletal effects of GC excess.
Invertebrate DNA methylation and gene regulation
Authors: Dixon Groves , Matz Mikhail
Published: November 2021

As human activity alters the planet, there is a pressing need to understand how organisms adapt to environmental change. Of growing interest in this area is the role of epigenetic modifications, such as DNA methylation, in tailoring gene expression to fit novel conditions. Here, we reanalyzed nine invertebrate (Anthozoa and Hexapoda) datasets to validate a key prediction of this hypothesis: changes in DNA methylation in response to some condition correlate with changes in gene expression. While we detected both differential methylation and differential expression, there was no simple relationship between these differences. Hence, if changes in DNA methylation regulate invertebrate transcription, the mechanism does not follow a simple linear relationship.
Connexinplexity: The spatial and temporal expression of connexin genes during vertebrate organogenesis
Authors: Lukowicz-Bedford Rachel M. , Farnsworth Dylan R. , Miller Adam C.
Published: November 2021

Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is well appreciated in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we probed the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step towards understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.