bluuebunny/crossref_metadata_2025 huggingface dataset:
COVID-19 vaccine uptake, predictors of vaccination, and self-reported barriers to vaccination among primary school teachers in Poland
Authors: Malesza Marta , Sobolewska Karolina
Published: July 2021

It has been proposed that teachers, like healthcare workers, constitute a strategic target for COVID-19 vaccine programs as immunization is a key element in protecting both them and their pupils. The present study examined vaccine uptake among primary-school teachers and sought to identify factors associated with it. A sample was recruited from 553 Polish primary schools, and data were collected at two time points: December 2020 and March 2021. Associations between vaccine uptake among teachers and their attitudes toward COVID-19 vaccination were assessed through multivariate logistic regression. 6152 participants completed both baseline and follow-up surveys. Of these, 4502 (73.2%) reported their intention at baseline to receive a COVID-19 vaccination, if available; at follow-up, 3894 (86.5%) of the same 4502 reported having received the vaccination. A significant association was revealed between vaccine uptake and perceived severity, self-efficacy, and social norms. The principal driver for vaccine acceptance was the wish to avoid contracting the disease. Conversely, the principal driver for vaccine refusal was concern about side effects and safety. A strong association exists between intention to receive the COVID-19 vaccine and actual uptake. Future COVID-19 immunization programs may benefit from a stronger understanding of the factors associated with vaccine uptake among this cohort.
Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: gene duplication facilitates social evolution
Authors: Shigenobu Shuji , Hayashi Yoshinobu , Watanabe Dai , Tokuda Gaku , Hojo Masaru Y , Toga Kouhei , Saiki Ryota , Yaguchi Hajime , Masuoka Yudai , Suzuki Ryutaro , Suzuki Shogo , Kimura Moe , Matsunami Masatoshi , Sugime Yasuhiro , Oguchi Kohei , Niimi Teruyuki , Gotoh Hiroki , Hojo Masaru K , Miyazaki Satoshi , Toyoda Atsushi , Miura Toru , Maekawa Kiyoto
Published: July 2021

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing an intermediate taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome and methylome of the Japanese subterranean termite Reticulitermes speratus. The analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression is prevalent in the R. speratus genome. Such duplicated genes encompass diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense) and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but the expression patterns were highly variable, exhibiting caste biases. Some duplicated genes assayed were expressed in caste-specific organs, such as the accessory glands of the queen ovary and frontal glands in soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and subfunctionalization and/or neofunctionalization that confers caste-specialized functions.

Significance Statement

Termites are model social organisms characterized by a sophisticated caste system, where distinct castes arise from the same genome. Our genomics data of Japanese subterranean termite provides insights into the evolution of the social system, highlighting the significance of gene duplication. Gene duplication associated with caste-biased gene expression is prevalent in the termite genome. Many of the duplicated genes were related to social functions, such as chemical communication, social immunity and defense, and they often expressed in caste-specific organs. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and functional specialization. In addition, since subterranean termites are ecologically and economically important species including destructive pests in the world, our genomics data serves as a foundation for these studies.
Conditional ERK3 overexpression cooperates with PTEN deletion to promote lung adenocarcinoma formation in mice
Authors: Vallabhaneni Sreeram , Liu Jian , Morel Marion , Demayo Francesco J. , Long Weiwen
Published: July 2021

Extracellular signal-regulated kinase 3 (ERK3), also known as MAPK6, belongs to the atypical mitogen-activated protein kinases (MAPKs) subfamily. In comparison with the well-studied classical MAPKs ERK1 and ERK2, much less is known about the cellular and molecular actions of ERK3. Accumulating studies have revealed the upregulation of ERK3 expression and suggested an important role for ERK3 in promoting tumor cell growth and invasion in multiple cancers, in particular lung cancer. However, it is unknown whether or not ERK3 plays a role in spontaneous tumorigenesis. To determine the role of ERK3 in lung tumorigenesis, we created a conditional ERK3 transgenic mouse line in which ERK3 transgene expression is controlled by Cre recombinase. By crossing with a lung tissue-specific CCSP-iCre mouse line, we have found that conditional ERK3 overexpression cooperates with PTEN deletion to induce the formation of lung adenocarcinomas (LUADs). Mechanistically, ERK3 overexpression stimulates activating phosphorylations of ERBB3 and ERBB2 by upregulating SP1-mediated gene transcription of NRG1, a potent ligand for ERBB3/ERBB2. To our knowledge, our study is the first revealing a bona fide tumor-promoting role for ERK3 using genetically engineered mouse models. Together with previous findings showing important roles of ERK3 in cultured cells and in xenograft lung tumor model, our findings corroborate that ERK3 acts as an oncoprotein in promoting LUAD development and progression.
Proteomic Signatures of Microbial Adaptation to the Highest UV-Irradiation on Earth: Lessons from a Soil Actinobacterium
Authors: Zannier Federico , Portero Luciano Raúl , Douki Thierry , Gärtner Wolfgang , Farías María Eugenia , Albarracin Virginia Helena
Published: July 2021

In the Puna region, the total solar irradiation and the UV incidence is the highest on Earth, thus, restraining the physiology of individual microorganisms and the composition of microbial communities. UV-resistance of microbial strains thriving in High-Altitude Andean Lakes was demonstrated and their mechanisms were partially characterized by genomic analysis, biochemical and physiological assays. In this work, we present the molecular events involved in the adaptive response of the model HAAL extremophilic actinobacterium Nesterenkonia sp. Act20 under artificial UV-B radiation, herein called as UV-resistome. Proteomic profiles of cultures exposed to different UV-experimental conditions showed that the leading systems for adaptation to the UV-challenge in-vitro are DNA repair and antioxidant mechanisms.
Native MOWChIP-seq: Genome-wide profiles of key protein bindings reveal functional differences among various brain regions
Authors: Liu Zhengzhi , Naler Lynette B. , Zhu Yan , Deng Chengyu , Zhang Qiang , Zhu Bohan , Zhou Zirui , Sarma Mimosa , Murray Alexander , Xie Hehuang , Lu Chang
Published: July 2021

Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach, termed native MOWChIP-seq, produces high-quality binding profiles using 1000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.
ERK-mediated Curvature Feedback Regulates Branching Morphogenesis in Lung Epithelial Tissue
Authors: Hirashima Tsuyoshi , Matsuda Michiyuki
Published: July 2021

Intricate branching patterns emerge in internal organs because of the repetitive presence of simple deformations in epithelial tissues. During murine lung development, epithelial cells in distal tips of a single tube require fibroblast growth factor (FGF) signals generated by their surrounding mesenchyme to form repetitive tip bifurcations. However, it remains unknown how the cells employ FGF signaling to convert their behaviors to achieve the recursive branching processes. Here we show a self-sustained epithelial regulatory system during the murine lung branching morphogenesis, mediated by extracellular signal-regulated kinase (ERK), which acts as a downstream driver of FGF signaling. We found that tissue-scale curvature regulated ERK activity in the lung epithelium using two-photon live cell imaging and mechanical perturbations. ERK was activated specifically in epithelial tissues with a positive curvature, regardless of whether the change in curvature was attributable to morphogenesis or artificial perturbations. Moreover, ERK activation accelerated actin polymerization specifically at the apical side of cells, and mechanically contributed to the extension of the apical membrane, leading to a decrease in epithelial tissue curvature. These results indicate the existence of negative feedback loop between tissue curvature and ERK activity beyond scale. We confirmed that this regulation was sufficient to generate the recursive branching processes by a mathematical model. Taken together, we propose that ERK mediates the curvature feedback loop underlying the process of branching morphogenesis in developing lungs.
Isonicotinamide extends yeast chronological lifespan through a mechanism that diminishes nucleotides
Authors: Kalita Agata , Hesles Elisa Enriquez , Power Lindsey N. , Wang Dezhen , Singh Pankaj K. , Smith Jeffrey S.
Published: July 2021

Chronological lifespan (CLS) of budding yeast,Saccharomyces cerevisiae,is a commonly utilized model for cellular aging of non-dividing cells such as neurons. CLS is strongly extended by isonicotinamide (INAM), a non-metabolized isomer of the NAD+precursor nicotinamide (NAM), but the underlying mechanisms of lifespan extension remain uncharacterized. To identify potential biochemical INAM targets, we performed a chemical genetic screen with the yeast gene knockout (YKO) strain collection for INAM-hypersensitive mutants. Significantly enriched Gene Ontology terms that emerged included SWR1 and other transcription elongation factors, as well as metabolic pathways converging on one-carbon metabolism and contributing to nucleotide biosynthesis, together suggesting that INAM perturbs nucleotide pools. In line with this model, INAM effects on cell growth were synergistic with mycophenolic acid (MPA), which extends lifespan by reducing guanine nucleotide pools. Direct measurements of nucleotides and precursors by mass spectrometry indicated that INAM reduced nucleotides, including cAMP, at 24- and 96-hour time points post-inoculation. Taken together, we conclude that INAM extends CLS by perturbing nucleotide metabolism, which may be a common functional feature of multiple anti-aging interventions.
From telomere to telomere: the transcriptional and epigenetic state of human repeat elements
Authors: Hoyt Savannah J. , Storer Jessica M. , Hartley Gabrielle A. , Grady Patrick G. S. , Gershman Ariel , de Lima Leonardo G. , Limouse Charles , Halabian Reza , Wojenski Luke , Rodriguez Matias , Altemose Nicolas , Core Leighton J. , Gerton Jennifer L. , Makalowski Wojciech , Olson Daniel , Rosen Jeb , Smit Arian F. A. , Straight Aaron F. , Vollger Mitchell R. , Wheeler Travis J. , Schatz Michael C. , Eichler Evan E. , Phillippy Adam M. , Timp Winston , Miga Karen H. , O’Neill Rachel J.
Published: July 2021

Mobile elements and highly repetitive genomic regions are potent sources of lineage-specific genomic innovation and fingerprint individual genomes. Comprehensive analyses of large, composite or arrayed repeat elements and those found in more complex regions of the genome require a complete, linear genome assembly. Here we present the first de novo repeat discovery and annotation of a complete human reference genome, T2T-CHM13v1.0. We identified novel satellite arrays, expanded the catalog of variants and families for known repeats and mobile elements, characterized new classes of complex, composite repeats, and provided comprehensive annotations of retroelement transduction events. Utilizing PRO-seq to detect nascent transcription and nanopore sequencing to delineate CpG methylation profiles, we defined the structure of transcriptionally active retroelements in humans, including for the first time those found in centromeres. Together, these data provide expanded insight into the diversity, distribution and evolution of repetitive regions that have shaped the human genome.
Multiplex viral tropism assay in complex cell populations with single-cell resolution
Authors: Tat Keng Choong , Ke Guo , Shern Daryl Lim , Chew Wei Leong
Published: July 2021

Gene therapy constitutes one of the most promising modes of disease treatments. Two key properties for therapeutic delivery vectors are the transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into the other unintended cells within the body). Here we developed a novel technology that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. This library- on-library technology is important for determining the safety and efficacy of therapeutic delivery vectors.
IgG surface mobility promotes antibody dependent cellular phagocytosis by Syk and Arp2/3 mediated reorganization of Fcγ receptors in macrophages
Authors: Jo Seongwan , Cronin Nicholas M. , Dewi Nurmalasari Ni Putu , Kerkvliet Jason G. , Bailey Elizabeth M. , Anderson Robert B. , Scott Brandon L. , Hoppe Adam D.
Published: July 2021

By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fcγ receptors (FcγR) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to FcγR signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger redistribution of Rituximab-FcγR during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of FcγR-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and FcγR-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens.

Significance Statement

ADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange FcγR-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.
Disentangling microbial networks across pelagic zones in the global ocean
Authors: Deutschmann Ina M. , Delage Erwan , Giner Caterina R. , Sebastián Marta , Poulain Julie , Arístegui Javier , Duarte Carlos M. , Acinas Silvia G. , Massana Ramon , Gasol Josep M. , Eveillard Damien , Chaffron Samuel , Logares Ramiro
Published: July 2021

Microbial interactions underpin ocean ecosystem function, but they remain barely known. Multiple studies have analyzed microbial interactions using static association networks based on omics data, yet microbial interactions are dynamic and can change across spatiotemporal scales. Understanding the dynamics of microbial interactions is needed for a better comprehension of ocean ecosystems. Here, we explored associations between archaea, bacteria, and picoeukaryotes along the water column, from the surface to the deep ocean, across the northern subtropical to the southern temperate ocean and the Mediterranean Sea by defining sample-specific subnetworks, which allowed us to examine changes in microbial associations across space. We found that associations tend to change with depth as well as with geographical scale, with a few associations being global (i.e., present across regions within the same depth layer) and 11-36% being regional within specific water layers. The lowest fraction of global associations was found in the bathypelagic zone, while associations restricted to certain regions increased with depth. The majority of associations observed in surface waters disappeared with depth, suggesting that surface ocean associations are not transferred to the deep sea, despite microbial sinking. Altogether, our results suggest that microbial associations have highly heterogeneous distributions in the horizontal and vertical dimensions of the ocean and that such distributions do not mirror taxonomic distributions. Our work contributes to better understand the dynamics of microbial interactions in the global ocean, which is urgently needed in a context of global change.
Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer
Authors: Bell Emily S. , Shah Pragya , Zuela-Sopilniak Noam , Kim Dongsung , Varlet Alice-Anais , Morival Julien L.P. , McGregor Alexandra L. , Isermann Philipp , Davidson Patricia M. , Elacqua Joshua J. , Lakins Jonathan N. , Vahdat Linda , Weaver Valerie M. , Smolka Marcus B. , Span Paul N. , Lammerding Jan
Published: July 2021

Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.
Genome-resolved metagenomic analyses reveal the presence of a bacterial endosymbiont in an avian nasal mite (Rhinonyssidae; Mesostigmata)
Authors: Osuna-Mascaró Carolina , Doña Jorge , Johnson Kevin P. , Rojas Manuel de
Published: July 2021

Rhinonyssidae (Mesostigmata) is a family of nasal mites only found in birds. All species are hematophagous endoparasites, which may damage the nasal cavities of birds, and also could be potential reservoirs or vectors of other infections. However, the role of members of Rhinonyssidae as disease vectors in wild bird populations remains uninvestigated, with studies of the microbiomes of Rhinonyssidae being almost non-existent. In the nasal mite (Tinaminyssus melloi) from rock doves (Columba livia), a previous study found evidence of a highly abundant putatively endosymbiotic bacteria from Class Alphaproteobacteria. Here, we expanded the sample size of this species, incorporated contamination controls, and increased sequencing depth in shotgun sequencing and genome-resolved metagenomic analyses. Our goal was to increase the information regarding this mite species with its putative endosymbiont. Our results support the endosymbiotic nature of this bacterial taxon, which is the first described for bird’s nasal mites to date, and improve the overall understanding of the microbiota inhabiting these mites.
An integrated analysis tool reveals intrinsic biases in gene set enrichment
Authors: Thakur Nishant , Pujol Nathalie , van Helden Jacques , Waterston Robert H. , Hillier LaDeana W. , Tichit Laurent , Ewbank Jonathan J.
Published: July 2021

Generating meaningful interpretations of gene lists remains a challenge for all large-scale studies. Many approaches exist, often based on evaluating gene enrichment among pre-determined gene classes. Here, we conceived and implemented yet another analysis tool (YAAT), specifically for data from the widely-used model organism C. elegans. YAAT extends standard enrichment analyses, using a combination of co-expression data and profiles of phylogenetic conservation, to identify groups of functionally-related genes. It additionally allows class clustering, providing inference of functional links between groups of genes. We give examples of the utility of YAAT for uncovering unsuspected links between genes and show how the approach can be used to prioritise genes for in-depth study. Our analyses revealed several limitations to the meaningful interpretation of gene lists, specifically related to data sources and the “universe” of gene lists used. We hope that YAAT will represent a model for integrated analysis that could be useful for large-scale exploration of biological function in other species.
Joint analysis of microsatellites and flanking sequences enlightens complex demographic history of interspecific gene flow and vicariance in rear-edge oak populations
Authors: Lepais Olivier , Aissi Abdeldjalil , Véla Errol , Beghami Yassine
Published: July 2021

Inference of recent population divergence requires fast evolving markers and necessitates to differentiate shared genetic variation caused by ancestral polymorphism and gene flow. Theoretical research shows that the use of compound marker systems integrating linked polymorphisms with different mutational dynamics, such as a microsatellite and its flanking sequences, can improve estimation of population structure and inference of demographic history, especially in the case of complex population dynamics. However, empirical application in natural populations has so far been limited by lack of suitable methods for data collection. A solution comes from the development of sequence-based microsatellite genotyping which we used to study molecular variation at 36 sequenced nuclear microsatellites in sevenQuecus canariensisand fourQuercus faginearear-edge populations across Algeria, to decipher their taxonomic relationship, past evolutionary history and recent demographic trajectory. First, we compare the estimation of population genetics parameters and simulation-based inference of demographic history from microsatellite sequence alone, flanking sequence alone or the combination of linked microsatellite and flanking sequence variation. Second, we measure variable importance from random forest approximate Bayesian computation to identify which of these sequence types is most informative. Whereas the analysis of microsatellite variation alone indicates recent interspecific gene flow, additional information gained from combining with nucleotide variation in flanking sequences, by reducing homoplasy, suggests ancient interspecific gene flow followed by drift in isolation instead. The weight of each polymorphism in the inference also demonstrates the value of linked variations with contrasted mutation dynamic to improve estimation of both demographic and mutational parameters.
High throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments
Authors: Deger Teoman , Boers Ruben G , de Weerd Vanja , Angus Lindsay , van der Put Marjolijn MJ , Boers Joachim B , Azmani Zakia , Ijcken Wilfred FJ van , Grünhagen Dirk J , van Dessel Lisanne F , Lolkema Martijn PJK , Verhoef Cornelis , Sleijfer Stefan , Martens John WM , Gribnau Joost , Wilting Saskia M
Published: July 2021

Background

DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum-concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery.

Results

The MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QIAsymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (P<0.001). Vacuum-concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4, were validated by quantitative methylation specific PCR.

Conclusions

The MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.
In situ solid-liquid extraction enhances recovery of taxadiene from engineered S. cerevisiae cell factories
Authors: Santoyo-Garcia Jorge H. , Walls Laura E. , Nowrouzi Behnaz , Ochoa-Villareal Marisol , Loake Gary J. , Dimartino Simone , Rios-Solis Leonardo
Published: July 2021

A novel in situ solid phase adsorption strategy was investigated for enhanced recovery of taxadiene, a precursor to the blockbuster anticancer drug, paclitaxel, from engineered Saccharomyces cerevisiae. A synthetic adsorbent resin (HP-20) was employed to capture taxadiene across a range of cultivation scales. Cultivations from 12 % (w/v) resin concentration resulted in bead fragmentation which were found to be detrimental to cellular growth. After cultivation, the use of acetone for desorption captured intracellular and secreted taxadiene, achieving an integration of the bioprocess. Implementation of the proposed method at microscale (2 mL) and benchtop bioreactor scale (250 mL) resulted in 1.9-fold and 1.4-fold increments in taxadiene titer, respectively, compared to the extraction method using a dodecane overlay. Taxadiene was found to be distributed between resin beads and biomass in a ratio of 50 %. Finally, a maximum taxadiene titer of 76 ± 19 mg/L was achieved in the benchtop bioreactor cultivations.
The presence of corticomuscular coherence during unipedal stance
Authors: Alkaff Sylmina Dalily , Ushiyama Junichi
Published: July 2021

Standing in unipedal stance requires higher effort to maintain posture balance within narrow base-of-support. Although changes in cortical activity are known to occur as standing task difficulty increased, it is unclear whether it indicates a change in cortical control of muscle activity. To elucidate this point, this study examined corticomuscular coherence (CMC) between the sensorimotor cortex and ankle joint muscles during three tasks such as bipedal stance, unipedal stance, and isometric contraction tasks. For twenty-one healthy participants, we measured the maximal peak of CMC (CMCmax) between electroencephalograms overlying the foot representation area and surface electromyograms from the tibialis anterior (TA), medial gastrocnemius (MG), lateral gastrocnemius (LG), and soleus (SOL), respectively, for each task. We also measured the center of pressure (COP) during both stance tasks. Although there was no significant CMC during bipedal stance in all participants, most participants (n = 14) showed significant CMC for all muscles during unipedal stance with larger COP fluctuation in most participants. Indeed, there was significant difference in CMCmax between unipedal and bipedal stance tasks. The greater CMC would indicate increased cortical control of muscle activity to compensate for postural instability during unipedal stance.
The spatial landscape of clonal somatic mutations in benign and malignant tissue
Authors: Erickson Andrew , Berglund Emelie , He Mengxiao , Marklund Maja , Mirzazadeh Reza , Schultz Niklas , Bergenstråhle Ludvig , Kvastad Linda , Andersson Alma , Bergenstråhle Joseph , Larsson Ludvig , Shamikh Alia , Basmaci Elisa , De Ståhl Teresita Diaz , Rajakumar Timothy , Thrane Kim , Ji Andrew L , Khavari Paul A , Tarish Firaz , Tanoglidi Anna , Maaskola Jonas , Colling Richard , Mirtti Tuomas , Hamdy Freddie C , Woodcock Dan J , Helleday Thomas , Mills Ian G. , Lamb Alastair D , Lundeberg Joakim
Published: July 2021

Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. Here, we provide an unsupervised approach to study spatial genome integrity in situ to gain molecular insight into clonal relationships. We employed spatially resolved transcriptomics to infer spatial copy number variations in >120 000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
Drug-free nasal spray as a barrier against SARS-CoV-2 infection: safety and efficacy in human nasal airway epithelia
Authors: Fais Fabio , Juskeviciene Reda , Francardo Veronica , Mateos Stéphanie , Constant Samuel , Borelli Massimo , Hohenfeld Ilja P. , Meyer Thomas
Published: July 2021

Background

For SARS-CoV-2 and other respiratory viruses, the nasal epithelium is a key portal for infection. Therefore, the nose is an important target of prophylactic and therapeutic interventions against these viruses. We developed a nasal spray (AM-301, a medical device marketed as Bentrio) to protect against infection by SARS-CoV-2 and potentially other viruses.

Aims of the study

To test the safety and efficacy of AM-301 against SARS-CoV-2 infection.

Methods

AM-301 was tested on an in vitro 3D model of primary human nasal airway epithelium. Safety was assessed in assays for tight junction integrity, cytotoxicity and cilia beating frequency. Efficacy against SARS-CoV-2 infection was evaluated in prophylaxis and infection mitigation assays.

Results

AM-301 did not have any detrimental effect on the nasal epithelium. Prophylactic treatment with AM-301 reduced viral titer significantly vs. controls over 4 days, reaching a maximum reduction of 99%. When treatment with AM-301 was started 24 or 30 h after infection, epithelia that received the formulation had a 12- or 14-fold lower titer than controls.

Conclusion

AM-301 was found to be safe in vitro, and it significantly decelerated viral titer growth in experimental models of prophylaxis and mitigation. Its physical (non-pharmaceutical) mechanism of action, safety and efficacy pave the way for further investigation of its possible use against a broad spectrum of viruses, allergens and pollutants.
Serial dependence tracks objects and scenes in parallel and independently
Authors: Collins Thérèse
Published: July 2021

The visual world is made up of objects and scenes. Object perception requires both discriminating an individual object from others and binding together different perceptual samples of that object across time. Such binding manifests by serial dependence, the attraction of the current perception of a visual attribute towards values of that attribute seen in the recent past. Scene perception is subserved by global mechanisms like ensemble perception, the rapid extraction of the average feature value of a group of objects. The current study examined to what extent the perception of single objects in multi-object scenes depended on previous feature values of that object, or on the average previous attribute of all objects in the scene. Results show that serial dependence occurs independently on two simultaneously present objects, that ensemble perception depends only on previous ensembles, and that serial dependence of an individual object occurs only on the features of that particular object. These results suggest that the temporal integration of successive perceptual samples operates simultaneously at independent levels of visual processing.
Creating a bottleneck: Robust LC3B lipidation analysis by adjusting autophagic flux with low concentrations of Bafilomycin A1
Authors: Liebl Martina P. , Meister Sarah C. , Frey Lisa , Hendrich Kristina , Klemmer Anja , Pohl Christian , Lakics Viktor
Published: July 2021

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.
Therapeutic base and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa
Authors: Hong Sung-Ah , Kim Song-Ee , Lee A-young , Hwang Gue-Ho , Kim Jong Hoon , Iwata Hiroaki , Kim Soo-Chan , Bae Sangsu , Lee Sang Eun
Published: July 2021

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). We used base and prime editing to correct mutations in fibroblasts from two patients (Pat1, who carried a c.3631C>T mutation in one allele, and Pat2, who carried a c.2005C>T mutation in one allele). We applied adenine base editors (ABEs) to correct the pathogenic mutation or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that both ABE- and PE-mediated correction of COL7A1 mutations restored full-length C7 expression, reversed the impaired adhesion and proliferation exhibited by the patient-derived fibroblasts, and, following transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice, led to C7 deposition within the dermal-epidermal junction. These results suggest that base and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.
Arousal impacts distributed hubs modulating the integration of brain functional connectivity
Authors: Lee Kangjoo , Horien Corey , O’Connor David , Garand-Sheridan Bronwen , Tokoglu Fuyuze , Scheinost Dustin , Lake Evelyn M.R. , Constable R. Todd
Published: July 2021

Even when subjects are at rest, it is thought that brain activity is organized into distinct brain states during which reproducible patterns are observable. Yet, it is unclear how to define or distinguish different brain states. A potential source of brain state variation is arousal, which may play a role in modulating functional interactions between brain regions. Here, we use simultaneous resting state functional magnetic resonance imaging and pupillometry to study the impact of arousal levels indexed by pupil area on the integration of large-scale brain networks. We employ a novel sparse dictionary learning-based method to identify hub regions participating in between-network integration stratified by arousal, by measuring k-hubness, the number (k) of functionally overlapping networks in each brain region. We show evidence of a brain-wide decrease in between-network integration and inter-subject variability at low relative to high arousal, with differences emerging across regions of the frontoparietal, default mode, motor, limbic, and cerebellum networks. State-dependent changes in k-hubness relate to the actual patterns of network integration within these hubs, suggesting a brain state transition from high to low arousal characterized by global synchronization and reduced network overlaps. We demonstrate that arousal is not limited to specific brain areas known to be directly associated with arousal regulation, but instead has a brain-wide impact that involves high-level between-network communications.
Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease
Authors: Mouali Youssef El , Ponath Falk , Scharrer Vinzent , Wenner Nicolas , Hinton Jay C. D. , Vogel Jörg
Published: July 2021

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

SIGNIFICANCE

Proteins that interact with RNA play a vital role in controlling key functions in pathogenic bacteria. RNA-binding proteins regulate how, when and where bacteria feed, swim or interact with a host, and it is critical that we understand how RNAs associate with these proteins. ProQ is one of the three major RNA-binding proteins (RBPs) in Gram-negative bacteria. In this study, we mapped the amino acid residues of ProQ that are essential for function. We successfully identified residue substitutions that rendered the ProQ RBP both non-functional and unable to interact with RNA. Our findings raise the possibility that the Lon protease mediates a quality control mechanism of ProQ that targets this RBP in the absence of RNA. A posttranslational quality control mechanism of this type could prevent the accumulation of nonfunctional RBPs in the bacterial cytoplasm.
Determining molecular archetype composition and expression from bulk tissues with unsupervised deconvolution
Authors: Wu Chiung-Ting , Chen Lulu , Herrington David M. , Shen Minjie , Yu Guoqiang , Clarke Robert , Liu Chunyu , Wang Yue
Published: July 2021

Complex tissues are composite ecological systems whose components interact with each other to create a unique physiological or pathophysiological state distinct from that found in other tissue microenvironments. To explore this ground yet dynamic state, molecular profiling of bulk tissues and mathematical deconvolution can be jointly used to characterize heterogeneity as an aggregate of molecularly distinct tissue or cell subtypes. We first introduce an efficient and fully unsupervised deconvolution method, namely the Convex Analysis of Mixtures – CAM3.0, that may aid biologists to confirm existing or generate novel scientific hypotheses about complex tissues in many biomedical contexts. We then evaluate the CAM3.0 functional pipelines using both simulations and benchmark data. We also report diverse case studies on bulk tissues with unknown number, proportion and expression patterns of the molecular archetypes. Importantly, these preliminary results support the concept that expression patterns of molecular archetypes often reflect the interactive not individual contributions of many known or novel cell types, and unsupervised deconvolution would be more powerful in uncovering novel multicellular or subcellular archetypes.
Substrate recognition determinants of human eIF2α phosphatases
Authors: Hodgson George , Andreeva Antonina , Bertolotti Anne
Published: July 2021

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.