bluuebunny/crossref_metadata_2025 huggingface dataset:
Aquatic long-term persistence ofFrancisella tularensisssp.holarcticais driven by water temperature and transition to a viable but non-culturable state
Authors: Brunet Camille D. , Peyroux Julien , Pondérand Léa , Bouillot Stéphanie , Girard Thomas , Faudry Éric , Maurin Max , Caspar Yvan
Published: February 2022

Francisella tularensisis a highly virulent bacterium causing tularemia zoonosis. An increasing proportion of infections occur through contaminated hydro-telluric sources, especially for the subspeciesholarctica(Fth). Although this bacterium has been detected in several aquatic environments, the mechanisms of its long-term persistence in water are not yet elucidated. We evaluated the culturability and the viability of a virulentFthstrain in independent microcosms filled with nutrient-poor water. At 37°C, the bacteria remained culturable for only one week, while culturability was extended to 6 weeks at 18°C and up to 11 weeks at 4°C. However, while the viability of the bacteria declined similarly to culturability at 37°C, the viability of the bacteria remained stable overtime at 18°C and 4°C for more than 24 months, long after loss of culturability. We identified water temperature as one of the major factors driving the aquatic survival ofFththrough a transition of the wholeFthpopulation in a viable but non-culturable (VBNC) state. Low temperature of water (≤18°C) favors the persistence of the bacteria in a VBNC state, while a temperature above 30°C kills culturable and VBNCFthbacteria. These findings provide new insights into the environmental cycle ofFrancisella tularensisthat suggest that the yet unidentified primary reservoir of the subspeciesholarcticamay be the aquatic environment itself in which the bacteria could persist for months or years without the need for a host.
SARS-CoV-2 has not emerged in roe, red or fallow deer in Germany or Austria during the COVID 19 pandemic
Authors: Moreira-Soto Andres , Walzer Christian , Czirják Gábor Á. , Richter Martin , Marino Stephen F. , Posautz Annika , de Yebra Pau Rodo , McEwen Gayle K. , Drexler Jan Felix , Greenwood Alex D.
Published: February 2022

Spillover of SARS-CoV-2 to North American white tailed deer (Odocoileus virginianus) has been documented. We evaluated pre and pandemic exposure of German and Austrian deer species using a SARS-CoV-2 pseudoneutralization assay. In stark contrast to North American white tailed deer, we found no evidence of SARS-CoV-2 exposure.

Article Summary Line

Using a sensitive serological assay, 433 pre and pandemic deer samples from Germany and Austria tested negative for SARS-CoV-2-specific antibodies highlighting a major difference between central European and North American deer exposure and in their epidemiologic roles.
Unsupervised physiological noise correction of fMRI data using phase and magnitude information (PREPAIR)
Authors: Bancelin David , Bachrata Beata , Bollmann Saskia , de Lima Cardoso Pedro , Szomolanyi Pavol , Trattnig Siegfried , Robinson Simon Daniel
Published: February 2022

Of the sources of noise which affect BOLD fMRI, respiration and cardiac fluctuations are responsible for the largest part of the variance, particularly at high and ultra-high field. Existing approaches to removing physiological noise either use external recordings, which can be unwieldy and unreliable, or attempt to identify physiological noise from the magnitude fMRI data. Data-driven approaches are limited by sensitivity, temporal aliasing and the need for user interaction. In the light of the sensitivity of the phase of the MR signal to local changes in the field stemming from physiological processes, we have developed an unsupervised physiological noise correction method which uses the information carried in both the phase and the magnitude of EPI data. Our technique, Physiological Regressor Estimation from Phase and mAgnItude, sub-tR (PREPAIR) derives time series signals which are sampled at the slice TR from both phase and magnitude images. It allows physiological noise to be captured without aliasing, and efficiently removes other sources of signal fluctuations which are not related to physiology, prior to regressor estimation. We demonstrate that the physiological signal time courses identified with PREPAIR not only agree well with those from external devices, but also retrieve challenging cardiac dynamics. The removal of physiological noise was as effective as that achieved with the most commonly used approach based on external recordings, RETROICOR. In comparison with widely used physiological noise correction tools which do not use external signals, PESTICA and FIX, PREPAIR removed more respiratory and cardiac noise and achieved a larger increase in tSNR at both 3 T and 7 T.
Alternative c-MYC mRNA transcripts as an additional tool for c-Myc2 and c-MycS production in BL60 tumors
Authors: Ibrahim Dina , Faumont Nathalie , Troutaud Danielle , Feuillard Jean , Diab-Assaf Mona , Oulmouden Ahmad
Published: February 2022

While studying c-Myc protein expression in several Burkitt lymphoma cell lines and in lymph nodes from a mouse model bearing a translocated c-MYC gene from the human BL line IARC-BL60, we surprisingly discovered a complex electrophoretic profile. Indeed, the BL60 cell line carrying the t(8;22) c-MYC translocation exhibits a simple pattern, with a single c-Myc2 isoform. Analysis of the c-MYC transcripts expressed by tumor lymph nodes in the mouse λc-MYC (Avy/a) showed for the first time five transcripts associated with t(8;22) c-MYC translocation. The five transcripts were correlated with the production of c-Myc2 and c-MycS, and loss of c-Myc1. The contribution of these transcripts to the oncogenic activation of the t(8;22) c-MYC is discussed.
Cardiac and gastric interoception have distinct neural substrates
Authors: Haruki Yusuke , Ogawa Kenji
Published: February 2022

Interoception, or an awareness of the internal body state, guides agents in adaptive behavior by informing them of ongoing bodily signals, such as heart rate or energy status. However, it is still unclear whether the human brain represents the differences in the subjective experience of interoception differently. Hence, we directly compared the neural activation for cardiac (awareness related to heartbeats) and gastric (awareness related to the stomach) interoception in the same population (healthy human, N = 31). Participants were asked to focus on their heart and stomach sensations to be aware of interoception in a magnetic resonance imaging scanner. The results indicated that neural activation underlying gastric interoception encompassed larger brain regions, including the occipitotemporal visual cortices, bilateral primary motor cortex, primary somatosensory cortex, left orbitofrontal cortex, and bilateral hippocampal regions. Cardiac interoception, however, selectively activated the right anterior insula extending to the frontal operculum more compared to gastric interoception. Moreover, our detailed analyses focusing on the insula, the most relevant region for interoception, revealed that the left dorsal middle insula encoded cardiac and gastric interoception in different activation patterns but not the posterior insula. Our results demonstrate that cardiac and gastric interoception have distinct neural substrates; in particular, the selective brain activation may reflect differences in the functional roles of cardiac and gastric interoception.

Significance statement

Interoception, subjective senses that arise from within the body, plays a critical role in maintaining adaptive behavior by informing of the ongoing bodily states, such as heart rate and energy status. Although interoception has various characteristics depending on its source signals, previous neuroimaging studies have extensively used cardiac interoception (senses related to heartbeats), making it unclear whether the brain differently encodes diverse experiences of interoception. Here, we demonstrate that cardiac interoception and gastric interoception (senses related to the stomach) have distinct neural substrates by combining mass-univariate analysis with multivoxel pattern analysis for fMRI data. Our findings suggest that the selective brain activation may reflect differences in the functional roles of cardiac and gastric interoception.
MicroRNA-155 regulates osteogenesis and bone mass phenotype via targeting S1PR1 gene
Authors: Zheng Zhichao , Wu Lihong , Li Zhicong , Tang Ruoshu , Li Hongtao , Huang Yinyin , Ye Zhitong , Xiao Dong , Lin Xiaolin , Wu Gang , Jaspers Richard T , Pathak Janak L.
Published: February 2022

MicroRNA-155 (miR155) is overexpressed in various inflammatory diseases and cancer, in which bone resorption and osteolysis are frequently observed. However, the role of miR155 on osteogenesis and bone mass phenotype is still unknown. Here, we report a low bone mass phenotype in the long bone of miR155-Tg mice compared with control mice. In contrast, miR155-KO mice showed a high bone mass phenotype. miR155-KO mice showed robust bone regeneration in the ectopic and orthotopic model, but miR155-Tg mice showed compromised bone regeneration compared with the control mice. Similarly, the osteogenic differentiation potential of bone marrow stromal stem cells (BMSCs) from miR155-KO mice was robust and miR155-Tg was compromised compared with that of control mice. Moreover, miR155 knockdown in BMSCs from control mice showed higher osteogenic differentiation potential, supporting the results from miR155-KO mice. TargetScan analysis predicted S1PR1 as a target gene of miR155, which was further confirmed by luciferase assay and miR155 knockdown. S1PR1 overexpression in BMSCs robustly promoted osteogenic differentiation without affecting cell viability and proliferation. Thus, miR155 showed a catabolic effect on osteogenesis and bone mass phenotype via interaction with the S1PR1 gene, suggesting inhibition of miR155 as a potential strategy for bone regeneration and bone defect healing.
Hinokitiol alters Gene Expression in Aspergillus fumigatus, protects against fungal keratitis by Reducing Fungal Load, LOX-1, Proinflammatory cytokines and Neutrophil Infiltration
Authors: Ma Shuqin , Lin Hao , Peng Xudong , Li Cui , Wang Qian , Xu Qiang , He Mengting , Shao Dan , Liu Xing , Zhao Guiqiu , Lin Jing
Published: February 2022

Hinokitiol (HK), a tropolone-related compound found in cupressaceous plants, has antifungal and anti-inflammatory properties. But its roles in Aspergillus fumigatus ( A.F. ) keratitis remains unknown. This study examined the antifungal activity of HK against A.F. and investigated its possible mechanisms at the ultrastructural and transcriptional levels. We tested the minimum inhibitory concentration (MIC) of HK against A.F.. Our data showed that the MIC50 was 2 μg/ml and MIC90 was 8 μg/ml, and HK could significantly inhibit the spore germination and mycelial growth of A.F.. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations showed that HK induced significant changes in hyphal morphology and microstructure, including cell membrane rupture and intracellular structural disorder. Adhesion assay and biofilm assay experiments showed that HK reduced the adhesion of spores to human corneal epithelial cells (HCECs) and inhibited the formation of biofilms. The genetic changes of A.F. after HK treatment were analyzed by RNA sequencing, and a total of 2487 differentially expressed genes (DEGs) were identified, including the down-regulated DEGs related to carbohydrate and various amino acid metabolism, ribosome biogenesis, asexual reproduction, and up-regulated DEGs related to steroid biosynthesis through GO and KEGG enrichment analysis. RNA-seq results suggested that HK may play an antifungal effect by destroying the structural integrity of its wall and membrane, inhibiting protein biosynthesis and the growth and reproduction of A.F..10 μg/ml HK, which had no effect on proliferation and migration of HCECs, was used in the treatment of murine fungal keratitis (FK). We found that HK eyedrop treatment could reduce the severity of FK by reducing corneal fungal burden, neutrophil infiltration, and the expression of LOX-1 and pro-inflammatory factors. HK is expected to be a safe and effective new drug for FK treatment.

Author summary

FK is a serious common blind-causing eye disease. The current clinical antifungal drugs have the disadvantages of low bioavailability and high corneal toxicity, which greatly limit the clinical effectiveness. Therefore, there is an urgent need for a new, safe and effective treatment for FK. HK had shown great antifungal capacity against A.F., amazingly well anti-inflammatory effect and real improvement in murine FK in our research. These findings provide a theoretical basis for the application of HK as an antifungal drug and reveal the potential of HK to be applied in the clinical treatment of FK.
Systematic Evaluation of Different R-loop Mapping Methods: Achieving Consensus, Resolving Discrepancies and Uncovering Distinct Types of RNA:DNA Hybrids
Authors: Chen Jia-Yu , Lim Do-Hwan , Chen Liang , Zhou Yongli , Zhang Fangliang , Shao Changwei , Zhang Xuan , Li Hairi , Wang Dong , Zhang Dong-Er , Fu Xiang-Dong
Published: February 2022

R-loop, a three-stranded nucleic acid structure, has been recognized to play pivotal roles in critical physiological and pathological processes. Multiple technologies have been developed to profile R-loops genome-wide, but the existing data suffer from major discrepancies on determining genuine R-loop localization and its biological functions. Here, we experimentally and computationally evaluate eight representative R-loop mapping technologies, and reveal inherent biases and artifacts of individual technologies as key sources of discrepancies. Analyzing signals detected with different R-loop mapping strategies, we note that genuine R-loops predominately form at gene promoter regions, whereas most signals in gene body likely result from structured RNAs as part of repeat-containing transcripts. Interestingly, our analysis also uncovers two classes of R-loops: The first class consists of typical R-loops where the single-stranded DNA binding protein RPA binds both the template and non-template strands. By contrast, the second class appears independent of Pol II-mediated transcription and is characterized by RPA binding only in the template strand. These two different classes of RNA:DNA hybrids in the genome suggest distinct biochemical activities involved in their formation and regulation. In sum, our findings will guide future use of suitable technology for specific experimental purposes and the interpretation of R-loop functions.
ABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA mass spectrometry methods
Authors: Jones Hannah B. L. , Heilig Raphael , Davis Simon , Fischer Roman , Kessler Benedikt M. , Pinto-Fernández Adán
Published: February 2022

Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling), that in combination with DIA MS methods allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day.
Accurate staging of chick embryonic tissues via deep learning
Authors: Groves Ian , Holmshaw Jacob , Furley David , Towers Matthew , Evans Benjamin D. , Placzek Marysia , Fletcher Alexander G.
Published: February 2022

Recent work has indicated a need for increased temporal resolution for studies of the early chick brain. Over a 10-hour period, the developmental potential of progenitor cells in the HH10 brain changes, and concomitantly, the brain undergoes subtle changes in morphology. We asked if we could train a deep convolutional neural network to sub-stage HH10 brains from a small dataset (<200 images). By augmenting our images with a combination of biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (<250) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data.

SUMMARY STATEMENT

We train a deep convolutional network that can be generally applied to accurately classify chick embryos from images. Saliency analyses show that classification is based on biologically relevant features.
Representational dynamics of memories for real-life events
Authors: Jeunehomme Olivier , Heinen Rebekka , Stawarczyk David , Axmacher Nikolai , D’Argembeau Arnaud
Published: February 2022

The continuous flow of experience that characterizes real-life events is not recorded as such in episodic memory but is condensed as a succession of event segments separated by temporal discontinuities. To unravel the neural basis of this representational structure, we recorded real-life events using wearable camera technology and used fMRI to investigate brain activity during their temporal unfolding in memory. We found that, compared to the representation of static scenes in memory, dynamically unfolding memory representations were associated with greater activation of the posterior medial episodic network. Strikingly, by analyzing the autocorrelation of brain activity patterns at successive time points throughout the retrieval period, we found that this network showed higher temporal dynamics when recalling events that included a higher density of event segments. These results reveal the key role of the posterior medial network in representing the dynamic unfolding of the event segments that constitute real-world memories.
Self-organization of songbird neural sequences during social isolation
Authors: Mackevicius Emily L. , Gu Shijie , Denisenko Natalia I. , Fee Michale S.
Published: February 2022

Behaviors emerge via a combination of experience and innate predis-positions. As the brain matures, it undergoes major changes in cellular, network and functional properties that can be due to sensory experience as well as developmental processes. In normal birdsong learning, neural sequences emerge to control song syllables learned from a tutor. Here, we disambiguate the role of experience and development in neural sequence formation by delaying exposure to a tutor. Using functional calcium imaging, we observe neural sequences in the absence of tutoring, demonstrating that experience is not necessary for the formation of sequences. However, after exposure to a tutor, pre-existing sequences can become tightly associated with new song syllables. Since we delayed tutoring, only half our birds learned new syllables following tutor exposure. The birds that failed to learn were the birds in which pre-tutoring neural sequences were most ‘crystallized’, that is, already tightly associated with their (untutored) song.
Substrate stiffness impacts early biofilm formation by modulatingPseudomonas aeruginosatwitching motility
Authors: Ho Sofia Gomez , Bureau Lionel , John Karin , Débarre Delphine , Lecuyer Sigolène
Published: February 2022

Surface-associated lifestyles dominate in the bacterial world. Large multicellular assemblies, called biofilms, are essential to the survival of bacteria in harsh environments, and are closely linked to antibiotic resistance in pathogenic strains. Biofilms stem from the surface colonization of a wide variety of substrates encountered by bacteria, from living tissues to inert materials. Here, we demonstrate experimentally that the promiscuous opportunistic pathogenPseudomonas aeruginosaexplores substrates differently based on their rigidity, leading to striking variations in biofilm structure, exopolysaccharides (EPS) distribution, strain mixing during co-colonization and phenotypic expression. Using simple kinetic models, we show that these phenotypes arise through a mechanical interaction between the elasticity of the substrate and the type IV pilus (T4P) machinery, that mediates the surface-based motility called twitching. Together, our findings reveal a new role for substrate softness in the spatial organization of bacteria in complex microenvironments, with far-reaching consequences on efficient biofilm formation.
Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans
Authors: Setty Hagar , Salzberg Yehuda , Karimi Shadi , Berent-Barzel Elisheva , Krieg Michael , Oren-Suissa Meital
Published: February 2022

How sensory perception is processed by the two sexes of an organism is still only partially understood. Despite some evidence for sexual dimorphism in auditory and olfactory perception, whether touch is sensed in a dimorphic manner has not been addressed. Here we find that the neuronal circuit for tail mechanosensation in C. elegans is wired differently in the two sexes and employs a different combination of sex-shared sensory neurons and interneurons in each sex. Reverse genetic screens uncovered cell- and sex-specific functions of the alpha-tubulin mec-12 and the sodium channel tmc-1 in sensory neurons, and of the glutamate receptors nmr-1 and glr-1 in interneurons, revealing the underlying molecular mechanisms that mediate tail mechanosensation. Moreover, we show that only in males, the sex-shared interneuron AVG is strongly activated by tail mechanical stimulation, and accordingly is crucial for their behavioral response. Importantly, sex reversal experiments demonstrate that the sexual identity of AVG determines both the behavioral output of the mechanosensory response and the molecular pathways controlling it. Our results present for the first time extensive sexual dimorphism in a mechanosensory circuit at both the cellular and molecular levels.
Protein compactness and interaction valency define the architecture of a biomolecular condensate across scales
Authors: Polyansky Anton A. , Gallego Laura D. , Efremov Roman G. , Köhler Alwin , Zagrovic Bojan
Published: February 2022

Non-membrane-bound biomolecular condensates have been proposed to represent an important mode of subcellular organization in diverse biological settings. However, the fundamental principles governing the spatial organization and dynamics of condensates at the atomistic level remain unclear. TheS. cerevisiaeLge1 protein is required for histone H2B ubiquitination and its N-terminal intrinsically disordered fragment (Lge11-80) undergoes robust phase separation. This study connects single- and multi-chain all-atom molecular dynamics simulations of Lge11-80with thein vitrobehavior of Lge11-80condensates. Analysis of modelled protein-protein interactions elucidates the key determinants of Lge11-80condensate formation and links configurational entropy, valency and compactness of proteins inside the condensates. A newly derived analytical formalism, related to colloid fractal cluster formation, describes condensate architecture across length scales as a function of protein valency and compactness. In particular, the formalism provides an atomistically resolved model of Lge11-80condensates on the scale of hundreds of nanometers starting from individual protein conformers captured in simulations. The simulation-derived fractal dimensions of condensates of Lge11-80and its mutants agree with theirin vitromorphologies. The presented framework enables a multiscale description of biomolecular condensates and embeds their study in a wider context of colloid self-organization.
Structural insights into the assembly and activation of IL-27 signalling complex
Authors: Jin Yibo , Fyfe Paul K. , Gardner Scott , Wilmes Stephan , Bubeck Doryen , Moraga Ignacio
Published: February 2022

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Rα with GP130 to initiate signalling. While both receptors weakly bind the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Rα. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.
Furin-cleavage site is present in an antiparallel β-strand in SARS-CoV2 Spike protein
Authors: Bashir Arif , Shah Naveed Nazir
Published: February 2022

Furin cleavage-site (CS) present between the S1/S2 junction in SARS-CoV2 spike (S) protein is critical to drive the fusion of SARS-CoV2 with the host cell. SARS-CoV2 falls in the sarbecovirus lineage that doesn’t comprise of furin CS and therefore makes its origin enigmatic. The available wild-type (Wt) SARS-CoV2 S protein with PDB ID: 6yvb lacks a stretch of amino acid including furin CS as well. All investigators till date have shown this stretch existing in the form of a loop. We are for the first time reporting that this stretch comprises of 14 amino acid residues (677QTNSPRRARSVASQ689), forming an antiparallel β-sheet comprising of PRRAR furin CS. We observed the presence of this antiparallel β-sheet in MERS spike protein as well. While switching over from Wt. SARS-CoV2 with PRRAR furin CS to B.1.1.7 variant with HRRAR furin CS, we found 3% increase in the percentage content of β stands. Interestingly, we found that the change of B.1.1.7 to B.1.617 variant comprising of RRRAR furin CS shifted the percentage secondary structure back to that found in Wt. SARS-CoV2. We anticipate that this β-sheet is used as a docking site by host cell proteases to act on furin-CS. Additionally, we studied the interaction of modeled SARS-CoV2 S protein with transmembrane protease, serine 2 (TMPRSS2), and furin proteases, which clearly highlighted that these proteases exclusively uses furin CS located in β-sheet to cleave the SARS-CoV2 S protein at its S1/S2 junction.
Hybridization and a mixture of small and large-effect loci facilitate adaptive radiation
Authors: De-Kayne Rishi , Selz Oliver M. , Marques David A. , Frei David , Seehausen Ole , Feulner Philine G. D.
Published: February 2022

Adaptive radiations represent some of the most remarkable explosions of diversification across the tree of life. However, the constraints to rapid diversification and how they are sometimes overcome, particularly the relative roles of genetic architecture and hybridization, remain unclear. Here, we address these questions in the Alpine whitefish radiation, using a whole-genome dataset that includes multiple individuals of each of the 22 species belonging to six ecologically distinct ecomorph classes across several lake-systems. We reveal that repeated ecological and morphological diversification along a common environmental axis is associated with both genome-wide allele frequency shifts and a specific, larger effect, locus, associated with the geneedar. Additionally, we highlight the role of introgression between species from different lake-systems in facilitating the evolution and persistence of species with unique phenotypic combinations and ecology. These results highlight the role of both genome architecture and secondary contact with hybridization in fuelling adaptive radiation.
Large-scale comparison of immunoassay- and aptamer-based plasma proteomics through genetics and disease
Authors: Hjorleifsson Eldjarn Grimur , Ferkingstad Egil , Lund Sigrun H. , Helgason Hannes , Magnusson Olafur Th. , Olafsdottir Thorunn A. , Halldorsson Bjarni V. , Olason Pall I. , Zink Florian , Gudjonsson Sigurjon A. , Sveinbjornsson Gardar , Magnusson Magnus I. , Helgason Agnar , Oddsson Asmundur , Halldorsson Gisli H. , Magnusson Magnus K. , Saevarsdottir Saedis , Eiriksdottir Thjodbjorg , Masson Gisli , Stefansson Hreinn , Jonsdottir Ingileif , Holm Hilma , Rafnar Thorunn , Melsted Pall , Saemundsdottir Jona , Norddahl Gudmundur L. , Thorleifsson Gudmar , Ulfarsson Magnus O. , Gudbjartsson Daniel F. , Thorsteinsdottir Unnur , Sulem Patrick , Stefansson Kari
Published: February 2022

The authors have withdrawn this manuscript because this paper was posted prematurely in advance of a UK Biobank Pharma Proteomics Project consortium effort. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author
Borrowing ecological theory to infer interactions between sensitive and resistant breast cancer cell populations
Authors: Susswein Zachary , Sengupta Surojeet , Clarke Robert , Bansal Shweta
Published: February 2022

While some forms of breast cancer are highly responsive to treatment, endocrine therapy-resistant breast cancers are disproportionately lethal. There has been significant progress in understanding how endocrine therapy-resistant strains evolve from therapy-susceptible strains of cancer, but little is understood about the proliferation of resistance through cancer cell populations, or the interactions that occur between populations of resistant and sensitive cells. In this study, we characterize the nature of the ecological interaction between populations of resistant and susceptible breast cancer cells to reveal novel methods of controlling drug resistance.

Using in-vitro data on fluorescent-tagged resistant and susceptible cells, we use an image processing algorithm to identify and count cell growth till equilibrium. We then borrow theory from population biology to infer the type of ecological interaction that occurs between populations of resistant and sensitive cells. In particular, we use a Bayesian approach to fit single culture cell populations to infer density-dependent growth parameters (growth rate, carrying capacity) and a Generalized Lotka-Volterra model to understand how susceptible and resistant co-culture populations may be depressing or supporting growth of the other.

Our results identify a net mutualistic interaction between the susceptible and resistant cancer strains, demonstrating that there are ecological dynamics to cancer resistance. Our findings also suggest that ecological dynamics change in the presence of therapy, and that an adaptive treatment protocol can induce cycling behavior suggesting that heterogeneous ecological effects contribute to empirically observed adaptive-therapeutic dynamics.
The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds
Authors: Saha Itika , Yuste-Checa Patricia , Silva Padilha Miguel Da , Guo Qiang , Körner Roman , Holthusen Hauke , Trinkaus Victoria A. , Dudanova Irina , Fernández-Busnadiego Rubén , Baumeister Wolfgang , Sanders David W. , Gautam Saurabh , Diamond Marc I. , Ulrich Hartl F. , Hipp Mark S.
Published: February 2022

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimer’s disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the AAA+ chaperone VCP is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with Hsp70 and the ubiquitin-proteasome machinery. Inhibition of VCP activity stabilizes large Tau aggregates, and is accompanied by a reduction in the amount of Tau species competent of prion- like aggregate seeding in recipient cells. Thus, disaggregation by VCP generates seeding-active Tau as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.
Mural norrin/β-catenin signaling regulates Lama2 expression to promote neurovascular unit assembly
Authors: Biswas Saptarshi , Shahriar Sanjid , Giangreco Nicholas P. , Arvanitis Panos , Winkler Markus , Tatonetti Nicholas P. , Brunken William J. , Cutforth Tyler , Agalliu Dritan
Published: February 2022

Neurovascular unit (NVU) assembly and barrier maturation rely on vascular basement membrane (vBM) composition. Laminins, a major vBM component, are critical for these processes, yet which signaling pathway(s) regulate their expression remains unknown. Here we show that mural cells have active Norrin/β-catenin signaling during central nervous system development. Bulk RNA sequencing and validation using P10 and P14 wild-type versusApcdd1-/-retinas reveal that Lama2 (Laminin-α2 chain) mRNA and protein levels are increased in mutant vasculature undergoing higher Norrin/β-catenin signaling. Mural cells are the main source of Lama2, and β-catenin activation induces Lama2 expression in mural cellsin vitro. Markers of mature astrocytes including Aquaporin-4 (a water channel in astrocyte endfeet) and Integrin-α6 (a laminin receptor) are upregulated inApcdd1-/-retinas following higher Lama2 vBM deposition. Thus, the Norrin/β-catenin pathway regulates Lama2 expression in mural cells to promote NVU assembly and neurovascular barrier maturation.

Biswaset al., demonstrate that Norrin/β-catenin signaling is active in CNS mural cells and regulates Lama2 deposition in the vascular basement membrane, promoting neurovascular unit assembly and blood-CNS barrier maturation.
Deep learning accurately quantifies plasma cell percentages on CD138-stained bone marrow samples
Authors: Fu Fred , Guenther Angela , Sakhdari Ali , McKee Trevor D. , Xia Daniel
Published: February 2022

Background

The diagnosis of plasma cell neoplasms requires accurate, and ideally precise, percentages. This plasma cell percentage is often determined by visual estimation of CD138-stained bone marrow biopsies and clot sections. While not necessarily inaccurate, estimates are by definition imprecise. For this study, we hypothesized that deep learning can be used to improve precision.

Methods

We trained a semantic segmentation-based convolutional neural network (CNN) using annotations of CD138+ and CD138− cells provided by one pathologist on small image patches of bone marrow and validated the CNN on an independent test set of images patches using annotations from two pathologists and a non-deep-learning commercial software. Once satisfied with performance, we scaled-up the CNN to evaluate whole slide images (WSIs), and deployed the system as a workflow friendly web application to measure plasma cell percentages using snapshots taken from microscope cameras.

Results

On validation image patches, we found that the intraclass correlation coefficients for plasma cell percentages between the CNN and pathologist #1, a non-deep learning commercial software and pathologist #1, and pathologists #1 and #2 were 0.975, 0.892, and 0.994, respectively. The overall results show that CNN labels were almost as accurate pathologist labels at a cell-by-cell level. On WSIs from 10 clinical cases, the CNN continued to perform well, and identified two cases where the sign-out pathologist overestimated plasma cell percentages.

Conclusions

The high labeling accuracy of the CNN supports its eventually application as a computational second-opinion tool for the measurement of plasma cell percentages in clinical practice.
Transcriptome analysis of diverse Plasmodium falciparum clinical isolates identifies genes correlating with highly variable expression of merozoite surface protein MSPDBL2
Authors: Hocking Suzanne E. , Stewart Lindsay B. , Tarr Sarah J. , Freville Aline , Tetteh Kevin K. A. , Ahouidi Ambroise D. , Amambua-Ngwa Alfred , Diakite Mahamadou , Awandare Gordon A. , Conway David J.
Published: February 2022

The merozoite surface protein MSPDBL2 of Plasmodium falciparum is under strong balancing selection and is a target of naturally acquired antibodies. Remarkably, MSPDBL2 is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene transcription increases in response to overexpression of the gametocyte development inducer GDV1, so it is important to understand its natural expression. Here, MSPDBL2 in mature schizonts was analysed in the first ex vivo culture cycle of 96 clinical isolates from four populations with varying levels of infection endemicity in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as nine isolates had more than 3% schizonts positive and one had 73% positive. To investigate whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBL2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of genes previously implicated as likely to be involved in gametocytogenesis, but not including the gametocytogenesis master regulator ap2g. Although MSPDBL2 is apparently not directly involved in sexual commitment, it marks a co-occurring developmental subpopulation that may be functionally distinct within blood stage infections.
The Chlamydia protein CpoS modulates the inclusion microenvironment and restricts the interferon response by acting on Rab35
Authors: Meier Karsten , Jachmann Lana H. , Pérez Lucía , Kepp Oliver , Valdivia Raphael H. , Kroemer Guido , Sixt Barbara S.
Published: February 2022

The obligate intracellular bacterium Chlamydia trachomatis inserts into the membrane of its vacuole (the inclusion) a family of poorly characterized Inc proteins. While the Inc CpoS was recently revealed as a critical suppressor of host cellular immune surveillance, the underlying mechanism remained unknown. By complementing a cpoS mutant with modified variants of CpoS, we found that CpoS blocks distinct cellular defense responses through distinct mechanisms. Specifically, we show that the ability of CpoS to interact with Rab GTPases is not only instrumental to its ability to mediate lipid transport to the inclusion, but also key to CpoS-mediated inhibition of type I interferon responses. Indeed, depletion of Rab35 can phenocopy the respective defect of the cpoS mutant. Unexpectedly, we found that CpoS is also essential for the formation of inclusion microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. Overall, our findings highlight the modulation of membrane trafficking as a pathogenic immune evasion strategy and the role of Inc-Inc interactions in shaping the inclusion microenvironment.
Genomewide m6A mapping uncovers dynamic changes in the m6A epitranscriptome of cisplatin-treated apoptotic HeLa cells
Authors: Akçaöz Azime , Tüncel Özge , Bengisu Gelmez Ayşe , Sağlam Buket , Vatansever İpek Erdoğan , Akgül Bünyamin
Published: February 2022

Cisplatin, which is a traditional cancer therapeutic drug, induces apoptosis by modulating a diverse array of gene regulatory mechanisms. However, cisplatin-mediated changes in the m6A methylome is unknown. We employed m6A miCLIP-seq to investigate the effect of m6A methylation events under cisplatin-mediated apoptotic conditions in HeLa cells. Our high-resolution approach revealed numerous m6A marks on 972 target mRNAs with an enrichment on 132 apoptotic mRNAs. Following validation of site-specific m6A events on candidate RNAs, we tracked the fate of candidate mRNAs under METTL3 knockdown and cisplatin treatment conditions. We detected perturbations in the translational efficiency of PMAIP1 and PHLDA1 transcripts based on the polysome profile analyses. Congruently, PMAIP1 and p53 amounts were dependent on METTL3. Additionally, cisplatin-mediated apoptosis was sensitized by METTL3 knockdown. These results suggest that apoptotic pathways are modulated by m6A methylation events and METTL3-p53-PMAIP1 axis modulates cisplatin-mediated apoptosis in HeLa cells.
Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains
Authors: Chen Chuan , Saville James W. , Marti Michelle M. , Schäfer Alexandra , Cheng Mary Hongying , Mannar Dhiraj , Zhu Xing , Berezuk Alison M. , Banerjee Anupam , Sobolewski Michele D. , Kim Andrew , Treat Benjamin R. , Da Silva Castanha Priscila Mayrelle , Enick Nathan , McCormick Kevin D , Liu Xianglei , Adams Cynthia , Hines Margaret Grace , Sun Zehua , Chen Weizao , Jacobs Jana L. , Barratt-Boyes Simon M. , Mellors John W. , Baric Ralph S. , Bahar Ivet , Dimitrov Dimiter S. , Subramaniam Sriram , Martinez David R. , Li Wei
Published: February 2022

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.
Orthogonal Gene Engineering Enables CD8+ T Cells to Control Tumors through a Novel PD-1+TOX-indifferent Synthetic Effector State
Authors: Corria-Osorio Jesus , Carmona Santiago J. , Stefanidis Evangelos , Andreatta Massimo , Muller Tania , Ortiz-Miranda Yaquelin , Seijo Bili , Castro Wilson , Jimenez-Luna Cristina , Scarpellino Leonardo , Ronet Catherine , Spill Aodrenn , Lanitis Evripidis , Luther Sanjiv A. , Romero Pedro , Irving Melita , Coukos George
Published: February 2022

Adoptive immunotherapy offers opportunities to reprogram T cells and the tumor microenvironment. Orthogonal engineering of adoptively transferred T cells with an IL-2Rβγ-binding IL-2 variant, PD1-decoy and IL-33 led to cell-autonomous T-cell expansion, T-cell engraftment and tumor control in immunocompetent hosts through reprogramming of both transferred and endogenous CD8+ cells. Tumor-infiltrating lymphocytes adopted a novel effector state characterized by TOX suppression and specific expression of multiple effector molecules, most prominently granzyme C. While the IL-2 variant promoted CD8+ T-cell stemness and persistence, and was associated with downregulation of TOX, the combination with IL-33 was necessary to trigger the novel polyfunctional effector state. Rational T-cell engineering without host lymphodepletion enables optimal reprogramming of adoptively transferred T cells as well as mobilization of endogenous immunity into new functional CD8+ states mediating tumor control.
DJ-1 glyoxalase activity makes a modest contribution to cellular defense against methylglyoxal damage in neurons
Authors: Mazza Melissa Conti , Shuck Sarah , Lin Jiusheng , Moxley Michael A. , Termini John , Cookson Mark R. , Wilson Mark A.
Published: February 2022

Human DJ-1 is a cytoprotective protein whose absence causes Parkinson’s disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.