bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial multi-omic map of human myocardial infarction
Authors: Kuppe Christoph , Ramirez Flores Ricardo O. , Li Zhijian , Hannani Monica , Tanevski Jovan , Halder Maurice , Cheng Mingbo , Ziegler Susanne , Zhang Xiaoting , Preisker Fabian , Kaesler Nadine , Xu Yaoxian , Hoogenboezem Remco M. , Bindels Eric M.J. , Schneider Rebekka K. , Milting Hendrik , Costa Ivan G. , Saez-Rodriguez Julio , Kramann Rafael
Published: December 2020

Myocardial infarction is a leading cause of mortality. While advances in the acute treatment have been made, the late-stage mortality is still high, driven by an incomplete understanding of cardiac remodeling processes1,2. Here we used single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of different physiological zones and timepoints of human myocardial infarction and human control myocardium to generate an integrative high-resolution map of cardiac remodeling. This approach allowed us to increase spatial resolution of cell-type composition and provide spatially resolved insights into the cardiac transcriptome and epigenome with identification of distinct cellular zones of injury, repair and remodeling. We here identified and validated mechanisms of fibroblast to myofibroblast differentiation that drive cardiac fibrosis. Our study provides an integrative molecular map of human myocardial infarction and represents a reference to advance mechanistic and therapeutic studies of cardiac disease.
Constitutive activation of canonical Wnt signaling disrupts choroid plexus epithelial fate
Authors: Parichha Arpan , Suresh Varun , Chatterjee Mallika , Kshirsagar Aditya , Ben-Reuven Lihi , Olender Tsviya , Mark Taketo M. , Radosevic Velena , Bobic-Rasonja Mihaela , Trnski Sara , Holtzman Michael J. , Jovanov Milošević Nataša , Reiner Orly , Tole Shubha
Published: December 2020

The choroid plexus (ChP) secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the ChP epithelium (ChPe) arises from theWnt-expressing cortical hem. Embryonic mouse and human ChPe both express nuclear β-CATENIN, a canonical Wnt signaling pathway effector, indicating that this pathway is active during ChPe development. Point mutations in humanβ-CATENINresult in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of ChPe identity and an apparent transformation of the ChPe to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell (hESC)-derived organoids. The ChPe is also disrupted whenβ-Cateninis conditionally inactivated in the mouse. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal ChPe development in the mammalian brain.
A nanodomain anchored-scaffolding complex is required for PI4Kα function and localization in plants
Authors: Noack Lise C , Bayle Vincent , Armengot Laia , Frédérique Rozier F , Mamode-Cassim Adiilah , Stevens Floris D , Caillaud Marie-Cécile , Munnik Teun , Mongrand Sébastein , Pleskot Roman , Jaillais Yvon
Published: December 2020

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. PI4P accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers PI4Kα1 to the plasma membrane via a nanodomain-anchored scaffolding complex. We identified that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knock-out and knock-down strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens new perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.
Information transfer and recovery for the sense of touch
Authors: Huang Chao , Englitz Bernhard , Reznik Andrey , Zeldenrust Fleur , Celikel Tansu
Published: December 2020

Transformation of postsynaptic potentials (PSPs) into action potentials (APs) is the rate-limiting step of communication in neural networks. The efficiency of this intracellular information transfer also powerfully shapes stimulus representations in sensory cortices. Using whole-cell recordings and information-theoretic measures, we show herein that somatic PSPs accurately represent stimulus location on a trial-by-trial basis in single neurons even 4 synapses away from the sensory periphery in the whisker system. This information is largely lost during AP generation but can be rapidly (<20 ms) recovered using complementary information in local populations in a cell-type-specific manner. These results show that as sensory information is transferred from one neural locus to another, the circuits reconstruct the stimulus with high fidelity so that sensory representations of single neurons faithfully represent the stimulus in the periphery, but only in their PSPs, resulting in lossless information processing for the sense of touch in the primary somatosensory cortex.
3D printed scaffold combined to 2D osteoinductive coatings to repair a critical-size mandibular bone defect
Authors: Bouyer Michael , Garot Charlotte , Machillot Paul , Vollaire Julien , Fitzpatrick Vincent , Morand Sanela , Boutonnat Jean , Josserand Véronique , Bettega Georges , Picart Catherine
Published: December 2020

the reconstruction of large bone defects (12 cm3) remains a challenge for clinicians. We developed a new critical-size mandibular bone defect model on a mini-pig, close to human clinical issues. We analyzed the bone reconstruction obtained by a 3D printed scaffold made of clinical-grade PLA, coated with a polyelectrolyte film delivering an osteogenic bioactive molecule (BMP-2). We compared the results (CT-scan, μCT, histology) to the gold standard solution, bone autograft. We demonstrated that the dose of BMP-2 delivered from the scaffold significantly influenced the amount of regenerated bone and the repair kinetics, with a clear BMP-2 dose-dependence. Bone was homogeneously formed inside the scaffold without ectopic bone formation. The bone repair was as good as for the bone autograft. The BMP-2 doses applied in our study were reduced 20 to 75-fold compared to the commercial collagen sponges used in the current clinical applications, without any adverse effects. 3D printed PLA scaffolds loaded with reduced doses of BMP-2 can be a safe and simple solution for large bone defects faced in the clinic.
Does male mate choice select for female colouration in a promiscuous primate species?
Authors: Rigaill Lucie , Garcia Cécile
Published: December 2020

The traditional view of sex roles and sexual selection emphasises the evolution of male ornaments as a result of female mate choice and male-male competition. Female ornaments are now receiving more attention, although their roles in mating decision are still less well understood, especially considering cases in which colourful ornaments are expressed by both sexes. In this study, we analysed whether female skin colouration (luminance and redness of the face and hindquarters) influenced male mate choice and sexual behaviours in relation to intra-cycle (cycle phase), inter-cycle (number of consecutive cycles, conceptive nature of the cycle), and inter-individual (age, social rank, weight, and parity) variation in a captive population of Japanese macaques (Macaca fuscata). Males did not preferentially choose darker/redder females. Moreover, males did not appear to use female skin colouration to apportion their mating efforts on the most fertile period of the menstrual cycle or during cycles that lead to conception, or to discriminate between females. To our knowledge, our study is among the few to report a lack of male choice for female colouration in a species where both sexes potentially display ornamentation. While female colouration appeared to contain information about intra-cycle, inter-cycle, and inter-individual variation in fecundity, this study further demonstrates that this trait may not have been sexually selected and that males mated regardless of such variation across females. This study adds to the growing research on the role and evolution of female colouration in the context of sexual signalling and mate attraction.
Lightweight Reinforcement Algorithms for autonomous, scalable intra-cortical Brain Machine Interfaces
Authors: Shaikh Shoeb , So Rosa , Sibindi Tafadzwa , Libedinsky Camilo , Basu Arindam
Published: December 2020

Intra-cortical Brain Machine Interfaces (iBMIs) with wireless capability could scale the number of recording channels by integrating an intention decoder to reduce data rates. However, the need for frequent retraining due to neural signal non-stationarity is a big impediment. This paper presents an alternate paradigm of online reinforcement learning (RL) with a binary evaluative feedback in iBMIs to tackle this issue. This paradigm eliminates time-consuming calibration procedures. Instead, it relies on updating the model on a sequential sample-by-sample basis based on an instantaneous evaluative binary feedback signal. However, batch updates of weight in popular deep networks is very resource consuming and incompatible with constraints of an implant. In this work, using offline open-loop analysis on pre-recorded data, we show application of a simple RL algorithm - Banditron -in discrete-state iBMIs and compare it against previously reported state of the art RL algorithms – Hebbian RL, Attention gated RL, deep Q-learning. Owing to its simplistic single-layer architecture, Banditron is found to yield at least two orders of magnitude of reduction in power dissipation compared to state of the art RL algorithms. At the same time, post-hoc analysis performed on four pre-recorded experimental datasets procured from the motor cortex of two non-human primates performing joystick-based movement-related tasks indicate Banditron performing significantly better than state of the art RL algorithms by at least 5%, 10%, 7% and 7% in experiments 1, 2, 3 and 4 respectively. Furthermore, we propose a non-linear variant of Banditron, Banditron-RP, which gives an average improvement of 6%, 2% in decoding accuracy in experiments 2,4 respectively with only a moderate increase in power consumption.
Conformational diversity of CDR region during affinity maturation determines the affinity and stability of Sars-Cov-1 VHH-72 nanobody
Authors: George Suji
Published: December 2020

The affinity maturation of Sars-Cov-1 VHH-72 nanobody from its germline predecessor has been studied at the molecular level. The effect of somatic mutations accumulated during affinity maturation process on flexibility, stability and affinity of the germline and affinity matured nanobody was studied. Affinity maturation results in loss of local flexibility in CDR of H3 and this resulted in a gain of affinity towards the antigen. Further affinity maturation was found to destabilize the nanobody. Mechanistically the loss of flexibility of the CDR H3 is due to the redistribution of hydrogen bond network due to somatic mutation A50T, also this contributes significantly to the destability of the nanobody. Unlike antibody, in nanobody the framework region is highly conserved and structural diversity in CDR is the determining factor in diverse antigen binding and also a factor contributing to the stability. This study provide insights into the interrelationship between flexibility, stability and affinity during affinity maturation in a nanobody.
Voltage-based inhibitory synaptic plasticity: network regulation, diversity, and flexibility
Authors: Pedrosa Victor , Clopath Claudia
Published: December 2020

Neural networks are highly heterogeneous while homeostatic mechanisms ensure that this heterogeneity is kept within a physiologically safe range. One of such homeostatic mechanisms, inhibitory synaptic plasticity, has been observed across different brain regions. Computationally, however, inhibitory synaptic plasticity models often lead to a strong suppression of neuronal diversity. Here, we propose a model of inhibitory synaptic plasticity in which synaptic updates depend on presynaptic spike arrival and postsynaptic membrane voltage. Our plasticity rule regulates the network activity by setting a target value for the postsynaptic membrane potential over a long timescale. In a feedforward network, we show that our voltage-dependent inhibitory synaptic plasticity (vISP) model regulates the excitatory/inhibitory ratio while allowing for a broad range of postsynaptic firing rates and thus network diversity. In a feedforward network in which excitatory and inhibitory neurons receive correlated input, our plasticity model allows for the development of co-tuned excitation and inhibition, in agreement with recordings in rat auditory cortex. In recurrent networks, our model supports memory formation and retrieval while allowing for the development of heterogeneous neuronal activity. Finally, we implement our vISP rule in a model of the hippocampal CA1 region whose pyramidal cell excitability differs across cells. This model accounts for the experimentally observed variability in pyramidal cell features such as the number of place fields, the fields sizes, and the portion of the environment covered by each cell. Importantly, our model supports a combination of sparse and dense coding in the hippocampus. Therefore, our voltage-dependent inhibitory plasticity model accounts for network homeostasis while allowing for diverse neuronal dynamics observed across brain regions.
Paper-Thin Multilayer Microfluidic Devices with Integrated Valves
Authors: Kim Soohong , Dorlhiac Gabriel , Chaves Rodrigo Cotrim , Zalavadia Mansi , Streets Aaron
Published: December 2020

Integrated valve microfluidics has an unparalleled capability to automate the rapid delivery of fluids at the nanoliter scale for high-throughput biological experimentation. However, multilayer soft lithography, which is used to fabricate valve-microfluidics, produces devices with a minimum thickness of around five millimeters. This form-factor limitation prevents the use of such devices in experiments with limited sample thickness tolerance such as 4-pi microscopy, stimulated Raman scattering microscopy, and many forms of optical or magnetic tweezer applications. We present a new generation of integrated valve microfluidic devices that are less than 300 μm thick, including the cover-glass substrate, that resolves the thickness limitation. This "thin-chip" was fabricated through a novel soft-lithography technique that produces on-chip micro-valves with the same functionality and reliability of traditional thick valve-microfluidic devices despite the orders of magnitude reduction in thickness. We demonstrated the advantage of using our thin-chip over traditional thick devices to automate fluid control while imaging on a high-resolution inverted microscope. First, we demonstrate that the thin-chip provides improved signal to noise when imaging single cells with two-color stimulated Raman scattering (SRS). We then demonstrated how the thin-chip can be used to simultaneously perform on-chip magnetic manipulation of beads and fluorescent imaging. This study reveals the potential of our thin-chip in high-resolution imaging, sorting, and bead capture-based single-cell multi-omics applications.
In situ characterization of the 3D microanatomy of the pancreas and pancreatic cancer at single cell resolution
Authors: Kiemen Ashley , Braxton Alicia M. , Grahn Mia P. , Han Kyu Sang , Babu Jaanvi Mahesh , Reichel Rebecca , Amoa Falone , Hong Seung-Mo , Cornish Toby C. , Thompson Elizabeth D. , Wood Laura D. , Hruban Ralph H. , Wu Pei-Hsun , Wirtz Denis
Published: December 2020

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer. Accumulating evidence indicates the tumor microenvironment is highly associated with tumorigenesis through regulation of cellular physiology, signaling systems, and gene expression profiles of cancer cells. Yet the mechanisms by which the microenvironment evolves from normal pancreas architecture to precursor lesions and invasive cancer is poorly understood. Obtaining high-content and high-resolution information from a complex tumor microenvironment in large volumetric landscapes represents a key challenge in the field of cancer biology. To address this challenge, we established a novel method to reconstruct three-dimensional (3D) centimeter-scale tissues containing billions of cells from serially sectioned histological samples, utilizing deep learning approaches to recognize eight distinct tissue subtypes from hematoxylin and eosin stained sections at micrometer and single-cell resolution. Using samples from a range of normal, precancerous, and invasive pancreatic cancer tissue, we map in 3D modes of cancer invasion in the tumor microenvironment, and emphasize the need for further 3D quantification of biological systems.
Recruitment of Env to the HIV-1 T cell virological synapse by targeted and sustained Env recycling
Authors: Wang Lili , Sandmeyer Alice , Hübner Wolfgang , Li Hongru , Huser Thomas , Chen Benjamin K.
Published: December 2020

HIV-1 infection is enhanced by cell-cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Using surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, we observed targeted accumulation and sustained recycling of Env between the endocytic recycling compartment (ERC) and the VS. We observed dynamic exchange of Env at the VS while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse indicate that retention of Env is not likely to be maintained by entrapment into an immobile Gag lattice or through immobilizing interactions with CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus.
Uncovering the Organization of Neural Circuits with Generalized Phase Locking Analysis
Authors: Safavi Shervin , Panagiotaropoulos Theofanis I. , Kapoor Vishal , Ramirez-Villegas Juan F. , Logothetis Nikos K. , Besserve Michel
Published: December 2020

Despite the considerable progress ofin vivoneural recording techniques, inferring the biophysical mechanisms underlying large scale coordination of brain activity from neural data remains challenging. One obstacle is the difficulty to link high dimensional functional connectivity measures to mechanistic models of network activity. We address this issue by investigating spike-field coupling (SFC) measurements, which quantify the synchronization between, on the one hand, the action potentials produced by neurons, and on the other hand a mesoscopic “field” signals, reflecting subthreshold activities at possibly multiple recording sites. As the number of recording sites gets large, the amount of pairwise SFC measurements becomes overwhelmingly challenging to interpret. We developGeneralized Phase Locking Analysis(GPLA) as a dimensionality reduction of this multivariate SFC. GPLA describes the dominant coupling between field activity and neural ensembles across space and frequencies, thereby providing rich yet interpretable information. In particular, we show that GPLA features arebiophysically interpretablewhen used in conjunction with appropriate network models, such that we can identify the influence of underlying circuit properties on these features. We demonstrate the statistical benefits and interpretability of this approach in various computational models and Utah array recordings. The results suggest that GPLA used jointly with biophysical modeling can help uncover the contribution of recurrent microcircuits to the spatio-temporal dynamics observed in multi-channel experimental recordings.
Recognition of discrete export signals in early flagellar subunits during bacterial Type III secretion
Authors: Bryant Owain J. , Dhillon Paraminder , Hughes Colin , Fraser Gillian M.
Published: December 2020

Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhBCviathe GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgDshort), in which the distance between the N-terminal signal and the GRM was shortened, ‘stalled’ at the export machinery and was not exported. The attenuation of motility caused by FlgDshortwas suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate.
Cross-Platform Omics Prediction procedure: a game changer for implementing precision medicine in patients with stage-III melanoma
Authors: Wang Kevin Y.X. , Pupo Gulietta M. , Tembe Varsha , Patrick Ellis , Strbenac Dario , Schramm Sarah-Jane , Thompson John F. , Scolyer Richard A. , Mueller Samuel , Tarr Garth , Mann Graham J. , Yang Jean Y.H.
Published: December 2020

There is no consensus methodology that can account for the variation in omics signatures when they are acquired across different platforms and times. This poses a significant barrier to the implementation of valuable biomarkers into clinical practice. We present a novel procedure (Cross-Platform Omics Prediction) that accounts for these variations and demonstrate its utility in three risk models for different diseases that is suitable for prospective and multi-centre clinical implementation.
Blunting of insulin-stimulated glucose uptake in brown adipose tissue induces systemic metabolic dysregulation in female mice
Authors: Picatoste Belén , Yammine Lucie , Leahey Rosemary , Soares David , Cohen Paul , McGraw Timothy E.
Published: December 2020

The role of brown adipose tissue (BAT) in thermogenesis is widely appreciated, whereas its more recently described role in whole-body metabolism is not as well understood. Here we demonstrate that deletion of Rab10 from brown adipocytes reduces insulin-stimulated glucose transport by inhibiting translocation of the GLUT4 glucose transporter to the plasma membrane. This blunting of glucose uptake into brown adipocytes induces glucose intolerance and insulin-resistance in female but not male mice. The defect in glucose uptake does not affect the thermogenic function of BAT, and the dysregulation of whole-body metabolism is independent of the thermogenic function of BAT, thereby revealing a metabolism-specific role for BAT in female mice. The reduced glucose uptake induced by RAB10 deletion disrupts ChREBP regulation of the expression of de novo lipogenesis-related (DNL) genes, providing a link between DNL in BAT and whole-body metabolic regulation that is independent of thermogenesis.
Life-history dynamics: damping time, demographic dispersion and generation time
Authors: Jiang Sha , Jaggi Harman , Zuo Wenyun , Oli Madan K. , Gaillard Jean-Michel , Tuljapurkar Shripad
Published: December 2020

Transient dynamics are crucial for understanding ecological and life-history dynamics. In this study, we analyze damping time, the time taken by a population to converge to a stable (st)age structure following a perturbation, for over 600 species of animals and plants. We expected damping time to be associated with both generation timeTcand demographic dispersionσbased on previous theoretical work. Surprisingly, we find that damping time (calculated from the population projection matrix) is approximately proportional toTcacross taxa on the log-log scale, regardless ofσ. The result suggests that species at the slow end of fast-slow continuum (characterized with long generation time, late maturity, low fecundity) are more vulnerable to external disturbances as they take more time to recover compared to species with fast life-histories. The finding on damping time led us to next examine the relationship between generation time and demographic dispersion. Our result reveals that the two life-history variables are positively correlated on a log-log scale across taxa, implying long generation time promotes demographic dispersion in reproductive events. Finally, we discuss our results in the context of metabolic theory and contribute to existing allometric scaling relationships.
Exposure to Traffic-Related Air Pollution is Associated with Greater Bacterial Diversity in the Lower Respiratory Tract of Children
Authors: Niemeier-Walsh Christine , Ryan Patrick H , Meller Jaroslaw , Ollberding Nicholas J. , Adhikari Atin , Reponen Tiina
Published: December 2020

Background

Exposure to particulate matter has been shown to increase the adhesion of bacteria to human airway epithelial cells. However, the impact of traffic-related air pollution (TRAP) on the respiratory microbiome is unknown.

Methods

Forty children were recruited through the Cincinnati Childhood Allergy and Air Pollution Study, a longitudinal cohort followed from birth through early adolescence. Saliva and induced sputum were collected at age 14 years. Exposure to TRAP was characterized from birth through the time of sample collection using a previously validated land-use regression model. Sequencing of the bacterial 16S and ITS fungal rRNA genes was performed on sputum and saliva samples. The relative abundance of bacterial taxa and diversity indices were compared in children with exposure to high and low TRAP. We also used multiple linear regression to assess the effect of TRAP exposure, gender, asthma status, and socioeconomic status on the alpha diversity of bacteria in sputum.

Results

We observed higher bacterial alpha diversity indices in sputum than in saliva. The diversity indices for bacteria were greater in the high TRAP exposure group than the low exposure group. These differences remained after adjusting for asthma status, gender, and mother’s education. No differences were observed in the fungal microbiome between TRAP exposure groups.

Conclusion

Our findings indicate that exposure to TRAP in early childhood and adolescence may be associated with greater bacterial diversity in the lower respiratory tract. Asthma status does not appear to confound the observed differences in diversity. It is still unknown whether the development of asthma changes the lower respiratory tract microbiome or if an altered microbiome mediates a change in disease status. However, these results demonstrate that there may be a TRAP-exposure related change in the lower respiratory microbiota that is independent of asthma status.
High-throughput Single-cell CNV Detection Reveals Clonal Evolution During Hepatocellular Carcinoma Recurrence
Authors: Wu Liang , Wang Yuzhou , Jiang Miaomiao , Zhou Biaofeng , Sun Yunfan , Zhou Kaiqian , Xie Jiarui , Zhong Yu , Zhao Zhikun , Dean Michael , Hou Yong , Liu Shiping
Published: December 2020

Single-cell genomics provides substantial resources for dissecting cellular heterogeneity and cancer evolution, but classical DNA amplification-based methods are low-throughput and introduce coverage bias during sample preamplification. We developed asingle-cellDNA library preparation method withoutpreamplification innanolitre scale (scDPN). The method has a throughput of up to 1,800 cells per run for copy number variation (CNV) detection. Also, it has a lower level of amplification bias and noise than the multiple displacement amplification (MDA) method and showed high sensitivity and accuracy based on evaluation in cell lines and tumour tissues. We used this approach to profile the tumour clones in paired primary and relapsed tumour samples of hepatocellular carcinoma (HCC). We identified 3 clonal subpopulations with a multitude of aneuploid alterations across the genome. Furthermore, we observed that a minor clone of the primary tumour containing additional alterations in chromosomes 1q, 10q, and 14q developed into the dominant clone in the recurrent tumour, indicating clonal selection during recurrence in HCC. Overall, this approach provides a comprehensive and scalable solution to understand genome heterogeneity and evolution.
The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation
Authors: Pras Anita , Houben Bert , Aprile Francesco A. , Seinstra Renée , Gallardo Rodrigo , Janssen Leen , Hogewerf Wytse , De Vleeschouwer Matthias , Mata-Cabana Alejandro , Koopman Mandy , Stroo Esther , de Vries Minke , Edwards Samantha Louise , Vendruscolo Michele , Falsone S. Fabio , Rousseau Frederic , Schymkowitz Joost , Nollen Ellen A. A.
Published: December 2020

While aggregation-prone proteins are known to accelerate ageing and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A and SERF2 have recently been identified as cellular modifiers of such cytotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with specific patterns of negatively charged and hydrophobic, aromatic amino acids. The absence of such patterns, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In a protein aggregation model in the nematodeC. elegans, protein aggregation was suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that charge interactions are required for MOAG-4 and SERF2 to promote aggregation. Such charged interactions might accelerate the primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our finding that negatively charged segments are overrepresented in amyloid-forming proteins suggests that inhibition of charge interactions deserves exploration as a strategy to target age-related protein toxicity.

Significance Statement

How aging causes relatively common diseases such as Alzheimer’s and Parkinson’s is still a mystery. Since toxic structural changes in proteins are likely to be responsible, we investigated biological mechanisms that could drive such changes. We made use of a modifying factor called SERF2, which accelerates structural changes and aggregation of several disease-related proteins. Through a peptide-binding screen, we found that SERF2 acts on negatively charged protein regions. The abundance of such regions in the disease-related proteins explains why SERF has its effect. Removing positive charge in SERF was sufficient to suppress protein aggregation in models for disease. We propose that blocking charge-interactions with SERF or other modifiers could serve as a general approach to treat age-related protein toxicity.
Differential expression levels of Sox9 in early neocortical radial glial cells regulate the decision between stem cell maintenance and differentiation
Authors: Fabra-Beser Jaime , de Araujo Jessica Alves Medeiros , Marques-Coelho Diego , Goff Loyal A. , Müller Ulrich , Gil-Sanz Cristina
Published: December 2020

Radial glial progenitor cells (RGCs) in the dorsal forebrain directly or indirectly produce excitatory projection neurons and macroglia of the neocortex. Recent evidence shows that the pool of RGCs is more heterogeneous than originally thought and that progenitor subpopulations can generate particular neuronal cell types. Using single cell RNA sequencing, we have studied gene expression patterns of two subtypes of RGCs that differ in their neurogenic behavior. One progenitor type rapidly produces postmitotic neurons, whereas the second progenitor remains relatively quiescence before generating neurons. We have identified candidate genes that are differentially expressed between these RGCs progenitor subtypes, including the transcription factor Sox9. Usingin uteroelectroporation, we demonstrate that elevated Sox9 expression in progenitors prevents RGC division and leads to the generation of upper-layer cortical neurons from these progenitors at later ages. Our data thus reveal molecular differences between cortical progenitors with different neurogenic behavior and indicates that Sox9 is critical for the maintenance of RGCs to regulate the generation of upper layer neurons.

SIGNIFICANCE STATEMENT

The existence of heterogeneity in the pool of RGCs and its relationship with the generation of cellular diversity in the cerebral cortex has been an interesting topic of debate for many years. Here we describe the existence of a subpopulation of RGCs with reduced neurogenic behavior at early embryonic ages presenting a particular molecular signature. This molecular signature consists of differential expression of some genes including the transcription factor Sox9, found to be a specific master regulator of this subpopulation of progenitor cells. Functional experiments perturbing Sox9 expression’s levels reveal its instructive role in the regulation of the neurogenic behavior of RGCs and its relationship with the generation of upper layer projection neurons at later ages.
Thermodynamics and free energy landscape of BAR-domain dimerization from molecular simulations
Authors: Jhaveri Adip , Maisuria Dhruw , Varga Matthew , Mohammadyani Dariush , Johnson Margaret E
Published: December 2020

Nearly all proteins interact specifically with other proteins, often forming reversible bound structures whose stability is critical to function. Proteins with BAR domains function to bind to, bend, and remodel biological membranes, where the dimerization of BAR domains is a key step in this function. Here we characterize the binding thermodynamics of homodimerization between the LSP1 BAR domain proteins in solution, using Molecular Dynamics (MD) simulations. By combining the MARTINI coarse-grained protein models with enhanced sampling through metadynamics, we construct a two-dimensional free energy surface quantifying the bound versus unbound ensembles as a function of two distance variables. Our simulations portray a heterogeneous and extraordinarily stable bound ensemble for these modeled LSP1 proteins. The proper crystal structure dimer has a large hydrophobic interface that is part of a stable minima on the free energy surface, which is enthalpically the minima of all bound structures. However, we also find several other stable nonspecific dimers with comparable free energies to the specific dimer. Through structure-based clustering of these bound structures, we find that some of these ‘nonspecific’ contacts involve extended tail regions that help stabilize the higher-order oligomers formed by BAR-domains, contacts that are separated from the homodimer interface. We find that the known membrane-binding residues of the LSP1 proteins rarely participate in any of the bound interfaces, but that both patches of residues are aligned to interact with the membrane in the specific dimer. Hence, we would expect a strong selection of the specific dimer in binding to the membrane. The effect of a 100mM NaCl buffer reduces the relative stability of nonspecific dimers compared to the specific dimer, indicating that it would help prevent aggregation of the proteins. With these results, we provide the first free energy characterization of interaction pathways in this important class of membrane sculpting domains, revealing a variety of interfacial contacts outside of the specific dimer that may help stabilize its oligomeric assemblies.
Genomic insights on DNase production inStreptococcus agalactiaeST17 and ST19 strains
Authors: Silvestre Inês , Nunes Alexandra , Borges Vítor , Isidro Joana , Silva Catarina , Vieira Luís , Gomes João Paulo , José Borrego Maria
Published: December 2020

Streptococcus agalactiaeevasion from the human defense mechanisms has been linked to the production of DNases. These were proposed to contribute to the hypervirulence ofS. agalactiaeST17/capsular-type III strains, mostly associated with neonatal meningitis. We performed a comparative genomic analysis between ST17 and ST19 human strains with different cell tropism and distinct DNase production phenotypes. AllS. agalactiaeST17 strains, with the exception of 2211-04, were found to display DNase activity, while the opposite scenario was observed for ST19, where 1203-05 was the only DNase(+) strain. The analysis of the genetic variability of the seven genes putatively encoding secreted DNases inS. agalactiaerevealed an exclusive amino acid change in the predicted signal peptide of GBS0661 (NucA) of the ST17 DNase(-), and an exclusive amino acid change alteration in GBS0609 of the ST19 DNase(+) strain. Further core-genome analysis identified some specificities (SNVs or indels) differentiating the DNase(-) ST17 2211-04 and the DNase(+) ST19 1203-05 from the remaining strains of each ST. The pan-genomic analysis evidenced an intact phage without homology inS. agalactiaeand a transposon homologous to TnGBS2.3 in ST17 DNase(-) 2211-04; the transposon was also found in one ST17 DNase(+) strain, yet with a different site of insertion. A group of nine accessory genes were identified among all ST17 DNase(+) strains, including the Eco47II family restriction endonuclease and the C-5 cytosine-specific DNA methylase. None of these loci was found in any DNase(-) strain, which may suggest that these proteins might contribute to the lack of DNase activity. In summary, we provide novel insights on the genetic diversity between DNase(+) and DNase(-) strains, and identified genetic traits, namely specific mutations affecting predicted DNases (NucA and GBS0609) and differences in the accessory genome, that need further investigation as they may justify distinct DNase-related virulence phenotypes inS. agalactiae.
Oligodendrocyte progenitor proliferation is disinhibited following traumatic brain injury in LIF heterozygous mice
Authors: Frondelli Michelle J. , Mather Marie. L. , Levison Steven W.
Published: December 2020

Traumatic brain injury (TBI) is a significant problem that affects ∼500,000 children each year. As cell proliferation is disturbed by injury and is required for normal brain development, we investigated how a pediatric closed head injury (CHI) would affect the progenitors of the subventricular zone (SVZ). Additionally, we evaluated the contribution of Leukemia Inhibitory Factor (LIF) using LIF-heterozygous mice (LIF Het), as LIF is an injury-induced cytokine, known to influence neurogenesis and gliogenesis. CHI’s were performed on P20 LIF Het and WT mice. Ki-67 staining and stereology revealed that cell proliferation increased ∼250% in injured LIF Het mice compared to the 30% increase observed in injured WT mice at 48 h post CHI. Furthermore, Olig2+ cell proliferation increased in the SVZ and white matter of LIF Het injured mice at 48 h recovery. Using an 8-color flow cytometry panel, the proliferation of three distinct multipotential progenitors were greater in LIF Het injured mice compared to WT injured mice. Early oligodendrocyte progenitor cell (OPC) proliferation was 6-fold higher in LIF Het injured mice compared to WT injured mice. In vitro, addition of LIF decreased overall cell proliferation and OPC proliferation compared to controls. Addition of LIF to OPC cultures induced an increase of phospho-Akt after 20 minutes and an increase of phospho-S6RP at 20 and 40 minutes of exposure, suggesting that LIF stimulates the mammalian target of Rapamycin pathway. Altogether, our data provide new insights into the regulatory role of LIF in suppressing neural progenitor cell proliferation after a mild TBI.

Main Points

OPC proliferation is dis-inhibited in LIF haplodeficient mice.

LIF directly inhibits glial progenitor cell proliferation.

LIF stimulates the mTOR pathway.
A plastid antiporter as a bioindicator ofThalassiosira pseudonanaresilience
Authors: Valenzuela Jacob J. , Ashworth Justin , Cusick Allison , Abbriano Raffaela M. , Armbrust E.Virginia , Hildebrand Mark , Orellana Mónica V. , Baliga Nitin S.
Published: December 2020

Acidification of the ocean due to high atmospheric CO2levels may increase the resilience of diatoms causing dramatic shifts in abiotic and biotic cycles with lasting implications on marine ecosystems. Here, we report a potential bioindicator of a shift in the resilience of a coastal and centric model diatomThalassiosira pseudonanaunder elevated CO2. Specifically, we have discovered, through EGFP-tagging, a plastid membrane localized putative Na+(K+)/H+antiporter that is significantly upregulated at > 800 ppm CO2, with a potentially important role in maintaining pH homeostasis. Notably, transcript abundance of this antiporter gene was relatively low and constant over the diel cycle under contemporary CO2conditions. In future acidified oceanic conditions, dramatic oscillations of >10-fold change between nighttime (high) and daytime (low) in transcript abundances of the antiporter gene were associated with increased resilience ofT. pseudonana. By analyzing metatranscriptomic data from the Tara Oceans project, we demonstrate that phylogenetically diverse diatoms express homologs of this antiporter across the globe. We propose that the differential between night- and daytime transcript levels of the antiporter could serve as a bioindicator of a shift in the resilience of diatoms in response to high CO2conditions in marine environments.
Betulonic acid derivatives inhibiting coronavirus replication in cell culture via the nsp15 endoribonuclease
Authors: Krasniqi Besir , Stevaert Annelies , Van Loy Benjamin , Nguyen Tien , Thomas Joice , Vandeput Julie , Jochmans Dirk , Thiel Volker , Dijkman Ronald , Dehaen Wim , Voet Arnout , Naesens Lieve
Published: December 2020

The lack of medication to suppress coronavirus infections is a main reason for the dramatic course of the COVID-19 pandemic. There is an urgent need to identify suitable coronavirus drug targets and corresponding lead molecules. Here we describe the discovery of a class of coronavirus inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. SAR exploration of these 1,2,3-triazolo fused betulonic acid derivatives yielded lead molecule5has a strong inhibitor (antiviral EC50: 0.6 μM) of human coronavirus 229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to5h, and selected resistance to the compound mapped to mutations in the N-terminal part of nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.
A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilizing immunity against SARS-CoV-2 infection in mice
Authors: Counoupas Claudio , Johansen Matt D. , Stella Alberto O. , Nguyen Duc H. , Ferguson Angela L. , Aggarwal Anupriya , Bhattacharyya Nayan D. , Grey Alice , Patel Karishma , Siddiquee Rezwan , Stewart Erica L. , Feng Carl G. , Hansbro Nicole G. , Palendira Umaimainthan , Steain Megan C. , Saunders Bernadette M. , Low Jason K. K. , Mackay Joel P. , Kelleher Anthony D. , Britton Warwick J. , Turville Stuart G , Hansbro Philip M. , Triccas James A.
Published: December 2020

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilized, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralizing antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralized B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.
The combined treatment of Molnupiravir and Favipiravir results in a marked potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model
Authors: Abdelnabi Rana , Foo Caroline S. , Kaptein Suzanne J. F. , Zhang Xin , Langendries Lana , Vangeel Laura , Breuer Judith , Pang Juanita , Williams Rachel , Vergote Valentijn , Heylen Elisabeth , Leyssen Pieter , Dallmeier Kai , Coelmont Lotte , Jochmans Dirk , Chatterjee Arnab K. , De Jonghe Steven , Weynand Birgit , Neyts Johan
Published: December 2020

Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV2. In recent days preliminary efficacy data have been reported in COVID-19 patients. We here studied the combined antiviral effect of the drugs in the SARS-CoV2 hamster infection model. We first demonstrate that Molnupiravir can reduce infectious virus titers in lungs of infected animals in a dose-dependent manner by up to 3.5 log10which is associated with a marked improvement of virus-induced lung pathology. When animals are treated with a combination of suboptimal doses of Molnupiravir and Favipiravir (that each alone result in respectively a 1.3 log10and 1.1 log10reduction of infectious virus titers in the lungs), a marked combined potency is observed. Infectious virus titers in the lungs of animals treated with the combo are on average reduced by 4.5 log10and infectious virus are no longer detected in the lungs of 60% of treated infected animals. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs. In the combo-treated hamsters an increased frequency of C-to-T and G-to-A mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir and Favipiravir in the treatment of COVID-19.