bluuebunny/crossref_metadata_2025 huggingface dataset:
Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in 3-Dimensional Models
Authors: Kukla David A. , Crampton Alexandra L. , Wood David K. , Khetani Salman R.
Published: December 2019

Human liver models that are 3-dimensional (3D) in architecture are proving to be indispensable for diverse applications, including compound metabolism and toxicity screening during preclinical drug development, to model human liver diseases for the discovery of novel therapeutics, and for cell-based therapies in the clinic; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months in vitro. Therefore, here we determined how microscale 3D collagen-I presentation and fibroblast interaction could affect the long-term functions of PHHs. High-throughput droplet microfluidics was utilized to rapidly generate reproducibly-sized (~300 μm diameter) microtissues containing PHHs encapsulated in collagen-I +/− supportive fibroblasts, namely 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as gold-standard controls. Hepatic functions (e.g. albumin and cytochrome-P450 or CYP activities) and gene expression were subsequently measured for up to 6 weeks. We found that collagen-based 3D microtissues rescued PHH functions within static multi-well plates at 2- to 30-fold higher levels than self-assembled spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Additionally, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically-relevant manner to drug-mediated CYP induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display hepatic functions for 6+ weeks without any fluid perfusion at higher levels than spheroids and macrogels, and such microtissues can be used to assess drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find broader utility for modeling liver diseases and as building blocks for cell-based therapies.
Neural Arbitration between Social and Individual Learning Systems
Authors: Diaconescu Andreea O. , Stecy Madeline , Kasper Lars , Burke Christopher J. , Nagy Zoltan , Mathys Christoph , Tobler Philippe N.
Published: December 2019

Decision making often requires integrating self-gathered information with information acquired from observing others. Depending on the situation, it may be beneficial to rely more on one than the other source, taking into account that either information may be imprecise or deceiving. The process by which one source is selected over the other based on perceived reliability, here defined as arbitration, has not been fully elucidated. In this study, we formalised arbitration as the relative reliability (precision) of predictions afforded by each learning system using hierarchical Bayesian models. In a probabilistic learning task, participants predicted the outcome of a lottery using recommendations from a more informed advisor and self-sampled outcomes. The number of points participants wagered on their predictions reflected arbitration: The higher the relative precision of one learning system over the other and the lower the intention volatility, the more points participants wagered on a given trial. Functional neuroimaging demonstrated that the arbitration signal was independent of decision confidence and involved modalityspecific brain regions. Arbitrating in favour of self-gathered information activated the dorsolateral prefrontal cortex and the midbrain whereas arbitrating in favour of social information engaged ventromedial prefrontal cortex and the temporoparietal junction. These findings are in line with domain specificity and indicate that relative precision captures arbitration between social and individual learning systems at both the behavioural and neural level.
A cross–nearest neighbor/Monte Carlo algorithm for single-molecule localization microscopy defines interactions between p53, Mdm2, and MEG3
Authors: Bauer Nicholas C. , Yang Anli , Wang Xin , Zhou Yunli , Klibanski Anne , Soberman Roy J.
Published: December 2019

The functions of long noncoding (lnc)RNAs such as MEG3 are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with Mdm2. To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule localization microscopy (SMLM) technique to detect and quantify the localizations of p53, Mdm2, and MEG3 in U2OS cells. We developed a new cross-nearest neighbor/Monte Carlo algorithm to quantify the association of these molecules. Proof of concept for our method was obtained by examining the association between FKBP1A and mTOR, MEG3 and p53, and Mdm2 and p53. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of the interaction with Mdm2.
Realistic Anatomically Detailed Open-Source Spinal Cord Stimulation (RADO-SCS) Model
Authors: Khadka Niranjan , Liu Xijie , Zander Hans , Swami Jaiti , Rogers Evan , Lempka Scott F. , Bikson Marom
Published: December 2019

Objective

Computational current flow models of spinal cord stimulation (SCS) are widely used in device development, clinical trial design, and patient programming. Proprietary models of varied sophistication have been developed. An open-source model with state-of-the-art precision would serve as a standard for SCS simulation.

Approach

We developed a sophisticated SCS modeling platform, named Realistic Anatomically Detailed Open-Source Spinal Cord Stimulation (RADO-SCS) model. This platform consists of realistic and detailed spinal cord and ancillary tissues anatomy derived based on prior imaging and cadaveric studies. Represented tissues within the T9-T11 spine levels include vertebrae, intravertebral discs, epidural space, dura, CSF, white-matter, gray-matter, dorsal and ventral roots and rootlets, dorsal root ganglion, sympathetic chain, thoracic aorta, epidural space vasculature, white-matter vasculature, and thorax. As an exemplary, a bipolar SCS montage was simulated to illustrate the model workflow from the electric field calculated from a finite element model (FEM) to activation thresholds predicted for individual axons populating the spinal cord.

Main Results

Compared to prior models, RADO-SCS meets or exceeds detail for every tissue compartment. The resulting electric fields in white and gray-matter, and axon model activation thresholds are broadly consistent with prior stimulations.

Significance

The RADO-SCS can be used to simulate any SCS approach with both unprecedented resolution (precision) and transparency (reproducibility). Freely available online, the RADO-SCS will be updated continuously with version control.
Morphine differentially alters the synaptic and intrinsic properties of D1R- and D2R-expressing medium spiny neurons in the nucleus accumbens
Authors: McDevitt Dillon S. , Jonik Benjamin , Graziane Nicholas M.
Published: December 2019

Exposure to opioids reshapes future reward and motivated behaviors partially by altering the functional output of medium spiny neurons (MSNs) in the nucleus accumbens shell. Here, we investigated how morphine, a highly addictive opioid, alters synaptic transmission and intrinsic excitability on dopamine D1-receptor (D1R) expressing and dopamine D2-receptor (D2R) expressing MSNs, the two main output neurons in the nucleus accumbens shell. Using whole-cell electrophysiology recordings, we show, that 24 h abstinence following repeated non-contingent administration of morphine (10 mg/kg, i.p.) in mice reduces miniature excitatory postsynaptic current (mEPSC) frequency and miniature inhibitory postsynaptic current (mIPSC) frequency on D2R-MSNs, with concomitant increases in D2R-MSN intrinsic membrane excitability. We did not observe any changes on synaptic or intrinsic changes on D1R-MSNs. Lastly, in an attempt to determine the integrated effect of the synaptic and intrinsic alterations on the overall functional output of D2R-MSNs, we measured the input-output efficacy by measuring synaptically-driven action potential firing. We found that both D1R-MSN and D2R-MSN output was unchanged following morphine treatment.
Integrity of the Uncinate Fasciculus Predicts Emotional Pattern Separation-Related fMRI Signals in the Hippocampal Dentate and CA3
Authors: Granger Steven J. , Leal Stephanie L. , Janecek John T. , McMillan Liv , Stern Hal , Yassa Michael A.
Published: December 2019

Alterations in white matter integrity have been demonstrated in a number of psychiatric disorders that involve disruptions in emotional processing. One such pathway – the uncinate fasciculus (UF) – connects the orbitofrontal cortex (OFC) to the medial temporal lobes (MTL) and has been associated with early life adversity, maltreatment, anxiety, and depression. While it is purported to play a role in episodic memory and discrimination, its exact function remains poorly understood. We have previously described the role of the amygdala and dentate (DG)/CA3 fields of the hippocampus in the mnemonic discrimination of emotional experiences (i.e. emotional pattern separation). However, how this computation may be modulated by connectivity between the medial temporal lobes and the orbitofrontal cortex remains unknown. Here we ask the question of whether the uncinate fasciculus plays a role in influencing MTL subregional activity during emotional pattern separation. By combining diffusion imaging with high-resolution functional MRI, we found that reduced integrity of the UF is related to higher activation in the DG/CA3 subregions of the hippocampus during an emotional pattern separation task. We additionally report that higher levels of DG/CA3 activity are associated with poorer memory performance, suggesting that hyperexcitability in this network (which may be driven by CA3 recurrent collaterals) is associated with memory errors and that the UF may allow the OFC to exert inhibitory control on this network and improve discrimination of emotional experiences. This work provides novel mechanistic insight into the role of prefrontal interactions with the MTL, particularly in the context of emotional memory.
Effect of Sequence Depth and Length in Long-read Assembly of the Maize Inbred NC358
Authors: Ou Shujun , Liu Jianing , Chougule Kapeel M. , Fungtammasan Arkarachai , Seetharam Arun , Stein Joshua , Llaca Victor , Manchanda Nancy , Gilbert Amanda M. , Wei Xuehong , Chin Chen-Shan , Hufnagel David E. , Pedersen Sarah , Snodgrass Samantha , Fengler Kevin , Woodhouse Margaret , Walenz Brian P. , Koren Sergey , Phillippy Adam M. , Hannigan Brett , Kelly Dawe R. , Hirsch Candice N. , Hufford Matthew B. , Ware Doreen
Published: December 2019

Recent improvements in the quality and yield of long-read data and scaffolding technology have made it possible to rapidly generate reference-quality assemblies for complex genomes. Still, generating these assemblies is costly, and an assessment of critical sequence depth and read length to obtain high-quality assemblies is important for allocating limited resources. To this end, we have generated eight independent assemblies for the complex genome of the maize inbred line NC358 using PacBio datasets ranging from 20-75x genomic depth and N50 read lengths of 11-21 kb. Assemblies with 30x or less depth and N50 read length of 11 kb were highly fragmented, with even the low-copy genic fraction of the genome showing degradation at 20x depth. Distinct sequence-quality thresholds were observed for complete assembly of genes, transposable elements, and highly repetitive genomic features such as telomeres, heterochromatic knobs and centromeres. This study provides a useful resource allocation reference to the community as long-read technologies continue to mature.
Charting the metabolic landscape of the facultative methylotroph Bacillus methanolicus
Authors: Delépine Baudoin , López Marina Gil , Carnicer Marc , Vicente Cláudia M. , Wendisch Volker F. , Heux Stéphanie
Published: December 2019

Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. This has motivated a number of omics studies of B. methanolicus at the genome, transcript, protein and metabolic levels. Intriguingly, the only substrates known to support B. methanolicus growth as sole source of carbon and energy are methanol, mannitol, and to a lesser extent glucose and arabitol. We hypothesized that comparing methylotrophic and non-methylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. 13C metabolic flux analysis (13C-MFA) is a powerful computational method to estimate carbon flows from substrate to biomass (i.e. the in vivo reaction rates of the central metabolic pathways) from experimental labeling data. In this study, we designed and performed a 13C-MFA of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol and arabitol to compare the associated metabolic states. The results obtained validate previous findings on the methylotrophy of B. methanolicus, allowed us to characterize the assimilation pathway of one of the studied carbon sources, and provide a better overall understanding of this strain.

IMPORTANCE

Methanol is cheap, easy to transport and can be produced both from renewable and fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as B. methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories, or to design methylotrophic capability in other strains already used by the industry.
Highly diverse Asgard archaea participate in organic matter degradation in coastal sediments
Authors: Cai Mingwei , Liu Yang , Yin Xiuran , Zhou Zhichao , Friedrich Michael W. , Richter-Heitmann Tim , Nimzyk Rolf , Kulkarni Ajinkya , Wang Xiaowen , Li Wenjin , Pan Jie , Yang Yuchun , Gu Ji-Dong , Li Meng
Published: December 2019

Asgard is an archaeal superphylum that might hold the key to understand the origin of eukaryotes, but its diversity and ecological roles remain poorly understood. Here, we reconstructed 15 metagenomic-assembled genomes (MAGs) from coastal sediments covering most known Asgard archaea and a novel group, which is proposed as a new Asgard phylum named as the “Gerdarchaeota”. Genomic analyses predict that Gerdarchaeota are facultative anaerobes in utilizing both organic and inorganic carbon. Unlike their closest relatives Heimdallarchaeota, Gerdarchaeota have genes encoding for cellulase and enzymes involving in the tetrahydromethanopterin-based Wood–Ljungdahl pathway. Transcriptomic evidence showed that all known Asgard archaea are capable of degrading organic matter, including peptides, amino acids and fatty acids, in different ecological niches in sediments. Overall, this study broadens the diversity of the mysterious Asgard archaea and provides evidence for their ecological roles in coastal sediments.
Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1
Authors: Ikemoto Yu , Miyashita Toshiyuki , Nasu Michiyo , Hatsuse Hiromi , Kajiwara Kazuhiro , Fujii Katsunori , Motojima Toshino , Toyoda Masashi , Umezawa Akihiro
Published: December 2019

Gorlin syndrome is a rare autosomal dominant hereditary disease with high incidence of tumors such as basal cell carcinoma and medulloblastoma. Disease-specific induced pluripotent stem cells (iPSCs) have now been used as a model to analyze disease pathogenesis as well as an animal model. In this study, we generated iPSCs derived from fibroblasts of four patients with Gorlin syndrome (Gln-iPSCs) with a heterozygous mutation of the PTCH1 gene. Gln-iPSCs from the four patients developed medulloblastoma in 100% (four out of four), a manifestation of Gorlin syndrome, in the teratomas after implantation into immunodeficient mice, but none (0/584) of the other iPSC-teratomas. One of the medulloblastomas had loss of heterozygosity in the PTCH1 gene while benign teratoma, i.e. non-medulloblastoma part, did not, indicating a close clinical correlation between tumorigenesis in Gorlin syndrome patients and Gln-iPSCs.
mTOR-RhoA signalling impairments in direct striatal projection neurons induce altered behaviours and striatal physiology in mice
Authors: Rial Daniel , Puighermanal Emma , Valjent Emmanuel , Schiffmann Serge , de Kerchove d’Exaerde Alban
Published: December 2019

As an integrator of molecular pathways, mTOR has been associated with diseases including neurodevelopmental, psychiatric and neurodegenerative disorders as autism, schizophrenia, and Huntington’s disease. An important brain area involved in all these diseases is the striatum. However, the mechanisms behind how mTOR is involved in striatal physiology and its relative role in distinct neuronal populations in these striatal-related diseases still remain to be clarified.

Taking advantage of the D1-mTOR KO mice (males), we combined behavioural, biochemical, electrophysiological and morphological analysis aiming to untangle the role of mTOR in direct pathway striatal projection neurons (dMSNs) and how this would impact on striatal physiology.

Our results indicate deep behavioural changes in absence of mTOR in dMSNs such as decreased spontaneous locomotion, impaired social interaction and repetitive behaviour. These were accompanied by a Kv1.1-induced increase in the fast phase of afterhyperpolarization and decreased distal spines density that were mechanistically independent of protein synthesis but dependent of RhoA activity.

These results identify mTOR RhoA signaling as an important regulator of striatal functions through an intricate mechanism involving RhoA and culminating in Kv1.1 overfunction, which could be targeted to treat striatal-related mTORopathies.
Identification of Genomic Insertion and Flanking Sequences of the Transgenic Drought-tolerant Maize Line “SbSNAC1-382” using the Single Molecular Real-Time (SMRT) Sequencing Method
Authors: Zeng Tingru , Zhang Dengfeng , Li Yongxiang , Li Chunhui , Liu Xuyang , Shi Yunsu , Song Yanchun , Li Yu , Wang Tianyu
Published: December 2019

Safety assessment of genetically modified (GM) crops is crucial in the phase of product development before the GM crops are put on the market. Characteristics of flanking sequences of exogenous insertion sequences are essential for the safety assessment and marking of transgenic crops. In this study, we used the methods of genome walking and whole genome sequencing (WGS) to identify the flanking sequence characteristics of aSbSNAC1transgenic drought-tolerant maize line “SbSNAC1-382”, but both of the methods failed. Then, we constructed a genomic fosmid library of the transgenic maize line, which contained 4.18×105clones with an average insertion fragment of 35 kb, covering 5.85 times of the maize genome. Subsequently, three positive clones were screened by pairs of specific primers and one of the three positive clones was sequenced by using the Single Molecule Real-Time (SMRT) sequencing technology. More than 1.95 Gb sequence data (∼105× coverage) for the sequenced clone was generated. The junction reads mapped to the boundaries of T-DNA and the flanking sequences in the transgenic line were identified by comparing all sequencing reads with the maize reference genome and the sequence of transgenic vector. Furthermore, the putative insertion loci and flanking sequences were confirmed by PCR amplification and Sanger sequencing. The results indicated that two copies of the exogenous T-DNA fragments were inserted in the same genomic site. And the exogenous T-DNA fragments were integrated at the position of Chromosome 5: 177155650 to 177155696 in the transgenic line 382. Herein, we have demonstrated the successful application of the SMRT technology for the characterization of genomic insertion and flanking sequences.
Quantifying colour difference in animals with variable patterning
Authors: Dračková Tereza , Smolinský Radovan , Hiadlovská Zuzana , Dolinay Matej , Martínková Natália
Published: December 2019

Colour pattern influences behaviour and modifies survival of organisms through perception of light reflectance. Spectrophotometric methods used to study colour navigate between precision and accuracy of reflectance across wavelengths, while photographic methods are generally used to assess the complexity of colour patterns. Here, we compare how colours characterised using point measurements (simulating spectrophotometry) on the skin of a sand lizard (Lacerta agilis) differ from colours estimated by clustering pixels in a photograph of the lizard’s body. We found that point measurements adequately represented the dominant colour of the lizard; however, where colour patterning influenced measurement geometry, image analysis outper-formed point measurement as regards stability between technical replicates on the same animal. The greater colour variation established from point measurements increased further under controlled laboratory illumination. Both methods revealed lateral colour asymmetry in sand lizards. We conclude that studies assessing the impact of colour on animal ecology and behaviour should utilise hyperspectral imaging, followed by image analysis that encompasses the whole colour pattern.
Differential propagation of ripples along the proximo-distal and septo-temporal axes of dorsal CA1 of rats
Authors: Kumar Mekhala , Deshmukh Sachin S.
Published: December 2019

The functional connectivity of the hippocampus with its primary cortical input, the entorhinal cortex, is organized topographically. In area CA1 of the hippocampus, this leads to different functional gradients along the proximo-distal and septo-temporal axes of spatial/sensory responsivity and spatial resolution respectively. CA1 ripples, a network phenomenon, allows us to test whether the hippocampal neural network shows corresponding gradients in functional connectivity along the two axes. We studied the occurrence and propagation of ripples across the entire proximo-distal axis along with a comparable spatial range of the septo-temporal axis of dorsal CA1. We observed that ripples could occur at any location, but their probability of co-occurrence and amplitude decreased with increasing distance from the reference tetrode. This reduction was greater along the proximo-distal axis than the septo-temporal axis. Furthermore, we found that ripples propagate primarily along the proximo-distal axis. Thus, over a spatial scale of ~1.5 mm, the network is anisotropic along the two axes, complementing the topographically organized cortico-hippocampal connections.
The fecal microbiota of piglets is influenced by the farming environment and is associated with piglet robustness at weaning
Authors: Luise Diana , Le Sciellour Mathilde , Buchet Arnaud , Resmond Rémi , Clement Charlène , Rossignol Marie-Noelle , Jardet Deborah , Zemb Olivier , Belloc Catherine , Merlot Elodie
Published: December 2019

This study describes the response of piglet microbiota to weaning in various environments, and investigates whether microbiota composition is associated with the robustness of piglets. Faecal samples and growth data were collected just before and 7 days after weaning in 288 piglets from 16 commercial farms characterised by their pathogenic status and antimicrobial use. The effect of weaning on the most abundant microbial families of faecal microbiota was roughly the same in all farms and in agreement with previous findings. Four enterotypes, ubiquitous in all farms, were identified, for which the most discriminating genera werePrevotella,Faecalibacterium,Roseburia, andLachnospira.They corresponded to a gradual maturational shift from pre to post-weaning microbiota. Besides antimicrobial use, the pathogenic status of the farm had a major influence on the post-weaning microbial evolution of apparently healthy piglets. Regarding individual characteristics, piglets whose growth was less perturbed by weaning had moreBacteroidetes(P < 0.01) and lessProteobacteria (P < 0.001).In response to weaning, they showed a greater increase inPrevotella(P < 0.01),Coprococcus(P< 0.01) andLachnospira(P < 0.05) than piglets that grew more slowly. Thus, the microbiota of robust piglets shares common characteristics regardless the living environment of animals.
Mathematical model for force and energy of virion-cell interactions during full engulfment in HIV: Impact of virion maturation and host cell morphology
Authors: Kruse Elizabeth , Abdalrahman Tamer , Selhorst Philippe , Franz Thomas
Published: December 2019

Viral endocytosis involves elastic cell deformation, driven by chemical adhesion energy, and depends on physical interactions between virion and cell membrane. These interactions are not easy to quantify experimentally. Hence, this study aimed to develop a mathematical model of the interactions of HIV particles with host cells and explore the effects of mechanical and morphological parameters during full virion engulfment. The invagination force and engulfment energy were described as viscoelastic and linear-elastic functions of radius and elastic modulus of virion and cell, ligand-receptor energy density and engulfment depth. The influence of changes in the virion-cell contact geometry representing different immune cells and ultrastructural membrane features and the decrease in virion radius and shedding of gp120 proteins during maturation on invagination force and engulfment energy was investigated. A low invagination force and high ligand-receptor energy are associated with high virion entry ability. The required invagination force was the same for immune cells of different sizes but lower for a local convex geometry of the cell membrane at the virion length scale. This suggests that localized membrane features of immune cells play a role in viral entry ability. The available engulfment energy decreased during virion maturation, indicating the involvement of additional biological or biochemical changes in viral entry. The developed mathematical model offers potential for the mechanobiological assessment of the invagination of enveloped viruses towards improving the prevention and treatment of viral infections.
The hematopoietic landscape at single-cell resolution reveals unexpected stem cell features in naked mole-rats
Authors: Emmrich Stephan , Mariotti Marco , Takasugi Masaki , Straight Maggie E. , Trapp Alexandre , Gladyshev Vadim N. , Seluanov Andrei , Gorbunova Vera
Published: December 2019

Naked mole-rats are the longest-lived rodents endowed with resistance to cancer and age-related diseases, yet their stem cell characteristics remain enigmatic. We profiled the naked mole-rat hematopoietic system down to single-cell resolution, and identified several unique features likely contributing to longevity. In adult naked mole-rats red blood cells are formed in spleen and marrow, a neotenic feature beneficial for hypoxic environments and to prevent anemia. Platelet numbers are lower compared to short-lived mice, which may preclude age-related platelet increase and thrombosis. T cells mature in thymus and lymph nodes, providing a supply of T cells after age-related thymus involution. The pool of quiescent stem cells is higher than in mice, and HSCs overexpress an oxidative phosphorylation signature, revealing a new paradigm of stem cell metabolism to benefit longevity and oppose oncogenesis. Our work provides a platform to study immunology and stem cell biology in an animal model of healthy aging.

HIGHLIGHTS

Flow cytometry labelling panel to purify viable naked mole-rat HSPCs

The spleen as the major site of erythropoiesis in the naked mole-rat

Naked mole-rats show extrathymic T-cell development under homeostatic conditions

Naked mole-rat hematopoietic stem cells (HSCs) have high OXPHOS activity
Multi-parametric quantitative spinal cord MRI with unified signal readout and image denoising
Authors: Grussu Francesco , Battiston Marco , Veraart Jelle , Schneider Torben , Cohen-Adad Julien , Shepherd Timothy M. , Alexander Daniel C. , Novikov Dmitry S. , Fieremans Els , Wheeler-Kingshott Claudia A. M. Gandini
Published: December 2019

Multi-parametric quantitative MRI (qMRI) of the spinal cord is a promising non-invasive tool to probe early microstructural damage in neurological disorders. It is usually performed by combining acquisitions with multiple signal readouts, which exhibit different thermal noise levels, geometrical distortions and susceptibility to physiological noise. This ultimately hinders joint multi-contrast modelling and makes the geometric correspondence of parametric maps challenging. We propose an approach to overcome these limitations, by implementing state-of-the-art microstructural MRI of the spinal cord with a unified signal readout. We base our acquisition on single-shot echo planar imaging with reduced field-of-view, and obtain data from two different vendors (vendor 1: Philips Achieva; vendor 2: Siemens Prisma). Importantly, the unified acquisition allows us to compare signal and noise across contrasts, thus enabling overall quality enhancement via Marchenko-Pastur (MP) Principal Component Analysis (PCA) denoising. MP-PCA is a recent method relying on redundant acquisitions, i.e. such that the number of measurements is much larger than the number of informative principal components. Here we used in vivo and synthetic data to test whether a unified readout enables more efficient denoising of less redundant acquisitions, since these can be denoised jointly with more redundant ones. We demonstrate that a unified readout provides robust multi-parametric maps, including diffusion and kurtosis tensors from diffusion MRI, myelin metrics from two-pool magnetisation transfer, and T1 and T2 from relaxometry. Moreover, we show that MP-PCA improves the quality of our multi-contrast acquisitions, since it reduces the coefficient of variation (i.e. variability) by up to 15% for mean kurtosis, 8% for bound pool fraction (BPF, myelin-sensitive), and 13% for T1, while enabling more efficient denoising of modalities limited in redundancy (e.g. relaxometry). In conclusion, multi-parametric spinal cord qMRI with unified readout is feasible and provides robust microstructural metrics with matched resolution and distortions, whose quality benefits from MP-PCA denoising, a useful pre-processing tool for spinal cord MRI.
Golgi anti-apoptotic proteins are evolutionarily conserved ion channels that regulate cell death in plants
Authors: Sierla Maija , Prole David L. , Saraiva Nuno , Carrara Guia , Dinischiotu Natalia , Vaattovaara Aleksia , Wrzaczek Michael , Taylor Colin W. , Smith Geoffrey L. , Feys Bart
Published: December 2019

Programmed cell death regulates developmental and stress responses in eukaryotes. Golgi anti-apoptotic proteins (GAAPs) are evolutionarily conserved cell death regulators. Human and viral GAAPs inhibit apoptosis and modulate intracellular Ca2+fluxes, and viral GAAPs form cation-selective channels. Although most mammalian cell death regulators are not conserved at the sequence level in plants, the GAAP gene family shows expansion, with five paralogues (AtGAAP1-5) in the Arabidopsis genome. We pursued molecular and physiological characterization of AtGAAPs making use of the advanced knowledge of their human and viral counterparts. Structural modeling of AtGAAPs predicted the presence of a channel-like pore, and electrophysiological recordings from purified AtGAAP3 reconstituted into lipid bilayers confirmed that plant GAAPs can function as ion channels. AtGAAP1 and AtGAAP4 localized exclusively to the Golgi within the plant cell, while AtGAAP2, AtGAAP3 and AtGAAP5 also showed tonoplastic localization. Gene expression analysis revealed differential spatial expression and abundance of transcript forAtGAAPparalogues in Arabidopsis tissues. We demonstrate that AtGAAP1-5 inhibit Bax-induced cell death in yeast. However, overexpression of AtGAAP1 induces cell death inNicotiana benthamianaleaves and lesion mimic phenotype in Arabidopsis. We propose that AtGAAPs function as Golgi-localized ion channels that regulate cell death by affecting ionic homeostasis within the cell.

Highlight

Arabidopsis Golgi anti-apoptotic proteins (GAAPs) share functional conservation with their human and viral counterparts in cell death regulation and ion channel activity

Abbreviations

AtGAAP,Arabidopsis thalianaGAAP; BI-1, Bax inhibitor-1; CFP, cyan fluorescent protein; CMLV, camelpox virus; ER, Endoplasmic reticulum; GAAP, Golgi anti-apoptotic protein; GFP, green fluorescent protein; hGAAP, human GAAP; LFG, Lifeguard; LMM, lesion mimic mutant; PCD, programmed cell death; TMBIM, transmembrane Bax inhibitor-1 motif-containing; TMDs, transmembrane domains; vGAAP, viral GAAP; YFP, yellow fluorescent protein
Alignment of time-course single-cell RNA-seq data with CAPITAL
Authors: Sugihara Reiichi , Kato Yuki , Mori Tomoya , Kawahara Yukio
Published: December 2019

Recent techniques on single-cell RNA sequencing have boosted transcriptome-wide observation of gene expression dynamics of time-course data at a single-cell scale. Typical examples of such analysis include inference of a pseudotime cell trajectory, and comparison of pseudotime trajectories between different experimental conditions will tell us how feature genes regulate a dynamic cellular process. Existing methods for comparing pseudotime trajectories, however, force users to select trajectories to be compared because they can deal only with simple linear trajectories, leading to the possibility of making a biased interpretation. Here we present CAPITAL, a method for comparing pseudotime trajectories with tree alignment whereby trajectories including branching can be compared without any knowledge of paths to be compared. Computational tests on time-series public data indicate that CAPITAL can align non-linear pseudotime trajectories and reveal gene expression dynamics.
Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression
Authors: Salgado Catarina , Roelse Celine , Nell Rogier , Gruis Nelleke , van Doorn Remco , van der Velden Pieter
Published: December 2019

Thetelomerase reverse transcriptase(TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. Contrary to common concepts, CpG methylation in theTERTpromoter (TERTp), was correlated withTERTmRNA expression. Furthermore, two hotspot mutations inTERTp, dubbed C228T and C250T, have been revealed to assist binding of transcription factor ETS/TCF and subsequentTERTexpression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and higher-order chromatin structure) and genetic (promoter mutations) mechanisms in regulatingTERTgene expression in healthy skin and in melanoma cell lines (n=61). We unexpectedly observed that the methylation ofTERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples,TERTmRNA was only expressed in the melanoma cell lines with high methylation or intermediate methylation in combination withTERTmutations.TERTp methylation was positively correlated with chromatin accessibility and expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in whichTERTexpression requires either a widely open chromatin state throughout the promoter inTERTp-WT samples due to high methylation or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T/C250T mutations.

Author summary

PvdV and RvD formulated research goals and aims and supervised the overall progress. Wet-lab experiments, preparation of the manuscript and statistical analysis were performed by CS and CR. CS designed the novel assays. RN was involved in the experimental setup. RvD, NG and PvdV were responsible for funding acquisition. CR, RN, NG, RvD and PvdV critically reviewed the manuscript.
wg-blimp: an end-to-end analysis pipeline for whole genome bisulfite sequencing data
Authors: Wöste Marius , Leitão Elsa , Laurentino Sandra , Horsthemke Bernhard , Rahmann Sven , Schröder Christopher
Published: December 2019

Background

Analysing whole genome bisulfite sequencing datasets is a data-intensive task that requires comprehensive and reproducible workflows to generate valid results. While many algorithms have been developed for tasks such as alignment, comprehensive end-to-end pipelines are still sparse. Furthermore, previous pipelines lack features or show technical deficiencies, thus impeding analyses.

Results

We developed wg-blimp (whole genome bisulfite sequencing methylation analysis pipeline) as an end-to-end pipeline to ease whole genome bisulfite sequencing data analysis. It integrates established algorithms for alignment, quality control, methylation calling, detection of differentially methylated regions, and methylome segmentation, requiring only a reference genome and raw sequencing data as input. Comparing wg-blimp to previous end-to-end pipelines reveals similar setups for common sequence processing tasks, but shows differences for post-alignment analyses. We improve on previous pipelines by providing a more comprehensive analysis workflow as well as an interactive user interface. To demonstrate wg-blimp’s ability to produce correct results we used it to call differentially methylated regions for two publicly available datasets. We were able to replicate 112 of 114 previously published regions, and found results to be consistent with previous findings. We further applied wg-blimp to a publicly available sample of embryonic stem cells to showcase methylome segmentation. As expected, unmethylated regions were in close proximity of transcription start sites. Segmentation results were consistent with previous analyses, despite different reference genomes and sequencing techniques.

Conclusions

wg-blimp provides a comprehensive analysis pipeline for whole genome bisulfite sequencing data as well as a user interface for simplified result inspection. We demonstrated its applicability by analysing multiple publicly available datasets. Thus, wg-blimp is a relevant alternative to previous analysis pipelines and may facilitate future epigenetic research.
On the prediction of DNA-binding preferences of C2H2-ZF domains using structural models: application on human CTCF
Authors: Meseguer Alberto , Årman Filip , Fornes Oriol , Molina Ruben , Bonet Jaume , Fernandez-Fuentes Narcis , Oliva Baldo
Published: December 2019

Cis2-His2 zinc finger (C2H2-ZF) proteins are the largest family of transcription factors in human and higher metazoans. However, the DNA-binding preferences of many members of this family remain unknown. We have developed a computational method to predict these DNA-binding preferences. We combine information from crystal structures composed by C2H2-ZF domains and from bacterial one-hybrid experiments to compute scores for protein-DNA binding based on statistical potentials. We apply the scores to compute theoretical position weight matrices (PWMs) of proteins with a DNA-binding domain composed by C2H2-ZF domains, with the only requirement of an input structure (experimentally determined or modelled). We have tested the capacity to predict PWMs of zinc finger domains, successfully predicting 3-2 nucleotides of a trinucleotide binding site for about 70% variants of single zinc-finger domains of Zif268. We have also tested the capacity to predict the PWMs of proteins composed by three C2H2-ZF domains, successfully matching between 60% and 90% of the binding-site motif according to the JASPAR database. The tests are used as a proof of the capacity to scan a DNA fragment and find the potential binding sites of transcription-factors formed by C2H2-ZF domains. As an example, we have tested the approach to predict the DNA-binding preferences of the human chromatin binding factor CTCF.
Cryo-OrbiSIMS for 3D molecular imaging of a bacterial biofilm in its native state
Authors: Zhang Junting , Brown James , Scurr David , Bullen Anwen , MacLellan-Gibson Kirsty , Williams Paul , Alexander Morgan R. , Hardie Kim R. , Gilmore Ian S. , Rakowska Paulina D.
Published: December 2019

We describe a method for label-free molecular imaging of biological materials, preserved in a native state, by using an OrbiSIMS instrument equipped with cryogenic sample handling and developing a high-pressure freezing protocol compatible with mass spectrometry. We studied the 3D distribution of quorum sensing signalling molecules, nucleobases and bacterial membrane molecules, in a mature Pseudomonas aeruginosa biofilm, with high spatial-resolution and high mass-resolution.
An Adaptive Cue Selection Model of Allocentric Spatial Reorientation
Authors: Negen James , Bird Laura , Nardini Marko
Published: December 2019

After becoming disoriented, an organism must use the local environment to reorient and recover vectors to important locations. Debates over how this happens have been extensive. A new theory, Adaptive Combination, suggests that the information from different spatial cues are combined with Bayesian efficiency. To test this further, we modified the standard reorientation paradigm to be more amenable to Bayesian cue combination analyses while still requiring reorientation, still requiring participants to recall goal locations from memory, and focusing on situations that require the use of the allocentric (world-based; not egocentric) frame. 12 adults and 20 children at 5-7 years old were asked to recall locations in a virtual environment after a disorientation. They could use either a pair of landmarks at the North and South, a pair at the East and West, or both. Results were not consistent with Adaptive Combination. Instead, they are consistent with the use of the most useful (nearest) single landmark in isolation. We term this Adaptive Selection. Experiment 2 suggests that adults also use the Adaptive Selection method when they are not disoriented but still required to use a local allocentric frame. This suggests that the process of recalling a location in the allocentric frame is typically guided by the single most useful landmark, rather than a Bayesian combination of landmarks – regardless of whether the use of the allocentric frame is forced by disorientation or another method. These failures to benefit from a Bayesian strategy accord with the broad idea that there are important limits to Bayesian theories of the cognition, particularly for complex tasks such as allocentric recall.
Quantifying subclinical and longitudinal microvascular changes following episcleral plaque brachytherapy (EPB) using spectral-domain OCT angiography
Authors: Green Kyle M. , Toy Brian C. , Ashimatey Bright S. , Mustafi Debarshi , Jennelle Richard L. , Astrahan Melvin A. , Chu Zhongdi , Wang Ruikang K. , Kim Jonathan , Berry Jesse L. , Kashani Amir H.
Published: December 2019

Background

I-125 episcleral plaque brachytherapy (EPB) is standard-of-care for globe-conserving treatment of medium-sized choroidal melanomas. Radiation retinopathy is a potential consequence of treatment, characterized by deleterious effects on retinal microvasculature. We investigated the application of Optical Coherence Tomography Angiography (OCTA) for detecting and longitudinally monitoring I-125 episcleral plaque brachytherapy induced radiation retinopathy.

Methods

High resolution OCTA of the central 3×3mm macula were obtained from I-25 episcleral plaque brachytherapy treated and untreated fellow eyes of 62 patients. Capillary density (vessel skeleton density, VSD) and caliber (vessel diameter index, VDI) were quantified using previously validated semi-automated algorithms. Nonperfusion was also quantified as flow impairment regions (FIR). Exams from treated and fellow eyes obtained pre-treatment and at 6-month, 1-year, and 2-year intervals were compared using generalized estimating equation linear models. Dosimetry maps were used to evaluate spatial correlation between radiation dose and microvascular metrics.

Results

Mean time from treatment to last follow-up was 10.8 months. Mean±SD and median radiation dose at the fovea were 64.5 ± 76 Gy and 32.0 Gy, respectively. Preoperative logMAR (Snellen) mean visual acuity was 0.26 ± 0.05 (∼20/35) and 0.08 ± 0.02 (∼20/25) in treated and fellow eyes, respectively. At 6 months, treated eyes had significantly lower VSD (0.147 ± 0.003 vs 0.155 ± 0.002;p= 0.023) and higher FIR (1.95 ± 0.176 vs 1.45 ± 0.099;p= 0.018) compared to fellow eyes. There was a significant decrease in VSD and a corresponding increase in FIR even for treated eyes without clinically identifiable retinopathy at 6 months. VDI was significantly higher in treated eyes than in fellow eyes at 2 years (2.93 ± 0.022 vs 2.84 ± 0.016;p= 0.002). Microvascular changes were spatially correlated with a radiation gradient of 85-250 Gy across the fovea.

Conclusions

OCTA can be used to quantify and monitor EPB induced radiation, and can detect vascular abnormalities even in the absence of clinically observable retinopathy. OCTA may therefore be useful in investigating treatment interventions that aim to delay EPB-induced radiation retinopathy.