bluuebunny/crossref_metadata_2025 huggingface dataset:
interpolatedXY: a two-step strategy to normalise DNA methylation microarray data avoiding sex bias
Authors: Wang Yucheng , Gorrie-Stone Tyler J. , Grant Olivia A. , Andrayas Alexandria D. , Zhai Xiaojun , McDonald-Maier Klaus D. , Schalkwyk Leonard C.
Published: October 2021

Motivation

Data normalization is an essential step to reduce technical variation within and between arrays. Due to the different karyotypes and the effects of X chromosome inactivation, females and males exhibit distinct methylation patterns on sex chromosomes, thus it poses a significant challenge to normalise sex chromosome data without introducing bias. Currently, existing methods do not provide unbiased solutions to normalise sex chromosome data, usually, they just process autosomal and sex chromosomes indiscriminately.

Results

Here, we demonstrate that ignoring this sex difference will lead to introducing artificial sex bias, especially for thousands of autosomal CpGs. We present a novel two-step strategy (interpolatedXY) to address this issue, which is applicable to all quantile-based normalisation methods. By this new strategy, the autosomal CpGs are first normalised independently by conventional methods, such as funnorm or dasen; then the corrected methylation values of sex chromosome linked CpGs are estimated as the weighted average of their nearest neighbours on autosomes. The proposed two-step strategy can also be applied to other non-quantile-based normalisation methods, as well as other array-based data types. Moreover, we propose a useful concept: the sex explained fraction of variance, to quantitatively measure the normalisation effect.

Availability

The proposed methods are available by calling the function ‘adjustedDasen’ or ‘adjustedFunnorm’ in the latest wateRmelon package (https://github.com/schalkwyk/wateRmelon), with methods compatible with all the major workflows, including minfi.

Contact

xzhai@essex.ac.uk; lschal@essex.ac.uk

Supplementary information

Supplementary data are available at...
Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability
Authors: Cibelli Antonio , Stout Randy , Timmermann Aline , de Menezes Laura , Guo Peng , Maass Karen , Seifert Gerald , Steinhäuser Christian , Spray David C. , Scemes Eliana
Published: October 2021

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if blood-brain-barrier integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.
Multivariate pattern analysis reveals location specific aftereffects of 10Hz motor cortex transcranial alternating current stimulation
Authors: Tzvi Elinor , Alizadeh Jalal , Schubert Christine , Classen Joseph
Published: October 2021

Background

Transcranial alternating current stimulation (tACS) may induce frequency-specific aftereffects on brain oscillations in the stimulated location, which could serve as evidence for region-specific neuroplasticity. Aftereffects of tACS on the motor system remain unknown.

Objective

To find evidence for aftereffects in short EEG segments following tACS to two critical nodes of the motor network, namely, left motor cortex (lMC) and right cerebellum (rCB). We hypothesized that aftereffects of lMC will be stronger in and around lMC compared to both active stimulation of rCB, as well as inactive (“sham”) control conditions.

Methods

To this end, we employed multivariate pattern analysis (MVPA), and trained a classifier to distinguish between EEG signals following each of the three stimulation protocols. This method accounts for the multitude facets of the EEG signal and thus is more sensitive to subtle modulation of the EEG signal.

Results

EEG signals in both theta (θ, 4-8Hz) and alpha (α, 8-13Hz) were better classified to lMC-tACS compared to rCB-tACS/sham, in and around lMC-tACS stimulation locations (electrodes FC3 and CP3). This effect was associated with a decrease in power following tACS. Source reconstruction revealed significant differences in premotor cortex but not in primary motor cortex as the computational model suggested. Correlation between classification accuracies in θ and α in lMC-tACS was stronger compared to rCB-tACS/sham, suggesting cross-frequency effects of tACS. Nonetheless, θ/α phase-coupling did not differ between stimulation protocols.

Conclusions

Successful classification of EEG signals to left motor cortex using MVPA revealed focal tACS aftereffects on the motor cortex, indicative of region-specific neuroplasticity.
Microevolution of Pieris butterfly genes involved in host-plant adaptation along a host-plant community cline
Authors: Okamura Yu , Sato Ai , Kawaguchi Lina , Nagano Atsushi J. , Murakami Masashi , Vogel Heiko , Kroymann Juergen
Published: October 2021

Herbivorous insects have evolved counteradaptations to overcome the chemical defenses of their host plants. Several of these counteradaptations have been elucidated at the molecular level, in particular for insects specialized on cruciferous host plants. While the importance of these counteradaptations for host plant colonization is well established,little is known about their microevolutionary dynamics in the field. In this study, we examine patterns of host plant use and insect counteradaptation in three Pieris butterfly species across Japan. The larvae of these butterflies express nitrile-specifier protein (NSP) and its paralog major allergen (MA) in their gut to overcome the highly diversified glucosinolate-myrosinase defense system of their cruciferous host plants. Pieris napi and Pieris melete colonize wild Brassicaceae whereas Pieris rapae typically uses cultivated Brassica as a host, regardless of the local composition of wild crucifers. As expected, NSP and MA diversity was independent of the local composition of wild Brassicaceae in P. rapae. In contrast, NSP diversity correlated with local host plant diversity in both species that preferred wild Brassicaceae. P. melete and P. napi both revealed two distinct major NSP alleles, which shaped diversity among local populations, albeit with different evolutionary trajectories. In comparison, MA showed no indication for local adaptation. Altogether, MA appeared to be evolutionary more conserved than NSP, suggesting that both genes play different roles in diverting host plant chemical defense.
Isotopic evidence for population dynamics in the Central Italian Copper Age and Bronze Age
Authors: Romboni Marco , Arienzo Ilenia , Di Vito Mauro Antonio , Lubritto Carmine , Piochi Monica , Di Cicco Maria Rosa , Rickards Olga , Rolfo Mario Federico , Sevink Jan , De Angelis Flavio , Alessandri Luca
Published: October 2021

The mobility patterns in the Italian peninsula during prehistory are still relatively unknown. The excavation of the Copper Age and Bronze Age deposits in La Sassa cave (Sonnino, Italy) allowed to broaden the knowledge about some local and regional dynamics. We employed a multi-disciplinary approach, including stable (carbon and nitrogen – C and N, respectively) and radiogenic (strontium-Sr) isotopes analyses and the identification of the cultural traits in the material culture to identify mobility patterns that took place in the region. The Sr isotopic analyses on the human bones show that in the Copper Age and at the beginning of the Bronze Age, the cave was used as a burial place by different villages, perhaps spread in a radius of no more than 5 km. Stable isotopes analyses suggest the introduction of C4 plants in the diet of the Middle Bronze Age (MBA) communities in the area. Remarkably, in the same period, the material culture shows increasing influxes coming from the North. This evidence is consistent with the recent genomic findings tracing the arrival of people carrying a Steppe-related ancestry in Central Italy in MBA.
Knockout of a singleSoxgene resurrects an ancestral cell type in the sea anemoneNematostella vectensis
Authors: Babonis Leslie S. , Enjolras Camille , Reft Abigail J. , Foster Brent M. , Hugosson Fredrik , Ryan Joseph F. , Daly Marymegan , Martindale Mark Q.
Published: October 2021

Cnidocytes are the explosive stinging cells found only in cnidarians (corals, jellyfish, etc). Specialized for prey capture and defense, cnidocytes are morphologically complex and vary widely in form and function across taxa; how such diversity evolved is unknown. Using CRISPR/Cas9-mediated genome editing in the burrowing sea anemoneNematostella vectensis, we show that a single transcription factor (NvSox2) acts as a binary switch between two alternative cnidocyte fates. Knockout ofNvSox2caused a complete transformation of nematocytes (piercing cells) into spirocytes (ensnaring cells). The type of spirocyte induced byNvSox2knockout (robust spirocyte) is not normally found inN. vectensisbut is common in sea anemones from other habitats. Homeotic control of cell fate provides a mechanistic explanation for the discontinuous distribution of cnidocyte types across cnidarians and demonstrates how simple counts of cell types can underestimate biodiversity.
MoSBi: Automated signature mining for molecular stratification and subtyping
Authors: Rose Tim Daniel , Bechtler Thibault , Ciora Octavia-Andreea , Lilian Le Kim Anh , Molnar Florian , Koehler Nikolai , Baumbach Jan , Röttger Richard , Pauling Josch Konstantin
Published: October 2021

The improving access to increasing amounts of biomedical data provides completely new chances for advanced patient stratification and disease subtyping strategies. This requires computational tools that produce uniformly robust results across highly heterogeneous molecular data. Unsupervised machine learning methodologies are able to discover de-novo patterns in such data. Biclustering is especially suited by simultaneously identifying sample groups and corresponding feature sets across heterogeneous omics data. The performance of available biclustering algorithms heavily depends on individual parameterization and varies with their application. Here, we developed MoSBi (Molecular Signature identification using Biclustering), an automated multi-algorithm ensemble approach that integrates results utilizing an error model-supported similarity network. We evaluated the performance of MoSBi on transcriptomics, proteomics and metabolomics data, as well as synthetic datasets covering various data properties. Profiting from multi-algorithm integration, MoSBi identified robust group and disease specific signatures across all scenarios overcoming single algorithm specificities. Furthermore, we developed a scalable network-based visualization of bicluster communities that support biological hypothesis generation. MoSBi is available as an R package and web-service to make automated biclustering analysis accessible for application in molecular sample stratification.
Climatic clustering and longitudinal analysis with impacts on food, bioenergy, and pandemics
Authors: Lagergren John , Cashman Mikaela , Melesse Vergara Verónica G. , Eller Paul R. , Gazolla Joao Gabriel Felipe Machado , Chhetri Hari B. , Streich Jared , Climer Sharlee , Thornton Peter , Joubert Wayne , Jacobson Daniel
Published: October 2021

Predicted growth in world population will put unparalleled stress on the need for sustainable energy and global food production, as well as increase the likelihood of future pandemics. In this work, we identify high-resolution environmental zones in the context of a changing climate and predict longitudinal processes relevant to these challenges. We do this using exhaustive vector comparison methods that measure the climatic similarity between all locations on earth at high geospatial resolution. The results are captured as networks, in which edges between geolocations are defined if their historical climates exceed a similarity threshold. We then apply Markov clustering and our novel Correlation of Correlations method to the resulting climatic networks, which provides unprecedented agglomerative and longitudinal views of climatic relationships across the globe. The methods performed here resulted in the fastest (9.37 × 1018 operations/sec) and one of the largest (168.7 × 1021 operations) scientific computations ever performed, with more than 100 quadrillion edges considered for a single climatic network. Correlation and network analysis methods of this kind are widely applicable across computational and predictive biology domains, including systems biology, ecology, carbon cycles, biogeochemistry, and zoonosis research.
The history of body size evolution in termites
Authors: Mizumoto Nobuaki , Bourguignon Thomas
Published: October 2021

Termites are social cockroaches. Because non-termite cockroaches are larger than basal termite lineages, which themselves include large termite species, it has been proposed that termites experienced a unidirectional body size reduction since they evolved eusociality. However, the validity of this hypothesis remains untested in a phylogenetic framework. Here, we reconstructed termite body size evolution using head width measurements of 1638 modern and fossil termite species. We found that the unidirectional body size reduction model was only supported by analyses excluding fossil species. Analyses including fossil species suggested that body size diversified along with speciation events and estimated that the size of the common ancestor of modern termites was comparable to that of modern species. Our analyses further revealed that body size variability among species, but not body size reduction, is associated with features attributed to advanced termite societies. Our results suggest that miniaturization took place at the origin of termites, while subsequent complexification of termite societies did not lead to further body size reduction.
Simulation-based inference of evolutionary parameters from adaptation dynamics using neural networks
Authors: Avecilla Grace , Chuong Julie N. , Li Fangfei , Sherlock Gavin , Gresham David , Ram Yoav
Published: October 2021

The rate of adaptive evolution depends on the rate at which beneficial mutations are introduced into a population and the fitness effects of those mutations. The rate of beneficial mutations and their expected fitness effects is often difficult to empirically quantify. As these two parameters determine the pace of evolutionary change in a population, the dynamics of adaptive evolution may enable inference of their values. Copy number variants (CNVs) are a pervasive source of heritable variation that can facilitate rapid adaptive evolution. Previously, we developed a locus-specific fluorescent CNV reporter to quantify CNV dynamics in evolving populations maintained in nutrient-limiting conditions using chemostats. Here, we use the observed CNV adaptation dynamics to estimate the rate at which beneficial CNVs are introduced through de novo mutation and their fitness effects using simulation-based Bayesian likelihood-free inference approaches. We tested the suitability of two evolutionary models: a standard Wright-Fisher model and a chemostat growth model. We evaluated two likelihood-free inference algorithms: the well-established Approximate Bayesian Computation with Sequential Monte Carlo (ABC-SMC) algorithm, and the recently developed Neural Posterior Estimation (NPE) algorithm, which applies an artificial neural network to directly estimate the posterior distribution. By systematically evaluating the suitability of different inference methods and models we show that NPE has several advantages over ABC-SMC and that a Wright-Fisher evolutionary model suffices in most cases. Using our validated inference framework, we estimate the CNV formation rate at the GAP1 locus in yeast as 10−4.7 -10−4 per cell division, and a selection coefficient of 0.04 - 0.1 per generation for GAP1 CNVs in glutamine-limited chemostats. We experimentally validated our estimates using barcode lineage tracking and pairwise fitness assays. Our results are consistent with a high beneficial CNV supply rate that is 10-fold greater than the estimated rates of beneficial single-nucleotide mutations, explaining their outsized importance in rapid adaptive evolution. More generally, our study demonstrates the utility of novel simulation-based likelihood-free inference methods for inferring the rates and effects of evolutionary processes from empirical data.
Metacognition and the effect of incentive motivation in two compulsive disorders: gambling disorder and obsessive-compulsive disorder
Authors: Hoven Monja , de Boer Nina S. , Goudriaan Anna E. , Denys Damiaan , Lebreton Mael , van Holst Ruth J. , Luigjes Judy
Published: October 2021

Compulsivity is a common phenotype amongst various psychiatric disorders, such as obsessive-compulsive disorder (OCD) and gambling disorder (GD). Deficiencies in metacognition, such as the inability to properly estimate ones’ own performance via well-calibrated confidence judgments could contribute to pathological decision-making in these psychiatric disorders. Earlier research has indeed suggested that OCD and GD patients reside at opposite ends of the confidence spectrum, with OCD patients exhibiting underconfidence, and GD patients exhibiting overconfidence. Recently, several studies established that motivational states (e.g. monetary incentives) influence metacognition, with gain (respectively loss) prospects increasing (respectively decreasing) confidence judgments. Here, we reasoned that the OCD and GD symptomatology might correspond to an exacerbation of this interaction between metacognition and motivational states. We hypothesized GD’s overconfidence to be exaggerated during gain prospects, while OCD’s underconfidence to be worsened in loss context, which we expected to see represented in ventromedial prefrontal cortex (VMPFC) blood-oxygen-level-dependent (BOLD) activity. We tested those hypotheses in a task-based functional magnetic resonance imaging (fMRI) design. Our initial analyses showed increased confidence levels for GD versus OCD patients, that could partly be explained by sex and IQ. Although our primary analyses did not support the hypothesized interaction between incentives and groups, exploratory analyses did show increased confidence in GD patients specifically in gain context. fMRI analyses confirmed a central role for VMPFC in the processing of confidence and incentives, but with no differences between the clinical samples. The trial is registered in the Dutch Trial Register (Trial NL6171, registration number: NTR6318) (https://www.trialregister.nl/trial/6171).
Ecological Stability Emerges at the Level of Strains in the Human Gut Microbiome
Authors: Wolff Richard , Shoemaker William , Garud Nandita
Published: October 2021

The human gut microbiome harbors substantial ecological diversity at the species level, as well as at the strain level within species. In healthy hosts, species abundance fluctuations in the microbiome are thought to be stable, and these fluctuations can be described by macroecological laws. However, it is less clear how strain abundances change over time. An open question is whether individual strains behave like species themselves, exhibiting stability and following the macroecological relationships known to hold at the species level, or whether strains have different dynamics, perhaps due to the relatively close phylogenetic relatedness of co-colonizing lineages. Here, we analyze the daily dynamics of intra-specific genetic variation in the gut microbiomes of four healthy, densely longitudinally sampled hosts. First, we find that overall genetic diversity in a large majority of species is stationary over time, despite short-term fluctuations. Next, we show that fluctuations in abundances in approximately 80% of strains analyzed can be predicted with a stochastic logistic model (SLM)—an ecological model of a population experiencing environmental fluctuations around a fixed carrying capacity which has previously been shown to capture statistical properties of species abundance fluctuations. The success of this model indicates that strain abundances typically fluctuate around a fixed carrying capacity, suggesting that most strains are dynamically stable. Finally, we find that the strain abundances follow several empirical macroecological laws known to hold at the species level. Together, our results suggest that macroecological properties of the human gut microbiome, including its stability, emerge at the level of strains.
Greater spear nosed bats commute long distances alone, rest together, but forage apart
Authors: Teague O’Mara M. , Dechmann Dina K.N.
Published: October 2021

Animals frequently forage in groups on ephemeral resources to profit from social information and increased efficiency. Greater spear-nosed bats (Phyllostomus hastatus) develop group-specific social calls, which are hypothesized to coordinate social foraging to feed on patchily-distributed balsa flowers. To test this, we tagged all members of three social groups ofP. hastatuson Isla Colón, Panamá, using high frequency GPS during a season when balsa had begun to flower. We found commuting distances of 20-30 km to foraging sites, more than double of what has been previously reported. In contrast to our expectations, we found that tagged individuals did not commute together, but did join group members in small foraging patches with high densities of flowering balsas on the mainland. We hypothesized that close proximity to group members would increase foraging efficiency if social foraging were used to find flower clusters, but distance between tagged individuals did not predict foraging efficiency or energy expenditure. However, decreased distance among tagged bat positively influenced time outside of the cave and increased the duration and synchrony of resting. These results suggest that social proximity seemed to be more important during resting, and indicate that factors other than increased feeding efficiency may structure social relationships of group members while foraging. It appears that, depending on the local resource landscape, these bats have an excellent map even of distant resources and may use social information only for current patch discovery. They then may no longer rely on social information during daily foraging.

Highlights

Unlike in Trinidad, greater spear-nosed bats in Panamá do not commute or forage as a group

Foraging distances are double of previously known and across the sea

Bats rest near each other, but do not forage more efficiently when close

Social information may mainly be used for the discovery of feeding areas

Local resource landscapes may cause strong variation in social behaviour
A Biophysical Counting Mechanism for Keeping Time
Authors: Zemlianova Klavdia , Bose Amitabha , Rinzel John
Published: October 2021

The ability to estimate and produce appropriately timed responses is central to many behaviors including speaking, dancing, and playing a musical instrument. A classical framework for estimating or producing a time interval is the pacemaker-accumulator model in which pulses of a pacemaker are counted and compared to a stored representation. However, the neural mechanisms for how these pulses are counted remains an open question. The presence of noise and stochasticity further complicate the picture. We present a biophysical model of how to keep count of a pacemaker in the presence of various forms of stochasticity using a system of bistable Wilson-Cowan units asymmetrically connected in a one-dimensional array; all units receive the same input pulses from a central clock but only one unit is active at any point in time. With each pulse from the clock, the position of the activated unit changes thereby encoding the total number of pulses emitted by the clock. This neural architecture maps the counting problem into the spatial domain, which in turn translates count to a time estimate. We further extend the model to a hierarchical structure to be able to robustly achieve higher counts.
Role of Ring6 in the function of the E. coli MCE protein LetB
Authors: Vieni Casey , Coudray Nicolas , Isom Georgia L. , Bhabha Gira , Ekiert Damian C.
Published: October 2021

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein.

Highlights

Deleting MCE domains 3 or 6 from LetB confers increased resistance to membrane-perturbing agents

Cryo-EM structure of ΔRing6 LetB mutant highlights the modular nature of MCE domains

Mutations along LetB pore-lining loops modulate detergent resistance
EWS/FLI mediated reprogramming of 3D chromatin promotes an altered transcriptional state in Ewing sarcoma
Authors: Showpnil Iftekhar A. , Selich-Anderson Julia , Taslim Cenny , Boone Megann A. , Crow Jesse C. , Theisen Emily R. , Lessnick Stephen L.
Published: October 2021

Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or highly related fusions. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. EWS/FLI binding is primarily associated with compartment activation, establishment of topologically-associated domain (TAD) boundaries, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators through three-dimensional reprogramming of chromatin.
Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions
Authors: Karakis Victoria , McDonald Thomas , Cordiner Abigail , Mischler Adam , Miguel Adriana San , Rao Balaji M
Published: October 2021

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we do not utilize transforming growth factor-beta inhibitors or a passage step for EVT differentiation, or forskolin for STB formation. Strikingly, under these conditions, presence of a single additional extracellular cue – lam-inin-1 – switched the terminal differentiation of hTSCs from STB to the EVT lineage. Activation of the sphingosine-1 receptor 3 receptor (S1PR3) using a chemical agonist could drive EVT differentiation of hTSCs in the absence of exogenous laminin, albeit less efficiently. To illustrate the utility of a chemically defined culture system for mechanistic studies, we examined the role of protein kinase C (PKC) signaling during hTSC differentiation to the EVT lineage. Inhibition of PKCα/β signaling significantly reduced HLA-G expression and the formation of HLA-G+mesen-chymal EVTs during hTSC differentiation mediated by laminin exposure; however, it did not prevent commitment to the EVT lineage or STB differentiation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation, and will enable mechanistic studies in vitro.

Significance

Despite its importance to a healthy pregnancy, early human placental development remains poorly understood. Mechanistic studies are impeded by restrictions on research with human embryos and fetal tissues, and significant differences in placentation between humans and commonly used animal models. In this context, human trophoblast stem cells (hTSCs) have emerged as attractive in vitro models for the epithelial cytotrophoblast of the early gestation human placenta. Here we describe chemically defined culture conditions for differentiation of hTSCs to the two major differentiated cell types – extravillous trophoblast and syncytiotrophoblast. These culture conditions enable in vitro studies to reveal molecular mechanisms regulating hTSC differentiation.
Genetic control of the dynamic transcriptional response to immune stimuli and glucocorticoids at single cell resolution
Authors: Resztak Justyna A , Wei Julong , Zilioli Samuele , Sendler Edward , Alazizi Adnan , Mair-Meijers Henriette E , Wu Peijun , Wen Xiaoquan , Slatcher Richard B , Zhou Xiang , Luca Francesca , Pique-Regi Roger
Published: October 2021

Synthetic glucocorticoids, such as dexamethasone, have been used as treatment for many immune conditions, such as asthma and more recently severe COVID-19. Single cell data can capture more fine-grained details on transcriptional variability and dynamics to gain a better understanding of the molecular underpinnings of inter-individual variation in drug response. Here, we used single cell RNA-seq to study the dynamics of the transcriptional response to glucocorticoids in activated Peripheral Blood Mononuclear Cells from 96 African American children. We employed novel statistical approaches to calculate a mean-independent measure of gene expression variability and a measure of transcriptional response pseudotime. Using these approaches, we demonstrated that glucocorticoids reverse the effects of immune stimulation on both gene expression mean and variability. Our novel measure of gene expression response dynamics, based on the diagonal linear discriminant analysis, separated individual cells by response status on the basis of their transcriptional profiles and allowed us to identify different dynamic patterns of gene expression along the response pseudotime. We identified genetic variants regulating gene expression mean and variability, including treatment-specific effects, and demonstrated widespread genetic regulation of the transcriptional dynamics of the gene expression response.
An Approach to Measuring Protein Turnover in Human Induced Pluripotent Stem Cell Organoids by Mass Spectrometry
Authors: Duchesne Anthony , Dong Jing , Bayne Andrew N. , Mohamed Nguyen-Vi , Yi Wei , Mathur Meghna , Fon Edward A. , Durcan Thomas M. , Trempe Jean-François
Published: October 2021

Patient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson’s disease - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is simply lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain a critical need as organoid models evolve. To solve this problem, we have designed a workflow to extract proteins from organoids and measure global protein turnover using mass spectrometry and stable isotope labeling using amino acids in cell culture (SILAC). This allowed us to measure the turnover rates of 844 proteins and compare protein turnover to previously reported data in primary cell cultures and in vivo models. Taken together, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.
Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model
Authors: Housman Genevieve , Briscoe Emilie , Gilad Yoav
Published: October 2021

The evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human and six chimpanzee induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.

Author Summary

Primates display a range of skeletal morphologies and susceptibilities to skeletal diseases, but the molecular basis of these phenotypic differences is unclear. Studies of gene expression variation in primate skeletal tissues are extremely restricted due to the ethical and practical challenges associated with collecting samples. Nevertheless, the ability to study gene regulation in primate skeletal tissues is crucial for understanding how the primate skeleton has evolved. We therefore developed a comparative primate skeletal cell culture model that allows us to access a spectrum of human and chimpanzee cell types as they differentiate from stem cells into bone cells. While most gene expression patterns are conserved across species, we also identified hundreds of differentially expressed genes between humans and chimpanzees within and across stages of differentiation. We also classified cells by osteogenic stage and identified additional interspecific differentially expressed genes which may contribute to skeletal trait differences. We anticipate that this model will be extremely useful for exploring questions related to gene regulation variation in primate bone biology and development.
Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern
Authors: Jeong Gi Uk , Yoon Gun Young , Moon Hyun Woo , Lee Wooseong , Hwang Insu , Kim Hae Soo , Kim Kyun-Do , Kim Chonsaeng , Ahn Dae-Gyun , Kim Bum-Tae , Kim Seong-Jun , Kwon Young-Chan
Published: October 2021

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha’s small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

IMPORTANCE

The global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have resulted in the emergence of different variants. Many of these variants have become more virulent and infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological understanding of these variants remains poor. Here, we directly compared the plaque size, stability, and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at physiological temperatures. Our observations may help to characterize each variant in terms of their interactions with host factors and responses to environmental conditions. We also believe that our evaluations will improve understanding of the emergence of new variants and contribute to controlling their spread.
Stress response inEntamoeba histolyticais associated with robust processing of tRNA to tRNA-halves
Authors: Sharma Manu , Zhang Hanbang , Ehrenkaufer Gretchen , Singh Upinder
Published: October 2021

tRNA-derived fragments have been reported in many different organisms and have diverse cellular roles such as regulating gene expression, inhibiting protein translation, silencing transposable elements and modulating cell proliferation. In particular tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here we report the presence of tRNA-derived fragments inEntamoebawith tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite to cyst conversion with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments are associated withEntamoebaArgonaute proteins,EhAgo2-2, andEhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoeba. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses including encystation suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments inEntamoeba.

Importance

tRNA-derived fragments are small RNAs formed by the cleavage of tRNAs at specific positions. These have been reported in many organisms to modulate gene expression and thus regulate various cell functions. In the present study, we report for the first time the presence of tRNA-derived fragments inEntamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small RNA sequencing datasets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during developmental process of encystation. We also found that shorter tRNA-derived fragments are bound toEntamoebaArgonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway which mediates robust gene silencing inEntamoeba. Our results suggest that tRNA-derived fragments inEntamoebahave a possible role in regulating gene expression during environmental stress.
Identification of candidate genes for developmental colour agnosia in a single unique family
Authors: Nijboer Tanja C. , Hessel Ellen V.S. , van Haaften Gijs W. , van Zandvoort Martine J. , van der Spek Peter J. , Troelstra Christine , Kovel Carolien de , Koeleman Bobby P.C. , van der Zwaag Bert , Brilstra Eva H. , Burbach J. Peter H.
Published: October 2021

Colour agnosia is a disorder that impairs colour knowledge (naming, recognition) despite intact colour perception. Previously, we have identified the first and only-known family with hereditary developmental colour agnosia. The aim of the current study was to explore genomic regions and candidate genes that potentially cause this trait in this family. For three family members with developmental colour agnosia and three unaffected family members CGH-array analysis and exome sequencing was performed, and linkage analysis was carried out using DominantMapper, resulting in the identification of 19 cosegregating chromosomal regions. Whole exome sequencing resulted in 11 rare coding variants present in all affected family members with developmental colour agnosia and absent in unaffected members. These variants affected genes that have been implicated in neural processes and functions (CACNA2D4, DDX25, GRINA, MYO15A), that have a indirect link to brain function or development (MAML2, STAU1, TMED3), and a remaining group lacking brain expression or involved in non-neural traits (DEPDC7, OR1J1, OR8D4, RABEPK). Although this is an explorative study, the small set of candidate genes that could serve as a starting point for unravelling mechanisms of higher level cognitive functions and cortical specialization, and disorders therein such as developmental colour agnosia.
COVID-19 Pandemic Response in a Migrant Farmworker Community: Excess Mortality, Testing Access and Contact Tracing in Immokalee, Florida
Authors: Limaye Neha , Ninesling Brennan , Marcelin Frantzso , Nolan Cody , Sobba Walter , Hing Matthew , Ptaszek Emily , Léandre Fernet , Palazuelos Daniel
Published: October 2021

Introduction

We aim to estimate the impact of COVID-19 in Immokalee, FL and assess community experiences with workplace conditions, access to testing, sources of information, and contact tracing to inform and strengthen local public health sector efforts in reaching and providing high-quality care to the community.

Methods

We conducted a descriptive analysis of data on COVID-19 deaths for Collier County from May-August 2020. We surveyed a cross-sectional, randomized representative sample of 318 adults living in Immokalee from March-November 2020 to assess socio-demographics, sources of information, ability to follow guidelines, and experiences with local programs. Results were compared across language groups.

Results

Average excess mortality in Collier County was 108%. The majority surveyed in Immokalee had socio-demographic factors associated with higher COVID risk. Non-English speakers had higher workplace risk due to less ability to work from home. Haitian Creole speakers were less likely to be tested, though all participants were willing to get symptomatic testing and quarantine. Those participants who tested positive or had COVID-19 exposures had low engagement with the contact tracing program, and Spanish-speakers reported lower quality of contact tracing than English speakers.

Conclusions

The community of Immokalee, FL is a vulnerable population that suffered disproportionate deaths from COVID-19. This study reveals language inequities in COVID testing and contact tracing should be targeted in future pandemic response in Immokalee and other migrant farmworker communities.