bluuebunny/crossref_metadata_2025 huggingface dataset:
STEM CELL TECHNOLOGY PROVIDES NOVEL TOOLS TO UNDERSTAND HUMAN VARIATION IN Plasmodium falciparum MALARIA
Authors: Pance Alena , Ling Bee , Mwikali Kioko , Koutsourakis Manousos , Agu Chukwuma , Rouhani Foad , Ponstingl Hannes , Montandon Ruddy , Law Frances , Rayner Julian C.
Published: July 2021

Plasmodium falciparum interacts with several human cell types during their complex life cycle, including erythrocytes and hepatocytes. The enuclated nature of erythrocytes makes them inaccessible to genetic tools, which in turn makes studying erythrocyte proteins involved in malaria invasion and development particularly difficult. Here we overcome this limitation using stem cell technology to develop a universal differentiation protocol for in vitro derivation of erythrocytes from a variety of stem cell lines of diverse origin. This allows manipulation of erythrocytic genes and examination of their impact on the parasite by flow cytometric detection of parasite haemozoin. Deletion of Basigin, the essential receptor for P. falciparum, abrogates invasion, while other less studied proteins such as ATP2B4 have a minor effect. Reprogramming of induced pluripotent stem cells from α-thalassemia primary samples shows reduced infection levels, demonstrating this approach is useful for understanding the effect of natural human polymorphisms on the disease.
High-throughput single-cell TCR - pMHC dissociation rate measurements performed by an autonomous microfluidic cellular processing unit
Authors: Jammes Fabien , Schmidt Julien , Coukos George , Maerkl Sebastian J.
Published: July 2021

We developed an integrated microfluidic cellular processing unit (mCPU) capable of autonomously isolating single cells, perform, measure, and on-the-fly analyze cell-surface dissociation rates, followed by recovery of selected cells. We performed proof-of-concept, high-throughput single-cell experiments characterizing pMHC-TCR interactions on live CD8+ T cells. The mCPU platform analyzed TCR-pMHC dissociation rates with a throughput of 50 cells per hour and hundreds of cells per run, and we demonstrate that cells can be selected, enriched, and easily recovered from the device.
Proper migration of lymphatic endothelial cells requires survival and guidance cues from arterial mural cells
Authors: Peng Di , Ando Koji , Gloger Marleen , Skoczylas Renae , Mochizuki Naoki , Betsholtz Christer , Fukuhara Shigetomo , Koltowska Katarzyna
Published: July 2021

The migration of lymphatic endothelial cells (LECs) is key for the development of the complex and vast lymphatic vascular network that pervades most of the tissues in an organism. In zebrafish, arterial intersegmental vessels together with chemokines have been shown to promote lymphatic cell migration from the horizontal myoseptum (HM). Here we found that LECs departure from HM coincides with the emergence of mural cells around the intersegmental arteries, raising the possibility that arterial mural cells promote LEC migration. Our live imaging and cell ablation experiments revealed that LECs migrate slower and fail to establish the lymphatic vascular network in the absence of arterial mural cells. We determined that mural cells are a source for the C-X-C motif chemokine 12 (Cxcl12a and Cxcl12b) and vascular endothelial growth factor C (Vegfc). We showed that ERK, a downstream component of Vegfc-Vegfr3 singling cascade, is activated in migrating LECs and that both chemokine and growth factor signalling is required for the robust migration. Furthermore, Vegfc-Vegfr3 has a pro-survival role in LECs during the migration. Together, the identification of mural cells a source for signals that guide LEC migration and survival will be important in the future design for rebuilding lymphatic vessels in the disease contexts.
Epileptiform GluN2B–driven excitation in hippocampus as a therapeutic target against temporal lobe epilepsy
Authors: Gorlewicz Adam , Pijet Barbara , Orlova Krstina , Kaczmarek Leszek , Knapska Ewelina
Published: July 2021

NMDAR is an ionotropic glutamate receptor critically involved in excitatory synaptic transmission. The receptor properties are strongly determined by its subunit composition. One of the NMDAR subunits is GluN2B, which displays restricted and spatially different from other subunits expression in the mature brain. GluN2B–containing NMDARs are present in the hippocampus – a structure playing a major role in temporal lobe epilepsy (TLE). However, the contribution of GluN2B to pathophysiology of TLE has not been fully explored. Here, we report the functional alterations of GluN2B–containing NMDAR receptors in the hippocampus in distinct mouse models of temporal lobe epilepsy. In particular, we show the impact of GluN2B on excitatory feedback in granule cells. Based on these results, we propose a mechanism–oriented effective antiepileptic strategy that selectively antagonizes GluN2B–containing NMDARs with ifenprodil, a well–known GluN2B antagonist. Collectively, our research identifies GluN2B as one of the pivotal factors in pathogenesis of temporal lobe epilepsy and associated recurrent seizures. Furthermore, our study indicates the prospective antiepileptic properties of ifenprodil in TLE.
Evaluation of FRET X for Single-Molecule Protein Fingerprinting
Authors: de Lannoy Carlos , Filius Mike , van Wee Raman , Joo Chirlmin , de Ridder Dick
Published: July 2021

Single-molecule protein identification is a novel, as of yet unrealized concept with potentially groundbreaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands, and accumulated FRET efficiency values constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on a system of model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics.
Electrical synapse asymmetry results from, and masks, neuronal heterogeneity
Authors: Mendoza Austin , Haas Julie S.
Published: July 2021

Electrical synapses couple inhibitory neurons across the brain, underlying a variety of functions that are modifiable by activity. Despite recent advances, many basic functions and contributions of electrical synapses within neural circuitry remain underappreciated. Among these is the source and impact of electrical synapse asymmetry. Using multi-compartmental models of neurons coupled through dendritic electrical synapses, we investigated intrinsic factors that contribute to synaptic asymmetry and that result in modulation of spike time between coupled cells. We show that electrical synapse location along a dendrite, input resistance, internal dendritic resistance, or directional conduction of the electrical synapse itself each alter asymmetry as measured by coupling between cell somas. Conversely, true synapse asymmetry can be masked by each of these properties. Furthermore, we show that asymmetry alters the spiking timing and latency of coupled cells by up to tens of milliseconds, depending on direction of conduction or dendritic location of the electrical synapse. These simulations illustrate that causes of asymmetry are multifactorial, may not be apparent in somatic measurements of electrical coupling, influence dendritic processing, and produce a variety of outcomes on spike timing of coupled cells. Our findings highlight aspects of electrical synapses that should be considered in experimental demonstrations of coupling, and when assembling networks containing electrical synapses.
Characterization of the Brassica napus flavonol synthase gene family reveals bifunctional flavonol synthases
Authors: Schilbert Hanna Marie , Schöne Maximilian , Baier Thomas , Busche Mareike , Viehöver Prisca , Weisshaar Bernd , Holtgräwe Daniela
Published: July 2021

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyses the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.
Intratumoral Heterogeneity Promotes Collective Cancer Invasion Through NOTCH1 Variation
Authors: Torab Peter , Yan Yue , Ahmed Mona , Yamashita Hironobu , Warrick Joshua I. , Raman Jay D. , DeGraff David J. , Wong Pak Kin
Published: July 2021

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a multidisciplinary approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.

Summary statement

This study reveals a mechanism that Notch1 variation, instead of the average value, promotes the invasiveness of microtumor, providing a link between intratumoral heterogeneity and collective cancer invasion.
The development of ovine gastric and intestinal organoids for studying ruminant host-pathogen interactions
Authors: Smith David , Price Daniel R. G. , Burrells Alison , Faber Marc N. , Hildersley Katie A. , Chintoan-Uta Cosmin , Chapuis Ambre F. , Stevens Mark , Stevenson Karen , Burgess Stewart T. G. , Innes Elisabeth A. , Nisbet Alasdair J. , McNeilly Tom N.
Published: July 2021

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.
Bayesian calibration, process modeling and uncertainty quantification in biotechnology
Authors: Helleckes Laura Marie , Osthege Michael , Wiechert Wolfgang , von Lieres Eric , Oldiges Marco
Published: July 2021

Abstract

High-throughput experimentation has revolutionized data-driven experimental sciences and opened the door to the application of machine learning techniques. Nevertheless, the quality of any data analysis strongly depends on the quality of the data and specifically the degree to which random effects in the experimental data-generating process are quantified and accounted for. Accordingly calibration, i.e. the quantitative association between observed quantities with measurement responses, is a core element of many workflows in experimental sciences. Particularly in life sciences, univariate calibration, often involving non-linear saturation effects, must be performed to extract quantitative information from measured data. At the same time, the estimation of uncertainty is inseparably connected to quantitative experimentation. Adequate calibration models that describe not only the input/output relationship in a measurement system, but also its inherent measurement noise are required. Due to its mathematical nature, statistically robust calibration modeling remains a challenge for many practitioners, at the same time being extremely beneficial for machine learning applications. In this work, we present a bottom-up conceptual and computational approach that solves many problems of understanding and implementing non-linear, empirical calibration modeling for quantification of analytes and process modeling. The methodology is first applied to the optical measurement of biomass concentrations in a high-throughput cultivation system, then to the quantification of glucose by an automated enzymatic assay. We implemented the conceptual framework in two Python packages, with which we demonstrate how it makes uncertainty quantification for various calibration tasks more accessible. Our software packages enable more reproducible and automatable data analysis routines compared to commonly observed workflows in life sciences. Subsequently, we combine the previously established calibration models with a hierarchical Monod-like differential equation model of microbial growth to describe multiple replicates of Corynebacterium glutamicum batch microbioreactor cultures. Key process model parameters are learned by both maximum likelihood estimation and Bayesian inference, highlighting the flexibility of the statistical and computational framework.
Quantitative modeling of regular retinal microglia distribution
Authors: Endo Yoshie , Asanuma Daisuke , Namiki Shigeyuki , Hirose Kenzo , Uemura Akiyoshi , Kubota Yoshiaki , Miura Takashi
Published: July 2021

1

Microglia are resident immune cells in the central nervous system (CNS), showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia’s morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated.

First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.

Author summary

Microglia are resident immune cells in the central nervous system. It is known that the microglia cells show a regular distribution, but the mechanism underlying the regular distribution remains to be elucidated. In the present study, we quantitatively assayed the regularity of the microglia soma distribution using the image processing technique. Next, we formulated a mathematical model of cell distribution that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters using organ culture. As a result, we obtained parameters for cell migration, cell process dynamics, and extracellular ATP dynamics. Then we undertook numerical simulation of the mathematical model using the parameters obtained by the experiments. The resulting model simulations showed that direct cell-cell contact is the most important factor in generating regular spacing. Finally, we screened possible molecular pathways involved in the regular spacing of microglia, which confirmed the validity of the model.
Mechanistic model-driven exometabolomic characterisation of human dopaminergic neuronal metabolism
Authors: Preciat German , Wegrzyn Agnieszka B. , Moreno Edinson Lucumi , Willacey Cornelius C.W. , Modamio Jennifer , Monteiro Fatima L. , El Assal Diana , Schurink Alissa , Oliveira Miguel A.P. , Zhang Zhi , Cousins Ben , Haraldsdóttir Hulda S. , Hachi Siham , Zach Susanne , Leparc German , Lee Yin Tat , Hengerer Bastian , Vempala Santosh , Saunders Michael A. , Harms Amy , Glaab Enrico , Schwamborn Jens C. , Thiele Ines , Hankemeier Thomas , Fleming Ronan M.T.
Published: July 2021

Starting with a comprehensive generic reconstruction of human metabolism, we generated high-quality, constraint-based, genome-scale, cell-type and condition specific models of metabolism in human dopaminergic neurons, the cell type most vulnerable to degeneration in Parkinson’s disease. They are a synthesis of extensive manual curation of the biochemical literature on neuronal metabolism, together with novel, quantitative, transcriptomic and targeted exometabolomic data from human stem cell-derived, midbrainspecific, dopaminergic neuronsin vitro. Thermodynamic constraint-based modelling enabled qualitatively accurate and moderately quantitatively accurate prediction of dopaminergic neuronal metabolite exchange fluxes, including predicting the consequences of metabolic perturbations in a manner also consistent with literature on monogenic mitochondrial Parkinson’s disease. These dopaminergic neurons models provide a foundation for a quantitative systems biochemistry approach to metabolic dysfunction in Parkinson’s disease. Moreover, the plethora of novel mathematical and computational approaches required to develop them are generalisable to study any other disease associated with metabolic dysfunction.
Evolution of a minimal cell
Authors: Moger-Reischer RZ , Glass JI , Wise KS , Sun L , Bittencourt D , Lynch M , Lennon JT
Published: July 2021

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life. Here, we report on how a synthetically constructed minimal cell contends with the forces of evolution compared to a non-minimized cell from which it was derived. Genome streamlining was costly, but 80% of fitness was regained in 2000 generations. Although selection acted upon divergent sets of mutations, the rates of adaptation in the minimal and non-minimal cell were equivalent. The only apparent constraint of minimization involved epistatic interactions that inhibited the evolution of cell size. Together, our findings demonstrate the power of natural selection to rapidly optimize fitness in the simplest autonomous organism, with implications for the evolution of cellular complexity.
Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei within the tsetse fly vector
Authors: Dewar Caroline E. , Casas-Sanchez Aitor , Dieme Constentin , Crouzols Aline , Haines Lee R. , Acosta-Serrano Álvaro , Rotureau Brice , Schnaufer Achim
Published: July 2021

The single-celled parasite Trypanosoma brucei causes sleeping sickness in humans and nagana in livestock and is transmitted by hematophagous tsetse flies. Lifecycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonises the glucose-poor insect midgut, its ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation. This process involves respiratory chain complexes and the F1FO-ATP synthase, and it requires protein subunits of these complexes that are encoded in the parasite’s mitochondrial DNA (kinetoplast or kDNA). Here we show that a progressive loss of kDNA-encoded functions correlates with an increasingly impaired ability of T. brucei to initiate and complete its development in the tsetse. First, parasites with a mutated F1FO-ATP synthase with a reduced capacity for oxidative phosphorylation can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonise the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonising or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1FO-ATP synthase complex that is completely unable to produce ATP by oxidative phosphorylation can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, mutant parasites lacking kDNA entirely can initiate differentiation but die within 24 h. Together, these three scenarios show that efficient ATP production via oxidative phosphorylation is not essential for initial colonisation of the tsetse vector, but it is required to power trypanosome migration within the fly.
Microbial community composition of translocated ancient woodland soil: a case study
Authors: Borchers Nicolas , Hannam Jacqueline , Pawlett Mark
Published: July 2021

Soil translocation is an ecological habitat restoration technique which consists of moving the entire topsoil from a donor site to a chosen receptor site. We investigated changes in soil chemistry and microbiology three years following the salvage of semi-ancient woodland soil and materials (0.94 ha) to a nearby receptor pasture due to road widening works (Kent, UK). We sampled i) intact woodland soils adjacent to the area of soils that was translocated to represent the lost donor site, ii) the soil three years after it had been translocated, and iii) grassland soils adjacent to the translocated soil to represent the original receptor site. The intention was to ascertain if shifts in soil chemistry and microbial community composition (Phospholipid Fatty-acid analysis: PLFA) occurred due to soil translocation. PLFA signature biomarkers demonstrated the overall microbial community profile of the translocated and woodland soils were similar; however, salvaged soils had a 40% increase in the Arbuscular Mycorrhizal Fungi (AMF) bioindicator fatty acid 16:1ω5, a 10% decrease in the Gram-positive bacterial fatty acids, and increased pH (5.01-5.77) compared to the original donor woodland soil. The AMF bioindicator and the first Principal Component (PC1) of the PCA of PLFA data positively correlated with soil pH (r2=0.94 and r2=0.88 respectively) across all three experimental groups. Considering that soil pH increases with depth in this location, it is likely that mixing of soil horizons during translocation increased the topsoil pH causing changes in the soil microbial communities. We concluded that after three years, the chemical and microbial properties of the salvaged soil were characteristic of a woodland soil but showed signs of disturbance.

Implications for practice

Translocated semi-ancient woodland soils may still exhibit similar microbiological characteristics to the donor site three years after being moved to a pasture receptor site.

However, disturbances expressed as significant changes in chemistry and microbial communities can affect soils following their translocation.

Where soil pH varies with depth, translocation by loose-tipping may redistribute acidity within the soil profile, with important cascading impacts on the soil microbial communities.
Pain and the Emotional Brain: Affective Rather than Cognitive Processes Drive the Cortical Encoding of Pain
Authors: Stankewitz Anne , Mayr Astrid , Irving Stephanie , Witkovsky Viktor , Schulz Enrico
Published: July 2021

Background

The experience of pain has been dissociated into two interwoven aspects: a sensory-discriminative aspect and an affective-motivational aspect. We aimed to explore which of the pain descriptors is more deeply rooted in the human brain.

Findings

Participants were asked to evaluate applied cold pain. The majority of the trials showed distinct ratings: some were rated higher for unpleasantness and others for intensity. We compared the relationship between functional data recorded from 7 tesla MRI with unpleasantness and intensity ratings and revealed a stronger relationship between cortical data and unpleasantness ratings.

Conclusions

The present study underlines the importance of the emotional-affective aspects of pain-related cortical processes in the brain. The findings corroborate previous studies showing a higher sensitivity to pain unpleasantness compared to ratings of pain intensity. For the processing of pain in healthy subjects, this effect may reflect the more direct and intuitive evaluation of emotional aspects of the pain system, which is to prevent harm and to preserve the physical integrity of the body.
Force generation of KIF1C is impaired by pathogenic mutations
Authors: Siddiqui Nida , Roth Daniel , Toleikis Algirdas , Zwetsloot Alexander J. , Cross Robert A. , Straube Anne
Published: July 2021

Intracellular transport is essential for neuronal function and survival. The fastest neuronal transporter is the kinesin-3 KIF1C. Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients. However, neither the force generation of the KIF1C motor protein, nor the biophysical and functional properties of pathogenic mutant proteins have been studied thus far.

Here we show that full length KIF1C is a processive motor that can generate forces up to 5.7 pN. We find that KIF1C single molecule processivity relies on its ability to slip and re-engage under load and that its slightly reduced stall force compared to kinesin-1 relates to its enhanced probability to backslip. Two pathogenic mutations P176L and R169W that cause hereditary spastic paraplegia in humans maintain fast, processive single molecule motility in vitro, but with decreased run length and slightly increased unloaded velocity compared to the wildtype motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles.

Our results establish a baseline for the single molecule mechanics of Kif1C and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.
ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication
Authors: Pearson G.J , Mears H. , Broncel M. , Snijders A.P. , Bauer D.L.V. , Carlton J.G.
Published: July 2021

The β-coronavirus SARS-CoV-2 is the causative agent of the global Covid-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release and pathogenesis. We developed an inert tagging strategy for SARS-CoV-2 E and find that it localises to the Golgi and to lysosomes. We identify sequences in E, conserved acrossCoronaviridae,responsible for ER-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify host-cell factors that interact with E and identify an ARFRP1/AP-1 dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and by developing atrans-complementation assay, we show that both lysosomal trafficking of E and its viroporin activity are necessary for efficient viral replication and release.
Neuronal mechanism of a BK channelopathy in absence epilepsy and movement disorders
Authors: Dong Ping , Zhang Yang , Mikati Mohamad A. , Cui Jianmin , Yang Huanghe
Published: July 2021

A growing number of gain-of-function (GOF) BK channelopathy have been identified in patients with epilepsy and paroxysmal movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and movement disorders. We found that the BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits. BK-D434G mutation causes hyperexcitability of cortical pyramidal neurons and cerebellar Purkinje cells, which contributes to the pathogenesis of absence seizures and the motor defects, respectively. A BK channel blocker paxilline potently suppresses BK-D434G-induced hyperexcitability and effectively mitigates absence seizures in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and provided the evidence that BK inhibition is a promising therapeutic strategy to mitigate BK GOF-induced neurological disorders.

Significance

Dysfunction of BK channels or BK channelopathy has been increasingly implicated in diverse neurological disorders including epilepsy, movement, cognitive and neurodevelopmental disorders. However, precision medicine to treat BK channelopathy is lacking. Here we characterized a mouse model carrying a gain-of-function BK channelopathy D434G from a large family of patients with absence epilepsy and involuntary movement disorders. The BK-D434G mice resemble the clinical manifestations of absence seizures and exhibit severe motor defects. The hyperexcitability in BK-D434G cortical neurons and cerebellar Purkinje cells underscores the neuronal mechanism of BK gain-of-function induced absence epilepsy and movement disorders. The effectiveness of a BK channel blocker on preventing absence seizures suggests that BK inhibition is a promising strategy to treat gain-of-function BK channelopathy.
Detection of potential new SARS-CoV-2 Gamma-related lineage in Tocantins shows the spread and ongoing evolution of P.1 in Brazil
Authors: Souza U.J.B. , Santos R.N. , Belmok A. , Melo F.L. , Galvão J.D. , Damasceno S.B. , Rezende T.C.V. , Andrade M.S. , Ribeiro B.M. , Ribeiro Junior J.C. , Carvalho R.F. , Santos I.G.C. , Oliveira M.S. , Spilki F.R. , Campos F.S.
Published: July 2021

After more than a year of the pandemic situation of COVID-19, the United Kingdom (UK), South Africa, and Brazil became the epicenter of new lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Variants of Concern (VOCs) were identified through a continuous genomic surveillance global effort, the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and P.1 (Gamma) harboring a constellation set of mutations. This research aims to: (i) report the predominance of the Gamma (P.1) lineage presenting the epidemiological situation of the SARS-CoV-2 genomic surveillance at the state of Tocantins, and (ii) describe the emergence of possible new mutations and viral variants with the potential new lineage (P1-related) represented by 8 genomes from the Tocantins harboring the mutation L106F in ORF3a. At the moment, 6,687 SARS-CoV-2 genomes from GISAID carry this mutation. The whole-genome sequencing has an important role in understanding the evolution and genomic diversity of SARS-CoV-2, thus, the continuous monitoring will help in the control measures and restrictions imposed by the secretary of health of the state to prevent the spread of variants.
Computational modeling predicts acidic microdomains in the glutamatergic synaptic cleft
Authors: Feghhi Touhid , Hernandez Roberto X. , Stawarski Michal , Thomas Connon I. , Kamasawa Naomi , Lau A.W.C. , Macleod Gregory T.
Published: July 2021

At chemical synapses, synaptic vesicles release their acidic contents into the cleft leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization, rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction (NMJ) as glutamate, adenosine triphosphate (ATP) and protons (H+) are released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase (PMCA) activity and predicts substantial cleft acidification but only for fractions of a millisecond following neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 milliseconds, as the PMCA removes H+ from the cleft in exchange for calcium ions (Ca2+) from adjacent pre- and post-synaptic compartments; thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis has little influence on the magnitude of acidification. Phosphate, but not bicarbonate buffering is effective at ameliorating the magnitude and time course of the acid spike, while both buffering systems are effective at ameliorating cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH disturbances accompanying neurotransmission.

SIGNIFICANCE STATEMENT

Acid-base imbalances have surprisingly potent neurological effects highlighting the acute pH sensitivity of many neural mechanisms. Acid-Sensing Ion-Channels (ASICs), which open in response to acid shifts in extracellular pH, are an example of such a mechanism. However, while ASICs open during neurotransmission at conventional glutamatergic synapses, pH-sensitive electrodes and fluorophores show no signs of acidification at these synapses, only alkalinization. To resolve this paradox, we built a computational model which allows a glimpse beyond the experimental limitations of pH-sensitive electrodes and fluorophores. Our model reveals a highly dynamic pH landscape within the synaptic cleft, harboring deep but exceedingly rapid acid transients that give way to a prolonged period of alkalinization, thus reconciling ASIC activation with direct measurements of extracellular pH.
The mutual repression between Pax2 and Snail factors regulates the epithelial/mesenchymal state during intermediate mesoderm differentiation
Authors: Fons Juan M. , Ocaña Oscar H. , Nieto M. Angela
Published: July 2021

The pronephros is the first renal structure in the embryo, arising after mesenchymal to epithelial transition (MET) of the intermediate mesoderm, where Pax2 induces epithelialisation of the mesenchyme. Here we show that, in the early embryo, Snail1 directly represses Pax2 transcription maintaining the intermediate mesoderm in an undifferentiated state. Reciprocally, Pax2 directly represses Snail1 expression to induce MET upon receiving differentiation signals. We also show that BMP7 acts as one such signal by downregulating Snail1 and upregulating Pax2 expression. This, together with the Snail1/Pax2 reciprocal repression, establishes a regulatory loop in a defined region along the anteroposterior axis, the bistability domain within the transition zone, where differentiation of the neural tube and the somites are known to occur. Thus, we show that the antagonism between Snail1 and Pax2 determines the epithelial/mesenchymal state during the differentiation of the intermediate mesoderm and propose that the bistability zone extends to the intermediate mesoderm, synchronizing the differentiation of tissues aligned along the mediolateral embryonic axis.

The antagonism between Snail and Pax2 factors in the embryonic differentiation zone, tightly regulates the timing of mesodermal epithelialisation
Optimal balancing of clinical factors in large scale clinical RNA-Seq studies
Authors: Chiang Austin W.T. , Gazestani Vahid H. , Altieri Mia G. , Kellman Benjamin P. , Nalabolu Srinivsa , Pramparo Tiziano , Pierce Karen , Courchesne Eric , Lewis Nathan E.
Published: July 2021

Omics technologies are ubiquitous in biomedical research. However, improper sample selection is an often-overlooked complication with large omics studies, resulting in confounding effects that can disrupt the internal validity of a study and lead to false conclusions. Here, we present a method called BalanceIT, which uses a genetic algorithm to identify an optimal set of samples with balanced clinical factors for large-scale omics experiments. We apply our approach to two large RNA-Seq studies in autism (1) to find a post-hoc balanced sample set among an imbalanced study, and (2) to design an optimal study that allows for efficient batch correction. Our approach leads to near-perfect estimates of differential gene expression, superior performance of pathway-level enrichment analysis, and consistent network dysregulation patterns of autism symptom severity. These results provide empirical support for the importance of balanced experimental design, and BalanceIT will be invaluable for large-scale study design and batch effect correction.
The flying insect thoracic cuticle is heterogenous in structure and in thickness-dependent modulus gradation
Authors: Casey Cailin , Yager Claire , Jankauski Mark , Heveran Chelsea
Published: July 2021

The thorax is a specialized structure central to an insect’s ability to fly. In the thorax, flight muscles are surrounded by a thin layer of cuticle. The structure, composition, and material properties of this chitinous structure may influence the efficiency of the thorax in flight. However, these properties, as well as their variation throughout anatomical regions of the thorax or between insect taxa, are not known. In this work, we provide a multi-faceted assessment of thorax cuticle for fliers with asynchronous (honey bee; Apis mellifera) and synchronous (hawkmoth; Manduca sexta) muscles. We investigated cuticle structure using histology, material composition through confocal laser scanning microscopy, and modulus gradation with nanoindentation. Our results suggest that cuticle properties of the thorax are highly dependent on anatomical region and species. Modulus gradation, but not mean modulus, differed between the two types of fliers. In some regions, A. mellifera had a positive linear modulus gradient from cuticle interior to exterior of about 2 GPa. In M. sexta, the modulus gradients were variable and were not well represented by linear fits with respect to cuticle thickness. We utilized finite element modeling to assess how measured modulus gradients influenced maximum stress in cuticle. Stress was reduced when cuticle with a linear gradient was compressed from the high modulus side. These results support the protective role of the A. mellifera thorax cuticle. Our multi-faceted assessment advances our understanding of thorax cuticle structural and material heterogeneity and the potential benefit of material gradation to flying insects.

Graphical Abstract

Statement of Significance

The insect thorax is essential for efficient flight but questions remain about the contribution of exoskeletal cuticle. We assessed the thorax cuticle using a high resolution multi-faceted approach to determine how cuticle properties vary within thorax anatomical regions and between fliers with asynchronous (honey bee; Apis mellifera) and synchronous (hawkmoth; Manduca sexta) muscles. We examined structure using histological staining, modulus using nanoindentation, and material composition using confocal scanning light microscopy. We further utilized finite element modeling to understand the effect of the modulus gradations observed experimentally on stress accumulation. Cuticle properties vary through cuticle thickness, by thorax region, and between flight lineages.
Synthetic dual-function RNA reveals features necessary for target regulation
Authors: Aoyama Jordan J. , Raina Medha , Storz Gisela
Published: July 2021

Small base pairing RNAs (sRNAs) and small proteins comprise two classes of regulators that allow bacterial cells to adapt to a wide variety of growth conditions. A limited number of transcripts encoding both of these activities, regulation of mRNA expression by base pairing and a small regulatory protein, have been identified. Given that few have been characterized, little is known about the interplay between the two regulatory functions. To investigate the competition between the two activities, we constructed synthetic dual-function RNAs, hereafter referred to as MgtSR or MgtRS, comprised of the Escherichia coli sRNA MgrR and the open reading frame encoding the small protein MgtS. MgrR is a 98 nt base pairing sRNA that negatively regulates eptB encoding phosphoethanolamine transferase. MgtS is a 31 aa small inner membrane protein that is required for the accumulation of MgtA, a magnesium (Mg2+) importer. Expression of the separate genes encoding MgrR and MgtS is normally induced in response to low Mg2+ by the PhoQP two-component system. By generating various versions of this synthetic dual-function RNA, we probed how the organization of components and the distance between the coding and base pairing sequences contribute to the proper function of both activities of a dual-function RNA. By understanding the features of natural and synthetic dual-function RNAs, future synthetic molecules can be constructed to maximize their regulatory impact.

IMPORTANCE

Dual-function RNAs encode a small protein and also base pair with mRNAs to act as small, regulatory RNAs. Given that only a limited number of dual-function RNAs have been characterized, further study of these regulators is needed to increase understanding of their features. This study demonstrates that a functional synthetic dual-regulator can be constructed from separate components and used to study the functional organization of dual-function RNAs, with the goal of exploiting these regulators.
Hybridization alters the shape of the genotypic fitness landscape, increasing access to novel fitness peaks during adaptive radiation
Authors: Patton Austin H. , Richards Emilie J. , Gould Katelyn J. , Buie Logan K. , Martin Christopher H.
Published: July 2021

Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 SNPs that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed andde novovariants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression andde novomutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation.
A role for worm cutl-24 in background- and parent-of-origin-dependent ER stress resistance
Authors: Wang Wenke , Flury Anna G. , Rodriguez Andrew T. , Garrison Jennifer L. , Brem Rachel B.
Published: July 2021

Organisms in the wild can acquire disease- and stress-resistance traits that outstrip the programs endogenous to humans. Finding the molecular basis of such natural resistance characters is a key goal of evolutionary genetics. Standard statistical-genetic methods toward this end can perform poorly in organismal systems that lack high rates of meiotic recombination, like Caenorhabditis worms. Here we discovered unique ER stress resistance in a wild Kenyan C. elegans isolate, which in inter-strain crosses was passed by hermaphrodite mothers to hybrid offspring. We developed an unbiased version of the reciprocal hemizygosity test, RH-seq, to explore the genetics of this parent-of-origin-dependent phenotype. Among top-scoring gene candidates from a partial-coverage RH-seq screen, we focused on the neuronally-expressed, cuticlin-like gene cutl-24 for validation. In gene disruption and controlled crossing experiments, we found that cutl-24 was required in Kenyan hermaphrodite mothers for ER stress tolerance in their inter-strain hybrid offspring; cutl-24 was also a contributor to the trait in purebred backgrounds. These data establish the Kenyan strain allele of cutl-24 as a determinant of a natural stress-resistant state, and they set a precedent for the dissection of natural trait diversity in invertebrate animals without the need for a panel of meiotic recombinants.