bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordination through inhibition: control of stabilizing and updating circuits in spatial orientation working memory
Authors: Han Rui , Huang Hsuan-Pei , Chuang Chia-Lung , Yen Hung-Hsiu , Kao Wei-Tse , Chang Hui-Yun , Lo Chung-Chuan
Published: October 2019

Spatial orientation memory plays a crucial role in animal navigation. Recent studies of tethered Drosophila melanogaster (fruit fly) in a virtual reality setting showed that the head direction is encoded in the form of an activity bump, i.e. localized neural activity, in the torus-shaped ellipsoid body (EB). However, how this system is involved in orientation working memory is not well understood. We investigated this question using free moving flies (Drosophila melanogaster) in a spatial orientation memory task by manipulating two EB subsystems, C and P circuits, which are hypothesized for stabilizing and updating the activity bump, respectively. To this end, we suppressed or activated two types of inhibitory ring neurons (EIP and P) which innervate EB, and we discovered that manipulating the two inhibitory neuron types produced distinct behavioral deficits, suggesting specific roles of the inhibitory neurons in coordinating the stabilization and updating functions of the EB circuits. We further elucidate the neural mechanisms underlying such control circuits using a connectome-constrained spiking neural network model.

Significance statement

Head-direction (HD) system has been discovered in rodents for decades. But the detailed neural circuit mechanisms underlying the HD system were only described recently by studies of fruit flies on the similar HD system. However, how this fruit fly HD system involves in orientation memory was not well investigated. The present study addresses this question by investigating free moving flies in a spatial orientation working memory task. By combining neural functional experiments and neural circuit modelling, the study shows how disrupting either of the two subcircuits, one stabilizing and the other updating the neural activity, in the HD system leads to different behavioral impairments. The result suggests specific roles of the HD subcircuits in the spatial orientation working memory.

Visual Abstract
Carbon metabolism, transcriptome and RNA editome in developmental paths differentiation ofCoprinopsis cinerea
Authors: Xie Yichun , Chang Jinhui , Kwan Hoi Shan
Published: October 2019

The balance and interplay between sexual and asexual reproduction is one of the most attractive mysteries in fungi. The choice of developmental strategy reflects the ability of fungi to adapt to the changing environment. However, the evolution of developmental paths and the metabolic regulation during differentiation and morphogenesis are poorly understood. Here, we monitor the carbohydrate metabolism and gene expression regulation during the early differentiation process from the “fungal stem cell”, vegetative mycelium, to the highly differentiated tissue/cells, fruiting body, oidia or sclerotia, of a homokaryotic fruitingCoprinopsis cinereastrain A43mut B43mut pab1-1 #326, uncovering the systematic changes during morphogenesis and the evolutionary process of developmental strategies. Conversion between glucose and glycogen and conversion between glucose and beta-glucan are the main carbon flows in the differentiation processes. Genes related to carbohydrate transport and metabolism are significantly differentially expressed among paths. RNA editing, a novel layer of gene expression regulation, occurs in all four developmental paths and enriched in cytoskeleton and carbohydrate metabolic genes. It is developmentally regulated and evolutionarily conserved in basidiomycetes. Evolutionary transcriptomic analysis on four developmental paths showed that all transcriptomes are under purifying selection, and the more stressful the environment, the younger the transcriptome age. Oidiation has the lowest value of transcriptome age index (TAI) and transcriptome divergence index (TDI), while fruiting process has the highest of both indexes. These findings provide new insight to the regulations of carbon metabolism and gene expressions during fungal developmental paths differentiation.

Importance

Fungi is a group of species with high diversity and plays essential roles to the ecosystem. The life cycle of fungi is complex in structure and delicate in function. Choice of developmental strategies and internal changes within the organism are both important for the fungus to fulfill their ecological functions, reflecting the relationship between environment and the population. This study put the developmental process of vegetative growth, sexual and asexual reproduction, resistant structure formation of a classical model basidiomycetes fungus,C. cinerea, together for the first time to view the developmental paths differentiation process with physiology, transcriptomics and evolutionary prospects. Carbohydrate assays and RNA-seq showed the changes of the fungus. Our results fill the gaps on gene expression regulation during the early stage of developmental paths differentiation, and expand our understanding of the evolutionary process of life history and reproductive strategy in fungi.
Accelerated clearing and molecular labeling of large tissue samples using magnetohydrodynamic force
Authors: Dwyer Joseph , Ramirez M. Desmond , Katz Paul S. , Karlstrom Rolf O. , Bergan Joseph
Published: October 2019

Here we report a strategy to efficiently render opaque biological tissues transparent and demonstrate that this approach can be modified to rapidly label intact samples with antibodies for large volume fluorescence microscopy. This strategy applies a magnetohydrodynamic (MHD) force to accelerate the removal of lipids from and the introduction of antibodies into tissue samples as large as an intact adult mouse brain. This strategy complements a growing array of tools that enable high-resolution 3-dimensional anatomical analyses in intact tissues using fluorescence microscopy. MHD-accelerated clearing and MHD-accelerated antibody labeling are fast, reliable, inexpensive, and compatible with existing strategies for high-quality fluorescence microscopy of intact tissues.
Mating changes the genital microbiome in both sexes of the common bedbugCimex lectulariusacross populations
Authors: Bellinvia Sara , Johnston Paul R. , Mbedi Susan , Otti Oliver
Published: October 2019

Many bacteria live on host surfaces, in cells, and specific organ systems. Although gut microbiomes of many organisms are well-documented, the bacterial communities of reproductive organs, i.e. genital microbiomes, have received little attention. During mating, male and female genitalia interact and copulatory wounds can occur, providing an entrance for sexually transmitted microbes. Besides being potentially harmful to the host, invading microbes might interact with resident genital microbes and affect immunity. While sexual transmission of individual symbiont species is relatively well-understood, few studies have addressed how mating changes genital microbiomes. Here, we characterize male and female genital microbiomes in four different populations of the common bedbugCimex lectulariusand investigate mating-induced changes. We dissected reproductive organs from virgin and mated individuals to sequence their genital microbiomes. We show that mating changes genital microbiomes, suggesting bacteria are sexually transmitted. This raises the question how the host’s physiological and immunological mechanisms control mating-induced changes in their genital microbiomes. Also, genital microbiomes varied between populations and the sexes. This provides evidence for local and sex-specific adaptation of bacteria and hosts, again suggesting that bacteria might play an important role in shaping the evolution of reproductive traits. Coadaptation of genital microbiomes and reproductive traits might further lead to reproductive isolation between populations, giving reproductive ecology an important role in speciation. Future studies should investigate the transmission dynamics between the sexes and populations to uncover potential reproductive barriers.
Inference with selection, varying population size and evolving population structure: Application of ABC to a forward-backward coalescent process with interactions
Authors: Lepers Clotilde , Billiard Sylvain , Porte Matthieu , Méléard Sylvie , Tran Viet Chi
Published: October 2019

Genetic data are often used to infer history, demographic changes or detect genes under selection. Inferential methods are commonly based on models making various strong assumptions: demography and population structures are supposeda prioriknown, the evolution of the genetic composition of a population does not affect demography nor population structure, and there is no selection nor interaction between and within genetic strains. In this paper, we present a stochastic birth-death model with competitive interaction to describe an asexual population, and we develop an inferential procedure for ecological, demographic and genetic parameters. We first show how genetic diversity and genealogies are related to birth and death rates, and to how individuals compete within and between strains. This leads us to propose an original model of phylogenies, with trait structure and interactions, that allows multiple merging. Second, we develop an Approximate Bayesian Computation framework to use our model for analyzing genetic data. We apply our procedure to simulated and real data. We show that the procedure give accurate estimate of the parameters of the model. We finally carry an illustration on real data and analyze the genetic diversity of microsatellites on Y-chromosomes sampled from Central Asia populations in order to test whether different social organizations show significantly different fertility.
Capacitive coupling increases the accuracy of cell-specific tumour ablation by electric fields
Authors: Wimberger Terje , Köhler Verena K. , Ehmoser Eva K. , Wassermann Klemens J.
Published: October 2019

Irreversible electroporation holds great potential for cell-specific lysis due to the size-dependent susceptibility of cells to externally imposed electric fields. Previous attempts at selective cell lysis lead to significant overlap between affected populations and struggle with inconsistent biological outcome. We propose that charge transfer at the electrode-liquid interface is responsible by inducing multifactorial effects originating from both the electric field and electrochemical reactions. A promising remedy is the coating of electrodes with a high-k dielectric layer. The resulting capacitive coupling restores the selective potential of electric field mediated lysis in a microfluidic setup. Initial experiments show the consistent depletion of erythrocytes from whole blood while leaving leukocytes intact. The same is true for the reproducible and selective depletion of Jurkat and MCF-7 cells in a mixture with leukocytes. Unexpectedly, the observed order of lysis cannot be correlated with cell size. This implies that the cellular response to capacitive coupling features a selective characteristic that is different from conventional lysis configurations.
TET1-mediated DNA hydroxy-methylation regulates adult remyelination
Authors: Moyon Sarah , Frawley Rebecca , Marshall-Phelps Katy LH , Kegel Linde , Bøstrand Sunniva MK , Sadowski Boguslawa , Huang Dennis , Jiang Yong-Hui , Lyons David , Möbius Wiebke , Casaccia Patrizia
Published: October 2019

Adult myelination is essential for brain function and response to injury, but the molecular mechanisms remain elusive. Here we identify DNA hydroxy-methylation, an epigenetic mark catalyzed by Ten-Eleven translocation (TET) enzymes, as necessary for adult myelin repair.

While DNA hydroxy-methylation and high levels of TET1 are detected in young adult mice during myelin regeneration after demyelination, this process is defective in old mice. Constitutive or inducible lineage-specific ablation ofTet1(but not ofTet2) recapitulate the age-related decline of DNA hydroxy-methylation and inefficient remyelination. Genome-wide hydroxy-methylation and transcriptomic analysis identify numerous TET1 targets, including several members of the solute carrier (Slc) gene family. Lower transcripts forSlcgenes, includingSlc12a2, are observed inTet1mutants and old mice and are associated with swelling at the neuroglial interface, a phenotype detected also in zebrafishslc12a2bmutants.

We conclude that TET1-mediated DNA hydroxy-methylation is necessary for adult remyelination after injury.
Collateral fitness effects of mutations
Authors: Mehlhoff Jacob D. , Stearns Frank W. , Rohm Dahlia , Wang Buheng , Tsou Erh-Yeh , Dutta Nisita , Hsiao Meng-Hsuan , Gonzalez Courtney E. , Rubin Alan F. , Ostermeier Marc
Published: October 2019

The distribution of fitness effects (DFE) of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutation’s collateral fitness effects, which we define as effects that do not derive from changes in the protein’s ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in theE. coli TEM-1β-lactamase antibiotic resistance gene using growth competition experiments in theabsenceof antibiotic. At least 42% of missense mutations inTEM-1were deleterious, indicating that for some proteins, collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper post-translational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently inTEM-1than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly-expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against mutations with deleterious effects on protein activity and protein abundance.

Significance Statement

Mutations provide the source of genetic variability upon which evolution acts. Deleterious protein mutations are commonly thought of in terms of how they compromise the protein’s ability to perform its physiological function. However, mutations might also be deleterious if they cause negative effects on one of the countless other cellular processes. The frequency and magnitude of such collateral fitness effects is unknown. Our systematic study of mutations in a bacterial protein finds widespread collateral fitness effects that were associated with protein aggregation, improper protein processing, incomplete protein transport across membranes, incorrect disulfide-bond formation, induction of stress-response pathways, and unexpected changes in cell properties. Our results suggest that deleterious collateral fitness effects may be an important constraint on protein evolution.
Identification of neural oscillations and epileptiform changes in human brain organoids
Authors: Samarasinghe Ranmal A. , Miranda Osvaldo A. , Buth Jessie E. , Mitchell Simon , Ferando Isabella , Watanabe Momoko , Allison Thomas F. , Kurdian Arinnae , Fotion Namie N. , Gandal Michael J. , Golshani Peyman , Plath Kathrin , Lowry William E. , Parent Jack M. , Mody Istvan , Novitch Bennett G.
Published: October 2019

Brain organoids represent a powerful tool for the study of human neurological diseases, particularly those impacting brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes. This raises the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network behaviors reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from MECP2 mutant patients compared to isogenic controls accompanied by modest transcriptomic differences revealed by single cell analyses. We also rescue key physiological activities with an unconventional neuromodulatory drug, Pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.
New flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline
Authors: Wenzel Michaela , Dekker Marien P. , Wang Biwen , Burggraaf Maroeska J. , Bitter Wilbert , van Weering Jan R. T. , Hamoen Leendert W.
Published: October 2019

Transmission electron microscopy (TEM) is an important imaging technique in bacterial research and requires ultrathin sectioning of resin embedding of cell pellets. This method consumes milli- to deciliters of culture and results in sections of randomly orientated cells. For rod-shaped bacteria, this makes it exceedingly difficult to find longitudinally cut cells, which precludes large-scale quantification of morphological phenotypes. Here, we describe a new fixation method using either thin agarose layers or carbon-coated glass surfaces that enables flat embedding of bacteria. This technique allows for the observation of thousands of longitudinally cut rod-shaped cells per single section and requires only microliter culture volumes. We successfully applied this technique to Gram-positiveBacillus subtilis, Gram-negativeEscherichia coli, the tuberculosis vaccine strainMycobacterium bovisBCG, and the cell wall-lacking mycoplasmaAcholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we examined cellular changes induced by a panel of different antibiotics. Surprisingly, we found that the ribosome inhibitor tetracycline causes significant deformations of the cell membrane. Further investigations showed that the presence of tetracycline in the cell membrane changes membrane organization and affects the peripheral membrane proteins MinD, MinC, and MreB, which are important for regulation of cell division and elongation. Importantly, we could show that this effect is not the result of ribosome inhibition but is a secondary antibacterial activity of tetracycline that has defied discovery for more than 50 years.

Significance

Bacterial antibiotic resistance is a serious public health problem and novel antibiotics are urgently needed. Before a new antibiotic can be brought to the clinic, its antibacterial mechanism needs to be elucidated. Transmission electron microscopy is an important tool to investigate these mechanisms. We developed a flat embedding method that enables examination of many more bacterial cells than classical protocols, enabling large-scale quantification of phenotypic changes. Flat embedding can be adapted to most growth conditions and microbial species and can be employed in a wide variety of microbiological research fields. Using this technique, we show that even well-established antibiotics like tetracycline can have unknown additional antibacterial activities, demonstrating how flat embedding can contribute to finding new antibiotic mechanisms.
Atomic structures determined from digitally defined nanocrystalline regions
Authors: Gallagher-Jones Marcus , Bustillo Karen C. , Ophus Colin , Richards Logan S. , Ciston Jim , Lee Sangho , Minor Andrew M. , Rodriguez Jose A.
Published: October 2019

Nanocrystallography has transformed our ability to interrogate the atomic structures of proteins, peptides, organic molecules and materials. By probing atomic level details in ordered sub-10 nm regions of nanocrystals, approaches in scanning nanobeam electron diffraction extend the reach of nanocrystallography and mitigate the need for diffraction from large portions of one or more crystals. We now apply scanning nanobeam electron diffraction to determine atomic structures from digitally defined regions of beam-sensitive peptide nanocrystals. Using a direct electron detector, we record thousands of sparse diffraction patterns over multiple crystal orientations. We assign each pattern to a specific location on a single nanocrystal with axial, lateral and angular coordinates. This approach yields a collection of patterns that represent a tilt series across an angular wedge of reciprocal space: a scanning nanobeam diffraction tomogram. From this diffraction tomogram, we can digitally extract intensities from any desired region of a scan in real or diffraction space, exclusive of all other scanned points. Intensities from multiple regions of a crystal or from multiple crystals can be merged to increase data completeness and mitigate missing wedges. Merged intensities from digitally defined regions of two crystals of a segment from the OsPYL/RCAR5 protein produce fragment-based ab-initio solutions that can be refined to atomic resolution, analogous to structures determined by selected area electron diffraction. In allowing atomic structures to now be determined from digitally outlined regions of a nanocrystal, scanning nanobeam diffraction tomography breaks new ground in nanocrystallography.
Ancestral Regulatory Mechanisms Specify Conserved Midbrain Circuitry in Arthropods and Vertebrates
Authors: Bridi Jessika C. , Ludlow Zoe N. , Kottler Benjamin , Hartmann Beate , Broeck Lies Vanden , Dearlove Jonah , Göker Markus , Strausfeld Nicholas J. , Callaerts Patrick , Hirth Frank
Published: October 2019

Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. Here we identify a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) inDrosophila. We show this network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function is directed by cis-regulatory elements (CREs) of developmental control genes that include homologs of mammalianZinc finger of the cerebellumandPurkinje cell protein 4. Moreover,DrosophilaDTB-specific CREs correspond to regulatory sequences of humanENGRAILED-2, PAX-2andDACHSHUND-1that direct MHB-specific expression in the embryonic mouse brain. Together, these findings imply ancestral regulatory mechanisms mediating the genetic specification of midbrain-cerebellar circuitry for balance and motor control that may predated the radiation of cephalic nervous systems across the animal kingdom.
Another chemolithotrophic metabolism missing in nature—sulfur comproportionation
Authors: Amend Jan P. , Aronson Heidi S. , Macalady Jennifer , LaRowe Douglas E.
Published: October 2019

Chemotrophic microorganisms gain energy for cellular functions by catalyzing oxidation-reduction (redox) reactions that are out of equilibrium. Calculations of the Gibbs energy (ΔGr) can identify whether a reaction is thermodynamically favorable, and the accompanying energy yield at the temperature, pressure, and chemical composition in the system of interest. Based on carefully calculated values of ΔGr, we predict a novel microbial metabolism—sulfur comproportionation (3H2S + SO42-+ 2H+= 4S0+ 4H2O). We show that at elevated concentrations of sulfide and sulfate in acidic environments over a broad temperature range, this putative metabolism can be exergonic (ΔGr<0), yielding ∼30-50 kJ/mol. We suggest that this may be sufficient energy to support a chemolithotrophic metabolism currently missing in nature. Other versions of this metabolism, to thiosulfate (H2S + SO42-= S2O32-+ H2O) and to sulfite (H2S + 3SO42-= 4SO32-+ 2H+), are only moderately exergonic or endergonic even at ideal geochemical conditions. Natural and impacted environments, including sulfidic karst systems, shallow-sea hydrothermal vents, sites of acid mine drainage, and acid-sulfate crater lakes, may be ideal hunting grounds for finding microbial sulfur comproportionators.
The Genomic Stability at the Coding Regions of the Multidrug Transporter Gene ABCB1: Insights into the Development of Alternative Drug Resistance Mechanisms in Human Leukemia Cells
Authors: Chen Kevin G. , Duran George E. , Mogul Mark J. , Wang Yan C. , Ross Kevin L. , Jaffrézou Jean-Pierre , Huff Lyn M. , Fojo Tito , Lacayo Norman J. , Sikic Branimir I.
Published: October 2019

Despite considerable efforts in reversing clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) based on small molecule inhibitors has been hindered. This may be due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mechanisms that underlie this successful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, we report the development of two erythroleukemia variants, RVC and RDC, which were derived by step-wise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC, which retained P-gp expression, showed altered MDR phenotypes that were resistant to cyclosporine modulation. The ABCB1 coding regions were genetically stable even under long-term stringent drug selection. Genomically, ABCB1 is likely the most stable ABC transporter gene when comparing with several ABC superfamily members (such as ABCA1, ABCC1, CFTR, and ABCG2). Our findings suggested that non-P-gp mechanisms were likely responsible for the resistance to CsA modulation in both RVC and RDC cells. Moreover, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low level and unstable drug resistance, thus shedding some light on the benefits of CsA in treating certain types of AML patients.
Paternally expressed imprintedSnord116andPeg3regulate hypothalamic orexin neurons
Authors: Marta Pace , Matteo Falappa , Andrea Freschi , Edoardo Balzani , Chiara Berteotti , Viviana Lo Martire , Kaveh Fatemeh , Hovig Eivind , Giovanna Zoccoli , Matteo Cerri , Roberto Amici , Alfonso Urbanucci , Valter Tucci
Published: October 2019

Imprinted genes are highly expressed in the hypothalamus; however, whether specific imprinted genes affect hypothalamic neuromodulators and their functions is unknown. It has been suggested that Prader-Willi syndrome (PWS), a neurodevelopmental disorder caused by lack of paternal expression at chromosome 15q11-q13, is characterised by hypothalamic insufficiency. Here, we investigate the role of the paternally expressedSnord116gene within the context of sleep and metabolic abnormalities of PWS, and we report a novel role of this imprinted gene in the function and organisation of the two main neuromodulatory systems of the lateral hypothalamus (LH), namely, the orexin (OX) and melanin concentrating hormone (MCH) systems. We observe that the dynamics between neuronal discharge in the LH and the sleep-wake states of mice with paternal deletion ofSnord116(PWScrm+/p−) are compromised. This abnormal state-dependent neuronal activity is paralleled by a significant reduction in OX neurons in the LH of mutants. Therefore, we propose that an imbalance between OX- and MCH-expressing neurons in the LH of mutants reflects a series of deficits manifested in the PWS, such as dysregulation of rapid eye movement (REM) sleep, food intake and temperature control.

Highlights

Snord116regulates neuronal activity in the lateral hypothalamus (LH), which is time-locked with cortical states of sleep.

Loss ofSnord116reduces orexin neurons in the LH and affects sleep homeostasis and thermoregulation in mice.

Snord116andPeg3independently control orexin expression in the LH.

Paternally expressed alleles maximize the patrilineal effects in the control of REM sleep by the LH in mammals.
Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion
Authors: Menacho-Melgar Romel , Ye Zhixia , Moreb Eirik A. , Yang Tian , Efromson John P. , Decker John S. , Lynch Michael D.
Published: October 2019

We report the improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of total cellular protein. Initial use of the method in instrumented fed batch fermentations enables cell densities of ∼30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/−0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μL (384 well plates), 100 μL (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach ∼ 5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.

Highlights

Stationary phase protein expression results in high titers.

Autoinduction by phosphate depletion enables protein titers from 2-8 g/L.

Autoinduction has been validated from 384 well plates to instrumented bioreactors.
Stability of Two-quartet G-quadruplexes and Their Dimers in Atomistic Simulations
Authors: Islam Barira , Stadlbauer Petr , Vorlíčková Michaela , Mergny Jean-Louis , Otyepka Michal , Šponer Jiří
Published: October 2019

G-quadruplexes (GQs) are four-stranded non-canonical DNA and RNA architectures that can be formed by guanine-rich sequences. The stability of GQs increases with the number of G-quartets and three G-quartets generally form stable GQs. However, the stability of two-quartet GQs is an open issue. To understand the intrinsic stability of two-quartet GQ stems, we have carried out a series of unbiased molecular dynamics (MD) simulations (∼505 µs in total) of two- and four-quartet DNA and RNA GQs, with attention paid mainly to parallel-stranded arrangements. We used AMBER DNA parmOL15 and RNA parmOL3 force fields and tested different ion and water models. DNA two-quartet parallel-stranded GQs unfolded in all the simulations while the equivalent RNA GQ was stable in most of the simulations. GQs composed of two stacked units of two-quartet GQs were stable for both DNA and RNA. The simulations suggest that a minimum of three quartets are needed to form an intrinsically stable all-anti parallel-stranded DNA GQ. Parallel two-quartet DNA GQ may exist if substantially stabilized by another molecule or structural element, including multimerisation. On the other hand, we predict that isolated RNA two-quartet parallel GQs may form, albeit being weakly stable. We also show that ionic parameters and water models should be chosen with caution because some parameter combinations can cause spurious instability of GQ stems. Some in-so-far unnoticed limitations of force-field description of multiple ions inside the GQs are discussed, which compromise capability of simulations to fully capture the effect of increase of the number of quartets on the GQ stability.
Structure–function analyses of a keratin heterotypic complex identify specific keratin regions involved in intermediate filament assembly
Authors: Lee Chang-Hun , Kim Min-Sung , Li Shuang , Leahy Daniel J. , Coulombe Pierre A.
Published: October 2019

Intermediate filaments (IFs) provide vital mechanical support in a broad array of cell types. Interference with this role causes cell fragility and accounts for a large number of human diseases. Gaining an understanding IF structure is paramount to understanding their function and designing therapeutic agents for relevant diseases. Here, we report the 2.6 Å resolution crystal structure of a complex of interacting 2B domains of keratin 5 (K5) and K14. K5 and K14 form a long-range, left-handle coiled coil, with participating α-helices aligned in parallel and in register. Follow-up mutagenesis revealed that specific contacts between interacting 2B domains play a crucial role during 10-nm IF assembly, likely at the step of octamer–octamer association. The resulting structural model represents the first atomic-resolution visualization of 2B–2B interactions consistent with the A22dimer alignment and provide insight into the defects introduced by mutations in IF genes associated with human skin diseases.
Towards Staining Independent Segmentation of Glomerulus from Histopathological Images of Kidney
Authors: Liu Robin , Wang Lu , He Jim , Chen Wenfang
Published: October 2019

This paper introduces a detection-based framework to segment glomeruli from digital scanning image of light microscopic slide of renal biopsy specimens. The proposed method aims to better use the precise localization ability of Faster R-CNN and powerful segmentation ability of U-Net. We use a detector to localize the glomeruli from whole slide image to make the segmentation only focus on the most relevant area of the image. We explored the effectiveness of the network depth on its localization and segmentation ability in glomerular classification, and then propose to use the classification network with enhanced ability of localization and segmentation to construct and initialize a segmentation network. We also propose a weakly supervised training strategy to train the segmentation network by taking advantage of the unique morphology of the glomerulus. Both strong initialization and weakly supervised training are used to resolve the problem of insufficient and inaccurate data annotations and enhance the adaptability of the segmentation network. Experimental results demonstrate that the proposed framework is effective and robust.
Interindividual variability and lateralization of µ-opioid receptors in the human brain
Authors: Kantonen Tatu , Karjalainen Tomi , Isojärvi Janne , Nuutila Pirjo , Tuisku Jouni , Rinne Juha , Hietala Jarmo , Kaasinen Valtteri , Kalliokoski Kari , Scheinin Harry , Hirvonen Jussi , Vehtari Aki , Nummenmaa Lauri
Published: October 2019

The brain’s µ-opioid receptors (MORs) are involved in analgesia, reward and mood regulation. Several neuropsychiatric diseases have been associated with dysfunctional MOR system, and there is also considerable variation in receptor density among healthy individuals. Sex, age, body mass and smoking have been proposed to influence the MOR system, but due to small sample sizes the magnitude of their influence remains inconclusive. Here we quantifiedin vivoMOR availability in the brains of 204 individuals with no neurologic or psychiatric disorders using positron emission tomography (PET) with tracer [11C]carfentanil. We then used Bayesian hierarchical modeling to estimate the effects of sex, age, body mass index (BMI) and smoking on [11C]carfentanil binding potential. We also examined hemispheric lateralization of MOR availability. Age had regionally specific effects on MOR availability, with age-dependent increase in frontotemporal areas but decrease in amygdala, thalamus, and nucleus accumbens. The age-dependent increase was stronger in males. MOR availability was globally lowered in smokers but independent of BMI. Finally, MOR availability was higher in the right versus the left hemisphere. The presently observed variation in MOR availability may explain why some individuals are prone to develop MOR-linked pathological states, such as chronic pain or psychiatric disorders.
Region-specific expression of young small-scale duplications in the human central nervous system
Authors: Brohard-Julien Solène , Frouin Vincent , Meyer Vincent , Chalabi Smahane , Deleuze Jean-François , Floch Edith Le , Battail Christophe
Published: October 2019

Background

The duplication of genes is one of the main genetic mechanisms that led to the gain in complexity of biological tissue. Although the implication of duplicated gene expression in brain evolution was extensively studied through comparisons between organs, their role in the regional specialization of the adult human central nervous system has not yet been well described.

Results

Our work explored intra-organ expression properties of paralogs through multiple territories of the human central nervous system (CNS) using transcriptome data generated by the Genotype-Tissue Expression (GTEx) consortium. Interestingly, we found that paralogs were associated with region-specific expression in CNS, suggesting their involvement in the differentiation of these territories. Beside the influence of gene expression level on region-specificity, we observed the contribution of both duplication age and duplication type to the CNS region-specificity of paralogs. Indeed, we found that small scale duplicated genes (SSDs) and in particular ySSDs (SSDs younger than the 2 rounds of whole genome duplications) were more CNS region-specific than other paralogs. Next, by studying the two paralogs of ySSD pairs, we observed that when they were region-specific, they tend to be specific to the same region more often than for other paralogs, showing the high co-expression of ySSD pairs. Extension of this analysis to families of paralogs showed that the families with co-expressed gene members (i.e. homogeneous families) were enriched in ySSDs. Furthermore, these homogeneous families tended to be region-specific families, where the majority of their gene members were specifically expressed in the same region.

Conclusions

Overall, our study suggests the major involvement of ySSDs in the differentiation of human central nervous system territories. Therefore, we show the relevance of exploring region-specific expression of paralogs at the intra-organ level.
Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy
Authors: Zwettler Fabian U. , Spindler Marie-Christin , Reinhard Sebastian , Klein Teresa , Kurz Andreas , Sauer Markus , Benavente Ricardo
Published: October 2019

The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different ExM protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased expansion factor determination. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20-30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.
Cohesin and condensin extrude loops in a cell-cycle dependent manner
Authors: Golfier Stefan , Quail Thomas , Kimura Hiroshi , Brugués Jan
Published: October 2019

Chromatin undergoes a dramatic reorganization during the cell cycle1–3. In interphase, chromatin is organized into compartments and topological-associating domains (TADs) that are cell-type specific4–7, whereas in metaphase, chromosomes undergo large-scale compaction, leading to the loss of specific boundaries and the shutdown of transcription8–12. Loop extrusion by structural maintenance of chromosomes complexes (SMCs) has been proposed as a mechanism to organize chromatin in interphase and metaphase13–19. However, the requirements for chromatin organization in these cell phases are very different, and it is unknown whether loop extrusion dynamics and the complexes that extrude them also differ. Here, we used Xenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes asymmetric loops in metaphase, whereas cohesin extrudes symmetric loops in interphase. Our data show that loop extrusion is a general mechanism for the organization of DNA, with dynamic and structural properties that are molecularly regulated during the cell cycle.
Motor planning of vertical arm movements in healthy older adults: does effort optimization persist with aging?
Authors: Poirier Gabriel , Papaxanthis Charalambos , Mourey France , Gaveau Jeremie
Published: October 2019

Several sensorimotor modifications are known to occur with aging, possibly leading to adverse outcomes such as falls. Recently, some of those modifications have been proposed to emerge from motor planning deteriorations. Motor planning of vertical movements is thought to engage an internal model of gravity to anticipate its mechanical effects on the body-limbs and thus to genuinely produce movements that minimize muscle effort. This is supported, amongst other results, by direction-dependent kinematics where relative durations to peak accelerations and peak velocity are shorter for upward than for downward movements. The present study compares motor planning of fast and slow vertical arm reaching movements between eighteen young (24 ± 3 years old) and seventeen older adults (70 ± 5 years old). We found that older participants still exhibit directional asymmetries (i.e., differences between upward and downward movements), indicating that optimization processes during motor planning persist with healthy aging. However, the size of these differences was increased in older participants, indicating that gravity-related motor planning changes with age. We discuss this increase as the possible result of an overestimation of gravity torque or increased weight of the effort cost in the optimization process. Overall, these results support the hypothesis that feedforward processes and, more precisely, optimal motor planning, remain active with healthy aging.
6-O-galloylsalidroside, an active ingredient from Acer tegmentosum, ameliorates alcoholic steatosis and liver injury in a mouse model of chronic ethanol consumption
Authors: Kim Young Han , Woo Dong-Cheol , Ra Moonjin , Jung Sangmi , Ham Su Jung , Kim Ki Hyun , Lee Yongjun
Published: October 2019

We previously reported that Acer tegmentosum extract, which is traditionally used to treat liver disease in Korea, may help reduce fat accumulation, improve liver metabolism, and suppress inflammation in alcoholic liver disease. The active ingredient was found to be 6-O-galloylsalidroside, which was isolated from the methanol extract of A. tegmentosum. We hypothesized that 6-O-galloylsalidroside extracted from A. tegmentosum may help protect from liver damage and attenuate hepatic fat accumulation associated with chronic alcohol consumption. In the present study, we aimed to investigate whether 6-O-galloylsalidroside can regulate alcoholic fatty liver and liver injury in mice. For this purpose, mice were fed with Lieber-DeCarli 5% ethanol diet for 11 days to induce steatosis and liver injury. Oral 6-O-galloylsalidroside was administered once a day for 11 days. Intrahepatic lipid accumulation caused by alcohol consumption was measured using in vivo 1H magnetic resonance imaging. Hepatic steatosis was observed histologically in tissue samples stained with hematoxylin and eosin, as well as Oil Red O. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, as well as the triglyceride content in liver homogenates. On magnetic resonance spectroscopy, 6-O-galloylsalidroside appeared to alleviate alcohol-induced steatosis, which was reflected in decreased hepatic and serum triglyceride levels despite ethanol feeding. Furthermore, 6-O-galloylsalidroside treatment was associated with decreased RNA expression of Cd36, which plays an important role in the development of alcoholic steatosis through the hepatic de novo lipogenesis pathway. Furthermore, treatment with 6-O-galloylsalidroside inhibited the expression of cytochrome P4502E1 and attenuated hepatocellular damage, reflected in reduced ALT and AST levels. These findings suggest that 6-O-galloylsalidroside extracted from A. tegmentosum might serve as a bioactive agent for treating alcoholic fatty liver and liver damage.
Polynomial-Time Statistical Estimation of Species Trees under Gene Duplication and Loss
Authors: Legried Brandon , Molloy Erin K. , Warnow Tandy , Roch Sébastien
Published: October 2019

Phylogenomics—the estimation of species trees from multilocus datasets—is a common step in many biological studies. However, this estimation is challenged by the fact that genes can evolve under processes, including incomplete lineage sorting (ILS) and gene duplication and loss (GDL), that make their trees different from the species tree. In this paper, we address the challenge of estimating the species tree under GDL. We show that species trees areidentifiableunder a standard stochastic model for GDL, and that the polynomial-time algorithm ASTRAL-multi, a recent development in the ASTRAL suite of methods, isstatistically consistentunder this GDL model. We also provide a simulation study evaluating ASTRAL-multi for species tree estimation under GDL. All scripts and datasets used in this study are available on the Illinois Data Bank:https://doi.org/10.13012/B2IDB-2626814_V1.
Prospective and retrospective contributions to intention awareness in voluntary action
Authors: Schultze-Kraft Matthias , Parés-Pujolràs Elisabeth , Matić Karla , Haggard Patrick , Haynes John-Dylan
Published: October 2019

Spontaneous, voluntary actions are often accompanied by motor intentions, that is the experience of “being about to move”. The emergence of this experience is contentiously discussed, culminating in two opposing views of motor intention. While prospective theorists hold that an intention to move genuinely precedes the action, retrospective theorists suggest that an intention is reconstructed only after action execution. As such, both theories offer radically different predictions about the possibility of voluntary control over action intention. We assess here the respective contributions of prospective and retrospective factors in motor intention awareness in a real-time EEG setup. Participants performed self-paced movements with intermittent interruption by cues, signalling to either move or not to move. Cues were triggered by a brain-computer interface upon detection of either the presence or absence of a readiness potential (RP), a signal known to precede self-paced voluntary actions. Subsequently, participants reported their intention to move at the time of the cue. We found that participants were more likely to report an intention to move when the cue was followed by a movement than no movement, highlighting the effect of retrospection on awareness of intention. Interestingly, however, an intention to move was also more frequently reported when the cue was elicited by an RP, compared to when no RP was present, suggesting that awareness of intention is also informed by motor preparation processes, congruent with prospective theories. Overall, our findings show that intention awareness in voluntary action relies on both prospective and retrospective components, and we describe here how these two factors are dynamically integrated over time.