bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACTBrain 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

AbstractTransmission 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.SignificanceBacterial 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

AbstractNanocrystallography 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

ABSTRACTCorresponding 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

AbstractChemotrophic 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

ABSTRACTDespite 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

AbstractImprinted 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.HighlightsSnord116regulates 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

AbstractWe 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.HighlightsStationary 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

ABSTRACTG-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

SUMMARYIntermediate 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

AbstractThis 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

AbstractThe 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

ABSTRACTBackgroundThe 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.ResultsOur 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.ConclusionsOverall, 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.