bluuebunny/crossref_metadata_2025 huggingface dataset:
31° South: phenotypic flexibility in adaptive thermogenesis among conspecific populations of an arid-endemic bird - from organismal to cellular level
Authors: Ribeiro Ângela M. , Prats Clara , Pattinson Nicholas B. , Gilbert M. Thomas P. , Smit Ben
Published: November 2018

In north-temperate small passerines, overwinter survival is associated with a reversibly increased maximum cold-induced metabolism (Msum). This strategy may incur increased energy consumption. Therefore, species inhabiting ecosystems characterized by cold winters and low productivity (i.e., low available energy) may be precluded from displaying an increase in maximum metabolic rates. To examine whether Msumis a flexible phenotype in such challenging environments, and ultimately uncover its underpinning mechanisms, we studied an arid-endemic small bird (Karoo scrub-robin) whose range spans a primary productivity and minimum temperature gradient. We measured Msum, body condition, mass of thermogenic muscles and two indices of cellular aerobic capacity from populations living in three environmentally different regions. We found that Msumwas seasonally flexible, associated with aerobic capacity of limb muscles, but not increasing with lower temperatures, as predicted. Notwithstanding, the cold limit (temperature at which birds reached their maximum metabolic capacity) decreased in winter. These results indicate that birds from arid-zones may respond to cold conditions by altering thermosensation, rather than spending energy to produce heat in skeletal muscles.
Transcriptomic response to divergent selection for flowering times reveals convergence and key players of the underlying gene regulatory network
Authors: Tenaillon Maud I. , Seddiki Khawla , Mollion Maeva , Guilloux Martine Le , Marchadier Elodie , Ressayre Adrienne , Dillmann Christine
Published: November 2018

Artificial selection experiments are designed to investigate phenotypic evolution of complex traits and its genetic basis. Here we focused on flowering time, a trait of key importance for plant adaptation and life-cycle shifts. We undertook divergent selection experiments from two maize inbred lines. After 13 generations of selection, we obtained a time-lag of roughly two weeks between Early- and Late-populations. We used this material to characterize the genome-wide transcriptomic response to selection in the shoot apical meristem before, during and after floral transition in field conditions during two consecutive years. We validated the reliability of performing RNA-sequencing in uncontrolled conditions. We found that roughly half of maize genes were expressed in the shoot apical meristem, 59.3% of which were differentially expressed. We detected a majority of genes with differential expression between inbreds and across meristem status, and retrieved a subset of 2,451 genes involved in the response to selection. Among these, we found a significant enrichment for genes with known function in maize flowering time. Furthermore, they were more often shared between inbreds than expected by chance, suggesting convergence of gene expression. We discuss new insights into the expression pattern of key players of the underlying gene regulatory network including theZea maysgenesCENTRORADIALIS(ZCN8),RELATED TO AP2.7(RAP2.7),MADS4(ZMM4),KNOTTED1(KN1),GIBBERELLIN2-OXIDASE1(GA2ox1), as well as alternative scenarios for genetic convergence.
Fractional Deletion Of Compound Kushen Injection, A Natural Compound Mixture, Indicates Cytokine Signaling Pathways Are Critical For Its Perturbation Of The Cell Cycle
Authors: Aung TN , Nourmohammadi S , Qu Z , Harata-Lee Y , Cui J , Shen HY , Yool AJ , Pukala T , Hong Du , Kortschak RD , Adelson DL
Published: November 2018

We have used computational and experimental biology approaches to identify candidate mechanisms of action of a traditional Chinese medicine. Compound Kushen Injection (CKI), in a breast cancer cell line in which CKI causes apoptosis. Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis in MDA-MB-231 cells. Our initial fractionation separated major from minor compounds, and showed that the major compounds accounted for little of the activity of CKI. By systematically perturbing the major compounds in CKI we found that removal of no single major compound could alter the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical compounds with respect to CKI activity. We then used RNA sequencing and transcriptome analysis to correlate compound removal with gene expression and phenotype data. We determined that many compounds in CKI are required for its effectiveness in triggering apoptosis but that significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.
A curative combination therapy for lymphomas achieves high fractional cell killing through low cross-resistance and drug additivity but not synergy
Authors: Palmer Adam C , Chidley Christopher , Sorger Peter K
Published: November 2018

Curative cancer therapies are uncommon and nearly always involve multi-drug combinations developed by experimentation in humans; unfortunately, the mechanistic basis for the success of such combinations has rarely been investigated in detail, obscuring lessons learned. Here we use isobologram analysis to score pharmacological interaction, and clone tracing and CRISPR screening to measure cross-resistance among the five drugs comprising R-CHOP, a combination therapy that frequently cures Diffuse Large B-Cell Lymphomas. We find that drugs in R-CHOP exhibit very low cross-resistance but not synergistic interaction; together they achieve a greater fractional kill according to the null hypothesis for both the Loewe dose-additivity model and the Bliss effect-independence model. These data provide direct evidence for the 50-year old hypothesis that a curative cancer therapy can be constructed on the basis of independently effective drugs having non-overlapping mechanisms of resistance, without synergistic interaction, which has immediate significance for the design of new drug combinations.
The Time Course of Ineffective Sham Blinding During 1mA tDCS
Authors: Greinacher Robert , Buhôt Larissa , Möller Lisa , Learmonth Gemma
Published: November 2018

Background

Studies using transcranial direct current stimulation (tDCS) typically compare the effects of an active (10-30min) relative to a shorter sham (placebo) protocol. Both active and sham tDCS are presumed to be perceptually identical on the scalp, and thus represent an effective method of delivering double-blinded experimental designs. However, participants often show above-chance accuracy when asked which condition involved active/sham retrospectively.

Objective/Hypothesis

We aimed to assess the time course of sham-blinding during active and sham tDCS. We predicted that 1) Participants will be aware that the current is switched on for a longer duration in the active versus the sham protocol, 2) Active anodal tDCS will reduce reaction times more effectively than sham.

Methods

32 adults were tested in a pre-registered, double-blinded, within-subjects design. A forced-choice reaction time task was undertaken before, during and after active (10min 1mA) and sham (20s 1mA) tDCS. The anode was placed over the left primary motor cortex (C3) to target the right hand, and the cathode on the right forehead. Two probe questions were asked every 30s: “Is the stimulation on? “and “How sure are you?”.

Results

Distinct periods of non-overlapping confidence intervals were identified between the active and sham conditions, totalling 5min (57.1% of the total difference in stimulation time). These began immediately after sham ramp-down and lasted until the active protocol had ended. Active tDCS had no effect on reaction times compared to sham (ΔRT active vs sham p>0.38 in all blocks).

Conclusions

We show a failure of placebo control during low-intensity tDCS.
TAPBPR mediates peptide dissociation from MHC class I using a leucine lever
Authors: Ilca F. Tudor , Neerincx Andreas , Hermann Clemens , Marcu Ana , Stevanovic Stefan , Deane Janet E , Boyle Louise H
Published: November 2018

Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules, however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediate peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing of MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity.

Impact Statement

This work demonstrates for the first time that the K22-D35 loop of TAPBPR is the essential region for mediating peptide exchange and peptide selection on major histocompatibility complex class I molecules.
The invisibility cloak: Chitin binding protein ofVerticillium nonalfalfaedisguises fungus from plant chitinases
Authors: Volk Helena , Marton Kristina , Flajšman Marko , Radišek Sebastjan , Hein Ingo , Podlipnik Črtomir , Javornik Branka , Berne Sabina
Published: November 2018

During fungal infections, plant cells secrete chitinases that digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune receptors results in the activation of defence signalling pathways. We report here thatVerticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents this recognition process by secreting a CBM18 (carbohydrate binding motif 18)-chitin binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by theVna8.213gene which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however,Vna8.213knockout mutants exhibit wilting symptoms similar to the wild type fungus, suggesting thatVna8.213activity is functionally redundant during fungal infection of hop. In binding assay, recombinant VnaChtBP binds chitin and chitin oligomersin vitrowith submicromolar affinity and protects fungal hyphae from degradation by plant chitinases. Using a yeast-two-hybrid assay, homology modelling and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases.
A comparison of Alberta’s Infant Motor Scale and Brazelton Neonatal Behavior Assessment Scale
Authors: Perera Priyantha , Godamunne Pavithra , Nandasena Sumal , Tipre Meghan , Wimalasiri Udaya , Reader Mark , Beddage Thulani , Smith Tamika , Kasturiratne Anuradhani , Sathiakumar Nalini , Wickremasinghe Rajitha
Published: November 2018

Background and aims

The Neonatal Behavior Assessment Scale (NBAS) was introduced by Brazelton is a useful tool to assess neurodevelopment in newborn in research setting. However, it is complex, time-consuming and requires specialized training for administration and is therefore difficult tool to use in routine clinical settings. The Alberta Infant Motor Scale (AIMS) is a simple tool to assess of motor maturation of a child up to 18 months and can be administered with minimal training for the health care professionals. The study goal of this study was to evaluate the reliability and validity of the AIMS in comparison with the NBAS in assessing motor maturity at birth.

Methods

We administered the AIMS and NBAS to a total of 66 newborn babies delivered in three obstetric units at the Colombo North Teaching Hospital in Ragama, Sri Lanka. The subjects were selected from a sample of 545 newborn babies in an ongoing birth-cohort study evaluating effects of prenatal exposure to biomass smoke and infant neurodevelopment. Trained research assistants administered first the NBAS, followed by the AIMS one-hour later. Univariate and bivariate statistics were used to compare the two scales.

Results

Irrespective of maturity, sex, birth weight or socio-demographic characteristics, all babies had scored on the 75th percentile in the AIMS. In the NBAS, there was a significant variation in the Brazelton motor score scaled to 100. Low birth weight babies showed a narrower variation in the NABS score. None of the scales indicated a motor deficit in any of the children.

Conclusion

NBAS identifies subtle differences in motor maturity of full term babies that the AIMS fails to detect
Using transcriptome sequencing and pooled exome capture to study local adaptation in the giga-genome ofPinus cembra
Authors: Rellstab Christian , Dauphin Benjamin , Zoller Stefan , Brodbeck Sabine , Gugerli Felix
Published: November 2018

Despite decreasing sequencing costs, whole-genome sequencing for population-based genome scans for selection is still prohibitively expensive for organisms with large genomes. Moreover, the repetitive nature of large genomes often represents a challenge in bioinformatic and downstream analyses. Here we use in-depth transcriptome sequencing to design probes for exome capture in Swiss stone pine (Pinus cembra), a conifer with an estimated genome size of 29.3 Gbp and no reference genome available. We successfully applied around 55,000 self-designed probes, targeting 25,000 contigs, to DNA pools of seven populations from the Swiss Alps and identified > 140,000 SNPs in around 13,000 contigs. The probes performed equally well in pools of the closely related speciesPinus sibirica; in both species, more than 70% of the targeted contigs were sequenced at a depth ≥ 40x, i.e. the number of haplotypes in the pool. However, a thorough analysis of individually sequencedP. cembrasamples indicated that a majority of the contigs (63%) represented multi-copy genes. We therefore removed paralogous contigs based on heterozygote excess and deviation from allele balance. Without putatively paralogous contigs, allele frequencies of population pools represented accurate estimates of individually determined allele frequencies. Using population genetic and landscape genomic methods, we show that inferences of neutral and adaptive genetic variation may be biased when not accounting for such multi-copy genes. Future studies should therefore put more emphasis on identifying paralogous loci, which will be facilitated by the establishment of additional high-quality reference genomes.
An updated phylogeny of theAlphaproteobacteriareveals that the parasiticRickettsialesandHolosporaleshave independent origins
Authors: Muñoz-Gómez Sergio A. , Hess Sebastian , Burger Gertraud , Franz Lang B. , Susko Edward , Slamovits Claudio H. , Roger Andrew J.
Published: November 2018

TheAlphaproteobacteriais an extraordinarily diverse and ancient group of bacteria. Previous attempts to infer its deep phylogeny have been plagued with methodological artefacts. To overcome this, we analyzed a dataset of 200 single-copy and conserved genes and employed diverse strategies to reduce compositional artefacts. Such strategies include using novel dataset-specific profile mixture models and recoding schemes, and removing sites, genes and taxa that are compositionally biased. We show that theRickettsialesandHolosporales(both groups of intracellular parasites of eukaryotes) are not sisters to each other, but instead, theHolosporaleshas a derived position within theRhodospirillales. Furthermore, we find that theRhodospirillalesmight be paraphyletic and that theGeminicoccaceaecould be sister to all ancestrally free-living alphaproteobacteria. Our robust phylogeny will serve as a framework for future studies that aim to place mitochondria, and novel environmental diversity, within theAlphaproteobacteria.
OLIG2 Drives Abnormal Neurodevelopmental Phenotypes in Human iPSC-Based Organoid and Chimeric Mouse Models of Down Syndrome
Authors: Xu Ranjie , Brawner Andrew T , Li Shenglan , Liu JingJing , Kim Hyosung , Xue Haipeng , Pang Zhiping P. , Kim Woo-Yang , Hart Ronald P. , Liu Ying , Jiang Peng
Published: November 2018

Down syndrome (DS) is a common neurodevelopmental disorder, and cognitive defects in DS patients may arise form imbalances in excitatory and inhibitory neurotransmission. Understanding the mechanisms underlying such imbalances may provide opportunities for therapeutic intervention. Here, we show that human induced pluripotent stem cells (hiPSCs) derived from DS patients overproduce OLIG2+ventral forebrain neural progenitors. As a result, DS hiPSC-derived cerebral organoids excessively produce specific subclasses of GABAergic interneurons and cause impaired recognition memory in neuronal chimeric mice. Increased OLIG2 expression in DS cells directly upregulates interneuron lineage-determining transcription factors. shRNA-mediated knockdown ofOLIG2largely reverses abnormal gene expression in early-stage DS neural progenitors, reduces interneuron production in DS organoids and chimeric mouse brains, and improves behavioral deficits in DS chimeric mice. Thus, altered OLIG2 expression may underlie neurodevelopmental abnormalities and cognitive defects in DS patients.
Exploring neighborhoods in large metagenome assembly graphs reveals hidden sequence diversity
Authors: Brown C. Titus , Moritz Dominik , O’Brien Michael P. , Reidl Felix , Reiter Taylor , Sullivan Blair D.
Published: November 2018

Genomes computationally inferred from large metagenomic data sets are often incomplete and may be missing functionally important content and strain variation. We introduce an information retrieval system for large metagenomic data sets that exploits the sparsity of DNA assembly graphs to efficiently extract subgraphs surrounding an inferred genome. We apply this system to recover missing content from genome bins and show that substantial genomic sequence variation is present in a real metagenome. Our software implementation is available athttps://github.com/spacegraphcats/spacegraphcats under the 3-Clause BSD License.
Pan-cancer analysis on microRNA-mediated gene activation
Authors: Tan Hua , Huang Shan , Zhang Zhigang , Qian Xiaohua , Sun Peiqing , Zhou Xiaobo
Published: November 2018

While microRNAs (miRNAs) were widely considered to repress target genes at mRNA and/or protein levels, emerging evidence from in vitro experiments has shown that miRNAs can also activate gene expression in particular contexts. However, this counterintuitive observation has rarely been reported or interpreted in in vivo conditions. We systematically explored the positive correlation between miRNA and gene expressions and its potential implications in tumorigenesis, based on 8375 patient samples across 31 major human cancers from The Cancer Genome Atlas (TCGA). Results indicated that positive miRNA-gene correlations are surprisingly prevalent and consistent across cancer types, and show distinct patterns than negative correlations. The top-ranked positive correlations are significantly involved in the immune cell differentiation and cell membrane signaling related processes, and display strong power in stratifying patients in terms of survival rate, demonstrating their promising clinical relevance. Although intragenic miRNAs generally tend to co-express with their host genes, a substantial portion of miRNAs shows no obvious correlation with their host gene due to non-conservation. A miRNA can upregulate a gene by inhibiting its upstream suppressor, or shares transcription factors with that gene, both leading to positive correlation. The miRNA/gene sites associated with the top-ranked positive correlations are more likely to form super-enhancers compared to randomly chosen pairs, suggesting a potential epigenetics mechanism underlying the upregulation. Wet-lab experiments revealed that positive correlations partially remain in the in vitro condition. Our study provides the field with new perspectives on the critical role of miRNA in gene regulation and novel insights regarding the complex mechanisms underlying miRNA functions, and reveals the clinical significance of the potential positive regulation of gene expression by miRNA.
Physiological and transcriptomic analysis of yellow leaf coloration inPopulus deltoidesMarsh
Authors: Zhang Shuzhen , Wu Xiaolu , Cui Jie , Zhang Fan , Wan Xueqin , Liu Qinglin , Zhong Yu , Lin Tiantian
Published: November 2018

As important deciduous tree,Populus deltoidesMarsh possesses a high ornamental value for its leaves remaining yellow during the non-dormant period. However, little is known about the regulatory mechanism of leaf coloration inPopulus deltoidesMarsh. Thus, we analyzed physiological and transcriptional differences of yellow leaves (mutant) and green leaves (wild-type) ofPopulus deltoidesMarsh. Physiological experiments showed that the contents of chlorophyll (Chl) and carotenoid are lower in mutant, the flavonoid content is not differed significantly between mutant and wild-type. Transcriptomic sequencing was further used to identify 153 differentially expressed genes (DEGs). Functional classifications based on Gene Ontology enrichment and Genomes enrichment analysis indicated that the DEGs were involved in Chl biosynthesis and flavonoid biosynthesis pathway. Among these, geranylgeranyl diphosphate (CHLP) genes associated with Chl biosynthesis showed down-regulation, while chlorophyllase (CLH) genes associated with Chl degradation were up-regulated in yellow leaves. The expression levels of these genes were further confirmed using quantitative real-time PCR (RT-qPCR). Furthermore, the measurement of the main precursors of Chl confirmed that CHLP is vital enzymes for the yellow leaf color phenotype. Consequently, the formation of yellow leaf color is due to disruption of Chl synthesis and catabolism rather than flavonoid content. These results contribute to our understanding of mechanisms and regulation of leaf color variation in poplar at the transcriptional level.
Transmission of mechanical information by purinergic signaling
Authors: Mikolajewicz N. , Sehayek S. , Wiseman P. W. , Komarova S. V.
Published: November 2018

1

The human skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically-relevant mechanical forces lead to small, repairable membrane injuries in bone-forming osteoblasts, resulting in the release of ATP and stimulation of purinergic (P2) calcium responses in neighbouring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, extracellular diffusion and degradation, and purinergic responses in neighboring cells. The model was validated using experimental data obtained by measuring intracellular free calcium ([Ca2+]i) elevations following mechanical stimulation of a single osteoblast. The validated single-cell injury model was then scaled to a tissue-level injury to investigate how purinergic responses communicate information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed the maximal distance from the injury at which purinergic responses were stimulated, as well as the overall number of responders. The peak ATP concentration reflected the severity of an individual cell injury and determined signal propagation velocity and temporal synchrony of responses. Peak ATP concentrations also discriminated between minor and severe injuries that led to the release of similar total amounts of ATP due to differences in injury repair dynamics. The third component was ADP-mediated signaling which became relevant only in larger tissue-level injuries, and it conveyed information about the distance to the injury site and its geometry. Taken together, this study identified specific features of extracellular ATP/ADP spatiotemporal signals that encode the severity of the mechanical stimulus, the distance from the stimulus, as well as the mechano-resilient status of the tissue.
adductomicsR: A package for detection and quantification of protein adducts from mass spectra of tryptic digests
Authors: Hayes Josie , Edmands William M. B. , Yano Yukiko , Grigoryan Hasmik , Schiffman Courtney , Dudoit Sandrine , Rappaport Stephen M.
Published: November 2018

Liquid chromatography-high resolution mass spectrometry (LC-HRMS) has been used to establish a method, referred to as ‘adductomics’, for characterisation of putative protein adducts at selected loci in human serum albumin (HSA). Applications of this method have been limited by the lack of software for untargeted analysis of modified peptides in protein digests. Here we present adductomicsR, an open-source R package for processing LC-HRMS data from analysis of adducted HSA peptides. The software interrogates mass spectra to correct for retention-time drift, and to discover and quantify putative adducts along with those for a housekeeping peptide and internal standard.

Availability and implementation

adductomicsR is written in R and publicly available at https://github.com/JosieLHayes/adductomicsR, which includes a vignette with example data.

Supplementary information

mzXML files for the vignette and test dataset are available in an associated data package adductData (https://github.com/JosieLHayes/adductData).

Contact

josie.hayes@berkeley.edu

Issue Section

APPLICATIONS NOTE
A prebiotic protocell model based on dynamic protein membranes accommodating anabolic reactions
Authors: Schreiber Andreas , Huber Matthias C. , Schiller Stefan M.
Published: November 2018

Phospholipid membranes are essential constituents of extant cells rendering them preferred candidates as membrane components in origin of life scenarios. These models greatly neglect stability requirements and their problematic synthetic complexity necessary to access such lipid membrane constituents under early life conditions. Here we present an alternative protocell model, based on amphiphilic protein membranes constituted of prebiotic amino acids. These self-assembled dynamic Protein Membrane Based Compartments (PMBC) are impressively stable and compatible with prevalent protocell membrane constituents. PMBCs can enclose functional proteins, undergo membrane fusion, phase separate, accommodate anabolic ligation reactions and DNA encoded synthesis of their own membrane constituents. Our findings suggest that prebiotic PMBC represent a new type of protocell as plausible ancestor of current lipid-based cells. They can be used to design simple artificial cells important for the study of structural and catalytic pathways related to the emergence of life.
Repression of Dlx1/2 Signaling byNolz-1/Znf503is Essential for Parcellation of the Striatal Complex into Dorsal and Ventral Striatum
Authors: Lu Kuan-Ming , Chen Shih-Yun , Ko Hsin-An , Huang Ting-Hao , Hsin-Jou Hao Janice , Yan Yu-Ting , Li-Yun Chang Sunny , Evans Sylvia , Liu Fu-Chin
Published: November 2018

The division of the striatum into dorsal and ventral districts is of central clinical importance. The dorsal striatum is differentially affected in Huntington’s disease, dopamine in the ventral striatum is differentially spared in Parkinson’s disease, and human brain imaging studies implicate the ventral striatum in addictive disorders. If fits that the dorsal striatum contains the cells of origin of the direct and indirect basal ganglia pathways for motor control. The ventral striatum is a node in neural circuits related to motivation and affect. Despite these striking neurobiologic contrasts, there is almost no information about how the dorsal and ventral divisions of the striatum are set up during development. Here, we demonstrate that interactions between the two key transcription factors Nolz-1 and Dlx1/2 control the migratory paths of developing striatal neurons to the dorsal or ventral striatum. Moreover, these same transcription factors control the cell identity of striatal projection neurons in both the dorsal and ventral striatum including the cell origin of the direct and indirect pathways. We show that Nolz-1 suppresses Dlx1/2 expression. Deletion of Nolz-1 or over-expression of Dlx1/2 can produce a striatal phenotype characterized by withered dorsal striatum and a swollen ventral striatum, and that we can rescue this phenotype by manipulating the interactions between Nolz-1 and Dlx1/2 transcription factors. This evidence suggests that the fundamental basis for divisions of the striatum known to be differentially vulnerable at maturity is already encoded by the time embryonic striatal neurons begin their migrations into the developing striatum.
The transcription factors TFEB and TFE3 link the FLCN-AMPK signaling axis to innate immune response and pathogen resistance
Authors: El-Houjeiri Leeanna , Possik Elite , Vijayaraghavan Tarika , Paquette Mathieu , Martina José A , Kazan Jalal M. , Ma Eric H. , Jones Russell , Blanchette Paola , Puertollano Rosa , Pause Arnim
Published: November 2018

TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. UsingC. elegansand mammalian models, we report that the master metabolic modulator 5’-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK conferred pathogen resistanceviaactivation of TFEB/TFE3-dependent antimicrobial genes, while ablation of total AMPK activity abolished this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induced TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages was observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved and pharmacologically actionable mechanism coupling energy status with innate immunity.
Single-cell transcriptome analysis of Physcomitrella leaf cells during reprogramming using microcapillary manipulation
Authors: Kubo Minoru , Nishiyama Tomoaki , Tamada Yosuke , Sano Ryosuke , Ishikawa Masaki , Murata Takashi , Imai Akihiro , Lang Daniel , Demura Taku , Reski Ralf , Hasebe Mitsuyasu
Published: November 2018

Background

Next-generation sequencing technologies have made it possible to carry out transcriptome analysis at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) data provide insights into cellular dynamics, including intercellular heterogeneity as well as inter- and intra-cellular fluctuations in gene expression that cannot be studied using populations of cells. The utilization of scRNA-seq is, however, restricted to specific types of cells that can be isolated from their original tissues, and it can be difficult to obtain precise positional information for these cells in situ.

Results

Here, we established single cell-digital gene expression (1cell-DGE), a method of scRNA-seq that uses micromanipulation to extract the contents of individual living cells in intact tissue while recording their positional information. Furthermore, we employed a unique molecular identifier to reduce amplification bias in the cDNA libraries. With 1cell-DGE, we could detect differentially expressed genes (DEGs) during the reprogramming of leaf cells into stem cells in excised tissues of the moss Physcomitrella patens, identifying 6,382 DEGs between cells at 0 h and 24 h after excision. We found substantial variations in both the transcript levels of previously reported reprogramming factors and the overall expression profiles between cells, which appeared to be related to their different reprogramming abilities or the estimated states of the cells according to the pseudotime based on the transcript profiles.

Conclusions

We developed 1cell-DGE with microcapillary manipulation, a technique that can be used to analyze the gene expression of individual cells without detaching them from their tightly associated tissues, enabling us to retain positional information and investigate cell–cell interactions.
De novo clustering of long-read transcriptome data using a greedy, quality-value based algorithm
Authors: Sahlin Kristoffer , Medvedev Paul
Published: November 2018

Long-read sequencing of transcripts with PacBio Iso-Seq and Oxford Nanopore Technologies has proven to be central to the study of complex isoform landscapes in many organisms. However, current de novo transcript reconstruction algorithms from long-read data are limited, leaving the potential of these technologies unfulfilled. A common bottleneck is the dearth of scalable and accurate algorithms for clustering long reads according to their gene family of origin. To address this challenge, we develop isONclust, a clustering algorithm that is greedy (in order to scale) and makes use of quality values (in order to handle variable error rates). We test isONclust on three simulated and five biological datasets, across a breadth of organisms, technologies, and read depths. Our results demonstrate that isONclust is a substantial improvement over previous approaches, both in terms of overall accuracy and/or scalability to large datasets. Our tool is available at https://github.com/ksahlin/isONclust.
Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner to control cell cycle progression
Authors: Kuhn Jonathan , Dumont Sophie
Published: November 2018

The Spindle Assembly Checkpoint (SAC) maintains genomic integrity by preventing anaphase until all kinetochores attach to the spindle. What specific signals are required for SAC satisfaction at mammalian kinetochores, and in what magnitude, are not well understood and central to understanding SAC signal processing and function. Here, we directly and independently tune candidate input signals – spindle forces and Hec1-microtubule binding – and map SAC outputs. By detaching microtubules from the spindle, we first demonstrate that the SAC does not respond to changes in spindle pulling forces. We then tune and fix the fraction of Hec1 molecules capable of microtubule binding, and interpret SAC output changes as coming from changes in binding, and not spindle forces. While the speed of satisfaction reduces with fewer attached microtubules, the kinetochore turns off the SAC even with few – approximately four – such microtubules. Thus, the mammalian kinetochore responds specifically to microtubule binding, and does so as a single, switch-like, sensitive unit. This may allow the kinetochore to rapidly react to attachments and maintain a robust response despite dynamic microtubule numbers.
A uniformin vitroefficacy dataset to guide antimicrobial peptide design
Authors: Nagarajan Deepesh , Nagarajan Tushar , Nanajkar Neha , Chandra Nagasuma
Published: November 2018

Antimicrobial peptides are ubiquitous molecules that form the innate immune system of organisms across all kingdoms of life. Despite their prevalence and early origins, they continue to remain potent natural antimicrobial agents. Antimicrobial peptides are therefore promising drug candidates in the face of overwhelming multi-drug resistance to conventional antibiotics. Over the past few decades, thousands of antimicrobial peptides have been characterizedin vitro, and their efficacy data is now available in a multitude of public databases. Computational antimicrobial peptide design attempts typically use such data. However, utilizing heterogenous data aggregated from different sources presents significant drawbacks. In this report, we present a uniform dataset containing 20 antimicrobial peptides assayed against 30 organisms spanning gram positive, gram negative, fungal, and mycobacterial origin. We draw inferences from the results of 600 individual MIC assays, and discuss what characteristics are essential for antimicrobial peptide efficacy. We expect our uniform dataset to be useful for future projects involving computational antimicrobial peptide design.
Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures
Authors: Kamitani Mari , Kashima Makoto , Tezuka Ayumi , Nagano Atsushi J.
Published: November 2018

RNA-Seq is a whole-transcriptome analysis method used to research biological mechanisms and functions; its use in large-scale experiments is limited by costs and labour. In this study, we established a high-throughput and cost effective RNA-Seq library preparation method that did not require mRNA enrichment. The method adds unique index sequences to samples during reverse transcription (RT) that is conducted at a higher temperature (≥62°C) to suppress RT of A-rich sequences in rRNA, and then pools all samples into a single tube. Both single-read and paired end sequencing of libraries is enabled. We found that the pooled RT products contained large amounts of RNA, mainly rRNA, and caused over-estimations of the quantity of DNA, resulting in unstable tagmentation results. Degradation of RNA before tagmentation was necessary for the stable preparation of libraries. We named this protocol low-cost and easy RNA-Seq (Lasy-Seq), and used it to investigate temperature responses inArabidopsis thaliana. We analysed how sub-ambient temperatures (10–30°C) affected the plant transcriptomes, using time-courses of RNA-Seq from plants grown in randomly fluctuating temperature conditions. Our results suggest that there are diverse mechanisms behind plant temperature responses at different time scales.
Basal extrusion drives cell invasion and mechanical stripping of E-cadherin
Authors: Fadul John , Slattum Gloria M. , Redd Nadja M. , Jin Mauricio Franco , Redd Michael J. , Daetwyler Stephan , Hedeen Danielle , Huisken Jan , Rosenblatt Jody
Published: November 2018

Metastasis is the predominant reason that patients succumb to cancer, yet the mechanisms that drive initial tumor cell invasion are poorly understood. We previously discovered that crowding-induced apical extrusion drives most epithelial cell death, critical to maintaining constant cell densities. Oncogenic mutations can disrupt apical cell extrusion, instead causing masses to form and aberrant basal extrusion. Using transparent zebrafish epidermis to model simple epithelia, we can image invasion events live at high resolution. We find that KRas/p53-transformed cells form masses and, at completely independent sites, invade by basal extrusion. Basal extrusion also causes invading cells to simultaneously mechanically shed their entire apical membranes and E-cadherin. Once cells invade the underlying tissue, they migrate throughout the body, divide, enter the bloodstream, and become different cell types. KRas-transformation makes cells intrinsically invasive by increasing basal extrusion rates; collaborating mutations in p53 allow disseminated cells to survive at distant sites.
PETISCO is a novel protein complex required for 21U RNA biogenesis and embryonic viability
Authors: Cordeiro Rodrigues Ricardo J. , de Jesus Domingues António Miguel , Hellmann Svenja , Dietz Sabrina , de Albuquerque Bruno F. M. , Renz Christian , Ulrich Helle D. , Butter Falk , Ketting René F.
Published: November 2018

Piwi proteins are important for germ cell development in almost all animals studied thus far. These proteins are guided to specific targets, such as transposable elements, by small guide RNAs, often referred to as piRNAs, or 21U RNAs in C. elegans. In this organism, even though genetic screens have uncovered a number of potential 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1, we here define a novel protein complex, PETISCO, that is required for 21U RNA biogenesis. PETISCO contains both potential 5’-cap and 5’-phosphate RNA binding domains, suggesting involvement in 5’ end processing. We define the interaction architecture of PETISCO and reveal a second function for PETISCO in embryonic development. This essential function of PETISCO is not mediated by PID-1, but by TOST-1. Vice versa, TOST-1 is not involved in 21U RNA biogenesis. Both PID-1 and TOST-1 are small, intrinsically disordered proteins that interact directly with the PETISCO protein ERH-2 (enhancer of rudimentary homolog 2) using a conserved sequence motif. Finally, our data suggest an important role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes the first molecular platform for 21U RNA production in C. elegans, and strengthens the view that 21U RNA biogenesis is built upon a much more widely used, snRNA-related pathway.
Inferring causal connectivity from pairwise recordings and optogenetics
Authors: Lepperød Mikkel Elle , Stöber Tristan , Hafting Torkel , Fyhn Marianne , Kording Konrad Paul
Published: November 2018

To understand the neural mechanisms underlying brain function, neuroscientists aim to quantify causal interactions between neurons, for instance by perturbing the activity of neuron A and measuring the effect on neuron B. Recently, manipulating neuron activity using light-sensitive opsins, optogenetics, has increased the specificity of neural perturbation. However, using widefield optogenetic interventions, multiple neurons are usually perturbed, producing a confound – any of the stimulated neurons can have affected the postsynaptic neuron making it challenging to discern which neurons produced the causal effect. Here, we show how such confounds produce large biases in interpretations. We explain how confounding can be reduced by combining instrumental variables (IV) and difference in differences (DiD) techniques from econometrics. Combined, these methods can estimate (causal) effective connectivity by exploiting the weak, approximately random signal resulting from the interaction between stimulation and the absolute refractory period of the neuron. In simulated neural networks, we find that estimates using ideas from IV and DiD outperform naïve techniques suggesting that methods from causal inference can be useful to disentangle neural interactions in the brain.
Structure of a bacterial ATP synthase
Authors: Guo Hui , Suzuki Toshiharu , Rubinstein John L.
Published: November 2018

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.
Increased association between Epstein-Barr virus EBNA2 from type 2 strains and the transcriptional repressor BS69 restricts B cell growth
Authors: Ponnusamy Rajesh , Khatri Ritika , Correia Paulo B. , Mancini Erika , Farrell Paul J. , West Michelle J.
Published: November 2018

Natural variation separates Epstein-Barr virus (EBV) into type 1 and type 2 strains. Type 2 EBV is less transformingin vitrodue to sequence differences in the EBV transcription factor EBNA2. This correlates with reduced activation of the EBV oncogene LMP1 and some cell genes. Transcriptional activation by type 1 EBNA2 can be suppressed through the binding of two PXLXP motifs in its transactivation domain (TAD) to the dimeric coiled-coil MYND domain (CC-MYND) of the BS69 repressor protein (ZMYND11). We identified a third conserved PXLXP motif in type 2 EBNA2. We found that type 2 EBNA2 peptides containing this motif bound BS69CC-MYNDefficiently and that the type 2 EBNA2TADbound an additional BS69CC-MYNDmolecule. Full-length type 2 EBNA2 also bound BS69 more efficiently in pull-down assays. Molecular weight analysis and low-resolution structures obtained using small-angle X-ray scattering showed that three BS69CC-MYNDdimers bound two molecules of type 2 EBNA2TAD, in line with the dimeric state of full-length EBNA2in vivo. Importantly, mutation of the third BS69 binding motif in type 2 EBNA2 improved B-cell growth maintenance. Our data indicate that increased association with BS69 restricts growth promotion by EBNA2 and may contribute to reduced B-cell transformation by type 2 EBV.

Author summary

Epstein-Barr virus (EBV) drives the development of many human cancers worldwide including specific types of lymphoma and carcinoma. EBV infects B lymphocytes and immortalises them, thus contributing to lymphoma development. The virus promotes B lymphocyte growth and survival by altering the level at which hundreds of genes are expressed. The EBV protein EBNA2 is known to activate many growth-promoting genes. Natural variation in the sequence of EBNA2 defines the two main EBV strains: type 1 and type 2. Type 2 strains immortalise B lymphocytes less efficiency and activate some growth genes poorly, although the mechanism of this difference is unclear. We now show that sequence variation in type 2 EBNA2 creates a third site of interaction for the repressor protein (BS69, ZMYND11). We have characterised the complex formed between type 2 EBNA2 and BS69 and show that three dimers of BS69 form a bridged complex with two molecules of type 2 EBNA2. We demonstrate that mutation of the additional BS69 interaction site in type 2 EBNA2 improves its growth-promoting function. Our results therefore provide a molecular explanation for the different B lymphocyte growth promoting activities of type 1 and type 2 EBV. This aids our understanding of immortalisation by EBV.