bluuebunny/crossref_metadata_2025 huggingface dataset:
μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
Authors: Milon Nicolas , Chantry-Darmon Céline , Satge Carine , Fustier Margaux-Alison , Cauet Stephane , Moreau Sandra , Callot Caroline , Bellec Arnaud , Gabrieli Tslil , Saïas Laure , Boutonnet Audrey , Ginot Frédéric , Bergès Hélène , Bancaud Aurélien
Published: July 2019

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.
S-Nitrosothiol Signaling Is involved in Regulating Hydrogen Peroxide Metabolism of Zinc-Stressed Arabidopsis
Authors: Kolbert Zs , Molnï¿½r ï¿½ , Olï¿½h D , Feigl G , Horvï¿½th E , Erdei L , ï¿½rdï¿½g A , Rudolf E , Barth T , Lindermayr C
Published: July 2019

Accumulation of heavy metals such as zinc (Zn) disturbs the metabolism of reactive oxygen (e.g. hydrogen peroxide, H2O2) and nitrogen species (e.g. nitric oxide, NO; S-nitrosoglutathione, GSNO) in plant cells; however, their signal interactions are not well understood. Therefore, this study examines the interplay between H2O2 metabolism and GSNO signaling in Arabidopsis. Comparing the Zn tolerance of the wild type (WT), GSNO reductase (GSNOR) overexpressor 35S::FLAG-GSNOR1 and GSNOR-deficient gsnor1-3, we observed relative Zn tolerance of gsnor1-3, which was not accompanied by altered Zn accumulation capacity. Moreover, in gsnor1-3 plants Zn did not induce NO/S-nitrosothiol (SNO) signaling, possibly due to the enhanced activity of NADPH-dependent thioredoxin reductase. In WT and 35S::FLAG-GSNOR1, GSNOR was inactivated by Zn, and Zn-induced H2O2 is directly involved in the GSNOR activity loss. In WT seedlings, Zn resulted in a slight intensification of protein nitration detected by Western blot and protein S-nitrosation observed by resin-assisted capture of SNO proteins (RSNO-RAC). LC-MS/MS analyses indicate that Zn induces the S-nitrosation of ascorbate peroxidase 1. Our data collectively show that Zn-induced H2O2 may influence its own level, which involves GSNOR inactivation-triggered SNO signaling. These data provide new evidence for the interplay between H2O2 and SNO signaling in Arabidopsis plants affected by metal stress.
MaMYB4 Recruits Histone Deacetylase MaHDA2 and Modulates the Expression of ω-3 Fatty Acid Desaturase Genes during Cold Stress Response in Banana Fruit
Authors: Song Chunbo , Yang Yingying , Yang Tianwei , Ba Liangjie , Zhang Hui , Han Yanchao , Xiao Yunyi , Shan Wei , Kuang Jianfei , Chen Jianye , Lu Wangjin
Published: July 2019

Linoleic acid (LA; C18:2) and α-linolenic acid (ALA; C18:3) are two essential unsaturated fatty acids that play indispensable roles in maintaining membrane integrity in cold stress, and ω-3 fatty acid desaturases (FADs) are responsible for the transformation of LA into ALA. However, how this process is regulated at transcriptional and posttranscriptional levels remains largely unknown. In this study, an MYB transcription factor, MaMYB4, of a banana fruit was identified and found to target several ω-3 MaFADs, including MaFAD3-1, MaFAD3-3, MaFAD3-4 and MaFAD3-7, and repress their transcription. Intriguingly, the acetylation levels of histones H3 and H4 in the promoters of ω-3 MaFADs were elevated in response to cold stress, which was correlated with the enhancement in the transcription levels of ω-3 MaFADs and the ratio of ALA/LA. Moreover, a histone deacetylase MaHDA2 physically interacted with MaMYB4, thereby leading to the enhanced MaMYB4-mediated transcriptional repression of ω-3 MaFADs. Collectively, these data demonstrate that MaMYB4 might recruit MaHDA2 to repress the transcription of ω-3 MaFADs by affecting their acetylation levels, thus modulating fatty acid biosynthesis. Our findings provided new molecular insights into the regulatory mechanisms of fatty acid biosynthesis in cold stress in fruits.
Asymptotic Analysis of Frictional Contact Problem for Piezoelectric Shallow Shell
Authors: Mezabia M E , Ghezal A , Chacha D A
Published: July 2019

The objective of this work is to study the asymptotic justification of a new two-dimensional model for the equilibrium state of a piezoelectric linear shallow shell in frictional contact with a rigid foundation. More precisely, we consider the Signorini problem with Tresca friction of a piezoelectric linear shallow shell in contact with a rigid foundation. Then, we establish the convergence of the mechanical displacement and the electric potential as the thickness of the shallow shell goes to zero.
A rapid diagnostic multiplex PCR approach for xenomonitoring of human and animal schistosomiasis in a ‘One Health’ context
Authors: Schols Ruben , Carolus Hans , Hammoud Cyril , Mulero Stephen , Mudavanhu Aspire , Huyse Tine
Published: July 2019

Studying the epidemiology of schistosomiasis—the most prevalent gastropod-borne human disease and an economic burden for the livestock industry—relies on adequate monitoring tools. Here we describe a molecular assay for detecting human and animal African schistosome species in their planorbid gastropod host (xenomonitoring) using a two-step approach. First, schistosome infections are detected and discriminated from other trematode infections using a multiplex polymerase chain reaction (PCR) that includes a trematode-specific marker (in 18S rDNA), a Schistosoma genus-specific marker (in internal transcribed spacer 2 [ITS2]) and a general gastropod marker (in 18S rDNA) as an internal control. Upon Schistosoma sp. detection, a second multiplex PCR is performed to discriminate among Schistosoma haematobium, Schistosoma mansoni, Schistosoma mattheei and Schistosoma bovis/Schistosoma curassoni/Schistosoma guineensis using markers of differential lengths in the cytochrome c oxidase subunit 1 (COX1) gene. The specificity of these assays was validated with adult worms, naturally infected gastropods and human urine and stool samples. Sensitivity was tested on experimentally infected snail specimens that were sacrificed 10 and 40 days post-infection in order to mimic a natural prepatent and mature infection, respectively. The assay provides a diagnostic tool to support the xenomonitoring of planorbid gastropods for trematode infections in a One Health context, with a focus on the transmission monitoring of schistosomiasis.
A discussion on technologies for improving the operational speed of high-speed railway networks
Authors: Lu Chun-fang
Published: July 2019

China’s high-speed railway network has already achieved speeds of 350 km/h; however, this could be further increased to 400 km/h. After considering the development status and technical level of the high-speed railway system in China, this study indicates that there are four key technologies involved in improving its operational speed: the track, the electrical moving unit, the control system and the traction power supply. Through an experimental analysis, an evaluation index for the high-speed railway is then constructed based on four aspects: safety, comfort, intelligence and environmental protection. Using this system, the rationality of the high-speed railway speed-improvement plan can be scientifically evaluated. The results are of practical significance to the Chinese railway administration, as they can be used to formulate specific plans to increase rail speeds, and therefore promote the rapid development of the high-speed railway network in China.
Variation in Streptomycin Resistance Mechanisms in Clavibacter michiganensis
Authors: Lyu Qingyang , Bai Kaihong , Kan Yumin , Jiang Na , Thapa Shree P. , Coaker Gitta , Li Jianqiang , Luo Laixin
Published: July 2019

Clavibacter michiganensis is the causal agent of bacterial canker of tomato, which causes significant economic losses because of the lack of resistant tomato varieties. Chemical control with streptomycin or cupric bactericides is the last defensive line in canker disease management. Streptomycin is an aminoglycoside antibiotic that inhibits protein synthesis and targets the 30S ribosomal protein RpsL. Streptomycin has been used to control multiple plant bacterial diseases. However, identification and characterization of streptomycin resistance in C. michiganensis have remained unexplored. In this study, a naturally occurring C. michiganensis strain TX-0702 exhibiting spontaneous streptomycin resistance was identified, with a minimum inhibitory concentration of 128 μg/ml. Additionally, an induced streptomycin-resistant strain BT-0505-R was generated by experimental evolution of the sensitive C. michiganensis strain BT-0505. Genome sequencing and functional analyses were used to identify the genes conferring resistance. A point mutation at the 128th nucleotide in the rpsL gene of strain BT-0505-R is responsible for conferring streptomycin resistance. However, in TX-0702, resistance is not attributed to mutation of rpsL, streptomycin inactivation enzymes, or multidrug efflux pumps. The mechanism of resistance in TX-0702 is independent of previously reported bacterial loci. Taken together, these data highlight diverse mechanisms used by a Gram-positive plant pathogenic bacterium to confer antibiotic resistance.
Research transparency promotion by surgical journals publishing randomised controlled trials: a survey
Authors: Lombard N. , Gasmi A. , Sulpice L. , Boudjema K. , Naudet F. , Bergeat D.
Published: July 2019

Objective

To describe the surgical journal position statement on data-sharing policies (primary objective) and to describe the other features of their research transparency promotion.

Methods

Only “SURGICAL” journals with an impact factor superior to 2 (Web of Science) were eligible for the study. They were not included if there were no explicit instructions for clinical trial publication in the instructions for authors and if there were no RCT published between January 2016 and January 2019. The primary outcome was the existence of a data-sharing policy in the instructions for authors. Details on research transparency promotion were also collected, namely the existence of a “prospective registration of clinical trials requirement” policy; a “COIs” disclosure requirement and a specific reference to reporting guidelines such as CONSORT for RCT.

Results

Among the 87 surgical journals eligible, 82 (94%) were included in the analysis: 67 (77%) had explicit instructions for RCT and of the remaining, 15 (17.2%) had published at least one RCT between 2016-2019. The median impact factor was 2.98 [IQR=2.48-3.77] and in 2016 and 2017, the journals published a median of 11.5 RCT [IQR=5-20.75]. Data-sharing statement instructions (primary outcome) were ICMJE-compliant in four cases (4.88%), weaker in 45.12% (n=37) and inexistent in 50% (n=41) of the journals. As for data-sharing statements, no association was found between journal characteristics and the existence of data-sharing policies (ICMJE-compliant or weaker). A “prospective registration of clinical trials requirement” was associated with ICMJE allusion or affiliation and higher impact factors. Journals with specific RCT instructions in their OIA and journals referenced on the ICMJE website more frequently mandated the use of CONSORT guidelines.

Conclusion

Research transparency promotion is still limited in surgical journals. Uniformization of journal requirements vis-à-vis ICMJE guidelines could be a first step forward for research transparency promotion in surgery.
ChromSCape : a Shiny/R application for interactive analysis of single-cell chromatin profiles
Authors: Prompsy Pacôme , Kirchmeier Pia , Vallot Céline
Published: July 2019

Assessing chromatin profiles at single-cell resolution is now feasible thanks to recently published experimental methods such as single cell chromatin immunoprecipitation followed by sequencing (scChIP-seq) (Grosselin et al., 2019; Rotem et al., 2015) and single-cell assay for transposase-accessibility chromatin (scATAC-seq) (Buenrostro et al., 2015; Chen et al., 2018; Cusanovich et al., 2015; Lareau et al., 2019). With these methods, we can detect the heterogeneity of epigenomic profiles within complex biological samples. Yet, existing tools used to analyze bulk epigenomic experiments are not fit for the low coverage and sparsity of single-cell epigenomic datasets. Here, we present ChromSCape: a user-friendly Shiny/R application that processes single-cell epigenomic data to help the biological interpretation of epigenomic landscapes within cell populations. The user can identify different sub-populations within heterogeneous samples, find differentially enriched regions between subpopulations and identify associated genes and pathways. ChromSCape accepts multiple samples to allow comparisons of cell populations between and within samples. ChromSCape source code is written in Shiny/R, works as a stand-alone application and is freely downloadable athttps://github.com/vallotlab/ChromSCape. Here, using ChromSCape on multiple H3K27me3 scChIP-seq datasets, we deconvolve chromatin landscapes within the tumor microenvironment, identifying distinct H3K27me3 landscapes associated to cell identity and tumor subtype.

Contact

pacome.prompsy@curie.fr;celine.vallot@curie.fr
Performance of Complex Visual Tasks using Simulated Prosthetic Vision via Augmented-Reality Glasses
Authors: Ho Elton , Boffa Jack , Palanker Daniel
Published: July 2019

Purpose

Photovoltaic subretinal prosthesis is designed for restoration of central vision in patients with age-related macular degeneration (AMD). We investigated the utility of prosthetic central vision for complex visual tasks using augmented-reality (AR) glasses simulating reduced acuity, contrast and visual field.

Methods

AR glasses with blocked central 20° of visual field included an integrated video camera and software which adjusts the image quality according to three user-defined parameters: resolution, corresponding to the equivalent pixel size of an implant, field of view, corresponding to the implant size, and number of contrast levels. The real-time processed video was streamed on a screen in front of the right eye. Nineteen healthy participants were recruited to complete visual tasks including vision charts, sentence reading, and face recognition.

Results

With vision charts, letter acuity exceeded the pixel-sampling limit by 0.2 logMAR. Reading speed decreased with increasing pixel size and with reduced field of view (7-12°). In the face recognition task (4-way forced choice, 5° angular size) participants identified faces at >75% accuracy, even with 100 μm pixels and only 2 grey levels. With 60 μm pixels and 8 grey levels, the accuracy exceeded 97%.

Conclusions

Subjects with simulated prosthetic vision performed slightly better than the sampling limit on the letter acuity tasks, and were highly accurate at recognizing faces, even with 100 μm/pixel resolution. These results indicate feasibility of the reading and face recognition using prosthetic central vision even with 100 μm pixels, and performance improves further with smaller pixels.
Sex Differences in Predictors of Seizure in Contrast-Enhancing Gliomas at Clinical Presentation: A Network Approach
Authors: Johnston Sandra K , Khurana Aditya , Whitmire Paula , Ranjbar Sara , Sharma Akanksha , Hawkins-Daarud Andrea , Rubin Joshua B , Porter Alyx B , Canoll Peter D , Egan Kathleen , Hu Leland S , Mrugala Maciej M , Kumthekar Priya , Swanson Kristin R
Published: July 2019

Background

Brain tumor related epilepsy (BTE) is a major co-morbidity related to the management of patients with brain cancer. Despite published practice guidelines recommending against anti-epileptic drug (AED) utilization in patients with gliomas, there is heterogeneity in prescription practices of AEDs in these patients. In an attempt to impact BTE management, we statistically analyzed clinically relevant attributes (sex, age, tumor size, tumor growth kinetics, and tumor location) pertaining to seizure at presentation and used them to build a computational machine learning model to predict the probability of a seizure (at presentation).

Methods

From our clinical data repository, we identified 223 patients (females, n=86; males, n=137) with pathologically-determined glioma and known seizure status at clinical presentation. Non-parametric and Fisher’s Exact tests were used to identify statistical differences in clinical characteristics. We utilized a random forest machine learning method for generating our predictive models by entire cohort and separated by male and female.

Findings

Patients were divided into those that presented with seizure (SP, n=96, 43%; F, n= 28; M, n= 68) and those that presented without seizure (nSP, n=127, 57%, F n=58, M n=69). Females presented with seizures significantly less often than males (x2=6·28, p=0·01). SP patients had significantly smaller T1Gd radius compared to nSP (SP 11·30mm, nSP 18.66mm, p<0·0001). Tumor size and patient age were significant negative predictors for SP; patients with larger tumors, older age and less tumor diffusivity (p/D) were at lower risk for SP.

Interpretation

Despite heterogeneity across our patient cohort, there is strong evidence of a role for patient sex, tumor size, tumor invasion, and patient age in predicting the incidence of seizures at diagnosis. Future studies, with prospectively detailed data collection, may provide clearer insights into the incidence of seizures through a patient’s treatment course.
Diffusive Flux Analysis of Tumor Vascular Permeability for 3-Helix-Micelles in Comparison to Other Nanoparticles
Authors: Lim Marc , Dharmaraj Vishnu , Gong Boying , Xu Ting
Published: July 2019

Understanding the complex interplay of factors affecting nanoparticle accumulation in solid tumors is a challenge that must be surmounted to develop effective cancer nanomedicine. The tumor microenvironment is unique in comparison to healthy tissue, possessing elevated interstitial pressure that limits convective transport, hence leaving diffusive processes to predominate especially in the tumor’s necrotic core. Certain tumor types such as glioblastoma multiforme and pancreatic tumor are known to be poorly permeable, making them less accessible for nanoparticle drug delivery. Evidence indicates that small and long-circulating nanoparticles are ideal for taking advantage of the enhanced permeability and retention effect, even in such intractable tumor models. Three-helix-micelle (3HM) self-assembled nanoparticles possess these characteristics, and previous studies have shown that 3HM can achieve more favorable tumor accumulation and penetration than liposomes in several tumor models. The reason for its superior performance had yet to be determined, and thus we sought to examine its passive transport into tumors. In this paper we present a simple mathematical model based on diffusive flux to describe particle accumulation in tumors with respect to particle plasma pharmacokinetics. Fitting the diffusive flux equation to in vivo particle tumor concentration yields the particle effective permeability value, which for 3HM is 2.31 ± 0.18 x 10−8 cm/s in U87MG glioblastoma and 4.25 ± 0.91 x 10−8 cm/s in HT29 colon cancer murine models. Applying this diffusive flux model to other nanoparticles reported in literature enables the effect of plasma half-life to be decoupled from particle permeability in influencing tumor accumulation, reinforced trends reported in the field regarding the impact of particle size, and provided a semi-quantitative means of comparing various tumor models. This work is also the first demonstration, to the best our knowledge, of extracting particle permeability values from bulk biodistribution data obtained via positron emission tomography, as opposed to laborious tumor optical window intravital microscopy experiments. As such the analysis provided here presents a simple and accessible tool to further enhance nanomedicine development.

Abstract Figure

Graphical abstract
Description of Klebsiella indica sp. nov., isolated from the surface of tomato
Authors: Gujarati Sukriti , Chaudhari Diptaraj , Khairnar Mitesh , Shouche Yogesh , Rahi Praveen
Published: July 2019

A novel bacterial strain designated TOUT106T was isolated from the surface of a tomato collected from the local vegetable market in Pune, India. The cells were rod shaped, Gram-stain-negative, encapsulated and non-motile. The strain TOUT106T grows as mucoid and translucent colonies on blood agar medium and the best growth was observed at 28°C and at pH 7.0, and could tolerate up to 2% (w/v) NaCl. On the basis of 16S rRNA gene sequence analysis, strain TOUT106T was placed under Salmonella clade, with close similarity to Salmonella enterica subsp. arizonae strain NCTC 8297T (98.42%). Genome-based phylogenetic analysis revealed that the strain forms a distinct branch within the Klebsiella clade and K. michiganensis DSM25444T and K. oxytoca NBRC105695T were the closest neighbor. The genomic DNA G+C content of strain TOUT106T was 53.53 mol%. The average nucleotide identity of TOUT106T was less 86.4% with closely related members of the family Enterobacteriaceae. The major fatty acids of strain TOUT106T were C16:0, C17:0 cyclo, C14:0 3OH/C16:1 iso, C14:0, C19:0cyclow8c, C18:1 w6c/C18:1 w7c, C12:0 and C16:1 w7c/C16:1 w6c. The strain TOUT106T showed differences in physiological, phenotypic and protein profiles by MALDI-TOF MS to its closest relatives. Based on the phenotypic including chemotaxonomic properties and phylogenetic analysis the strain TOUT106T could be distinguished from the recognized species of the genus Klebsiella, was suggested to represent a novel species of this genus, for which the name Klebsiella indica sp. nov. is proposed. The type strain is TOUT106T (=MCC 2901T).
A method for the unbiased quantification of reassortment in segmented viruses
Authors: Hockman Megan R. , Phipps Kara , Lowen Anice C.
Published: July 2019

The diversification of segmented viruses via reassortment is important to understand due to the contributions of reassortment to viral evolution and emergence. Methods for the quantification of reassortment have been described, but are often cumbersome and best suited for the analysis of reassortment between highly divergent parental strains. While it is useful to understand the potential of divergent parents to reassort, outcomes of such heterologous reassortment are driven by differential selection acting on the progeny and are typically strain specific. To quantify reassortment, a system free of differential selection is needed. We have generated such a system for influenza A virus and for mammalian orthoreovirus by constructing well-matched parental viruses carrying small genetic tags. The method utilizes high-resolution melt technology for the identification of reassortant viruses. Ease of sample preparation and data analysis enables streamlined genotyping of a large number of virus clones. The method described here thereby allows quantification of the efficiency of unbiased reassortment and can be applied to diverse segmented viruses.

Highlights

Genetic tagging of viruses can be achieved without altering fitness

High-resolution melt can detect single nucleotide differences in viruses

Unbiased reassortment of influenza A virus and mammalian orthoreovirus can be quantified
Dopamine modulates dynamic decision-making during foraging
Authors: Le Heron Campbell , Kolling Nils , Plant Olivia , Kienast Annika , Janska Rebecca , Ang Yuen-Siang , Fallon Sean , Husain Masud , Apps Matthew A J
Published: July 2019

The mesolimbic dopaminergic system exerts a crucial influence on incentive processing. However, the contribution of dopamine in dynamic, ecological situations where reward rates vary, and decisions evolve over time, remains unclear. In such circumstances, current (foreground) reward accrual needs to be compared continuously with potential rewards that could be obtained by travelling elsewhere (background reward rate), in order to determine the opportunity cost of staying versus leaving. We hypothesised that dopamine specifically modulates the influence of background – but not foreground – reward information when making a dynamic comparison of these variables for optimal behaviour. On a novel foraging task based on an ecological account of animal behaviour (marginal value theorem), human participants were required to decide when to leave locations in situations where foreground rewards depleted at different rates, either in rich or poor environments with high or low background rates. In line with theoretical accounts, people’s decisions to move from current locations were independently modulated by both foreground and background reward rates. Pharmacological manipulation of dopamine D2 receptor activity using the agonist cabergoline significantly affected decisions to move on, specifically modulating the effect of background but not foreground rewards rates. In particular, when on cabergoline, people left patches in poor environments much earlier. These results demonstrate a role of dopamine in signalling the opportunity cost of rewards, not value per se. Using this ecologically derived framework we uncover a specific mechanism by which D2 dopamine receptor activity modulates decision-making when foreground and background reward rates are dynamically compared.

Significance statement

Many decisions, across economic, political and social spheres, involve choices to “leave”. Such decisions depend on a continuous comparison of a current location’s value, with that of other locations you could move on to. However, how the brain makes such decisions is poorly understood. Here, we developed a computerized task, based around theories of how animals make decisions to move on when foraging for food. Healthy human participants had to decide when to leave collecting financial rewards in a location, and travel to collect rewards elsewhere. Using a pharmacological manipulation, we show that the activity of dopamine in the brain modulates decisions to move on, with people valuing other locations differently depending on their dopaminergic state.
Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing
Authors: Arpat Alaaddin Bulak , Liechti Angélica , Matos Mara De , Dreos René , Janich Peggy , Gatfield David
Published: July 2019

Translation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of non-uniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites, independent of translation rate, is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs and nascent polypeptide structure, is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.
CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev
Authors: García-de-Gracia Francisco , Toro-Ascuy Daniela , Riquelme-Barrios Sebastián , Pereira-Montecinos Camila , Rojas-Araya Bárbara , Gaete-Argel Aracelly , Acevedo Mónica L. , Chnaiderman Jonás , Valiente-Echeverría Fernando , Soto-Rifo Ricardo
Published: July 2019

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) unspliced mRNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the unspliced mRNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF restricts HIV-1 replication by interfering with Gag synthesis from the unspliced mRNA. Our results indicate that CTIF associates with Rev through its N-terminal domain and is recruited onto the unspliced mRNA ribonucleoprotein complex in order to block translation. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that CTIF restricts HIV-2 but not MLV Gag synthesis indicating an inhibitory mechanism conserved in Rev-expressing human lentiviruses.
Global genome diversity of theLeishmania donovanicomplex
Authors: Franssen Susanne U. , Durrant Caroline , Stark Olivia , Moser Bettina , Downing Tim , Imamura Hideo , Dujardin Jean-Claude , Sanders Mandy , Mauricio Isabel , Miles Michael A. , Schnur Lionel F. , Jaffe Charles L. , Nasereddin Abdelmajeed , Schallig Henk , Yeo Matthew , Bhattacharyya Tapan , Alam Mohammad Z. , Berriman Matthew , Wirth Thierry , Schönian Gabriele , Cotton James A.
Published: July 2019

Protozoan parasites of theLeishmania donovanicomplex –L. donovaniandL. infantum– cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution.L. donovaniisolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority ofL. infantumsamples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of theLeishmania donovanicomplex.
Progressive shifts in the gut microbiome reflect prediabetes and diabetes development in a treatment-naive Mexican cohort
Authors: Diener Christian , Reyes Lourdes , Jimenez Lilia , Matus Mariana , Gomez Claudia , Chu Nathaniel D. , Zhong Vivian , Tejero Elizabeth , Alm Eric , Resendis-Antonio Osbaldo , Guardado-Mendoza Rodolfo
Published: July 2019

Type 2 diabetes (T2D) is a global epidemic that affects more than 8% of the world’s population and is a leading cause of death in Mexico. Diet and lifestyle are known to contribute to the onset of T2D. However, the role of the gut microbiome in T2D progression remains uncertain. Associations between microbiome composition and diabetes are confounded by medication use, diet, and obesity. Here we present data on a treatment-naive cohort of 405 Mexican individuals across varying stages of T2D severity. Associations between gut bacteria and more than 200 clinical variables revealed a defined set of bacterial genera that were consistent biomarkers of T2D prevalence and risk. Specifically, gradual increases in blood glucose levels, beta cell dysfunction, and the accumulation of measured T2D risk factors was correlated with the relative abundances of four bacterial genera. In a cohort of 25 individuals, T2D treatment - predominantly metformin - reliably returned the microbiome to the normoglycemic community state. Deep clinical characterization allowed us to broadly control for confounding variables, indicating that these microbiome patterns were independent of common T2D comorbidities, like obesity or cardiovascular disease. Thus, our work provides the first solid evidence for a direct link between the gut microbiome and T2D in a critically high-risk population. Whether or not these T2D-associated changes in the gut contribute to the etiology of T2D or its comorbidities remains to be seen.
Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene
Authors: Aparicio Tomas , Nyerges Akos , Nagy István , Pal Csaba , Martínez-García Esteban , de Lorenzo Víctor
Published: July 2019

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. On this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast’s URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G< C:C< G:A< C:A, A:A, G:G, T:T, T:G, A:C, C:T< G:T, T:C as the one prevalent in Pseudomonas putida. This information was vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.

Originality-Significance Statement

Single-stranded DNA (ssDNA) recombineering has emerged in recent years as one of the most powerful technologies of genome editing in E. coli and other Enterobacteria. However, the efforts to expand the concept and the methods towards environmental microorganisms such as Pseudomonas putida have been limited thus far by several gaps in our fundamental knowledge of how nucleotide mismatch repair (MMR) operates in such non-model species. One critical bottleneck is the hierarchy of recognition of different types of base mispairings as well as the need of setting up strategies for counteracting MMR and thus enabling tolerance to all types of changes. The work presented here tackles both issues and makes P. putida amenable to sophisticated genetic manipulations that were impossible before.
In Vivo Large Scale Mapping Of Protein Turnover In The Human Cerebrospinal Fluid
Authors: Lehmann Sylvain , Hirtz Christophe , Vialaret Jérôme , Ory Maxence , Combes Guillaume Gras , Le Corre Marine , Badiou Stéphanie , Cristol Jean-Paul , Hanon Olivier , Cornillot Emmanuel , Bauchet Luc , Gabelle Audrey , Colinge Jacques
Published: July 2019

The extraction of accurate physiological parameters from clinical samples provides a unique perspective to understand disease etiology and evolution, including under therapy. We introduce a new proteomics framework to map patient proteome dynamics in vivo, either proteome wide or in large targeted panels. We applied it to ventricular cerebrospinal fluid (CSF) and could determine the turnover parameters of almost 200 proteins, whereas a handful were known previously. We covered a large number of neuron biology- and immune system-related proteins including many biomarkers and drug targets. This first large data set unraveled a significant relationship between turnover and protein origin that relates to our ability to investigate the central nervous system physiology precisely in future studies. Our data constitute a reference in CSF biology as well as a repertoire of peptides for the community to design new proteome dynamics analyses. The disclosed methods apply to other fluids or tissues provided sequential sample collection can be performed.
Origination of the circadian clock system in stem cells regulates cell differentiation
Authors: Torii Kotaro , Inoue Keisuke , Bekki Keita , Haraguchi Kazuya , Kubo Minoru , Kondo Yuki , Suzuki Takamasa , Shimizu Hanako , Uemoto Kyohei , Saito Masato , Fukuda Hiroo , Araki Takashi , Endo Motomu
Published: July 2019

The circadian clock regulates various physiological responses. To achieve this, both animals and plants have distinct circadian clocks in each tissue that are optimized for that tissue’s respective functions. However, if and how the tissue-specific circadian clocks are involved in specification of cell types remains unclear. Here, by implementing a single-cell transcriptome with a new analytics pipeline, we have reconstructed an actual time-series of the cell differentiation process at single-cell resolution, and discovered that the Arabidopsis circadian clock is involved in the process of cell differentiation through transcription factor BRI1-EMS SUPPRESSOR 1 (BES1) signaling. In this pathway, direct repression of LATE ELONGATED HYPOCOTYL (LHY) expression by BES1 triggers reconstruction of the circadian clock in stem cells. The reconstructed circadian clock regulates cell differentiation through fine-tuning of key factors for epigenetic modification, cell-fate determination, and the cell cycle. Thus, the establishment of circadian systems precedes cell differentiation and specifies cell types.
Altered stomatal patterning accompanies a trichome dimorphism in a natural population ofArabidopsis
Authors: Simon Noriane M. L. , Sugisaka Jiro , Honjo Mie N. , Tunstad Sverre Aarseth , Tunna George , Kudoh Hiroshi , Dodd Antony N.
Published: July 2019

Trichomes are large epidermal cells on the surface of leaves that are thought to deter herbivores, yet the presence of trichomes can also negatively impact plant growth and reproduction. Stomatal guard cells and trichomes have shared developmental origins, and experimental manipulation of trichome formation can lead to changes in stomatal density. The influence of trichome formation upon stomatal development in natural populations of plants is currently unknown. Here, we show that a natural population ofArabidopsis hallerithat includes hairy (trichome-bearing) and glabrous (no trichomes) morphs has differences in stomatal density that are associated with this trichome dimorphism. We found that glabrous morphs had significantly greater stomatal density and stomatal index than hairy morphs. One interpretation is that this arises from a trade-off between the proportions of cells that have trichome and guard cell fates during leaf development. The differences in stomatal density between the two morphs might have impacts upon environmental adaptation, in addition to herbivory deterrence caused by trichome development.
Application of high-resolution landmark-free morphometrics to a mouse model of Down Syndrome reveals a tightly localised cranial phenotype
Authors: Toussaint Nicolas , Redhead Yushi , Liu Wei , Fisher Elizabeth M. C. , Hallgrimsson Benedikt , Tybulewicz Victor L.J. , Schnabel Julia A. , Green Jeremy B.A.
Published: July 2019

Characterising phenotypes often requires quantification of anatomical shapes. Quantitative shape comparison (morphometrics) traditionally uses anatomical landmarks and is therefore limited by the number of landmarks and operator accuracy when landmarks are located manually. Here we apply a landmark-free method to characterise the craniofacial skeletal phenotype of the Dp1Tyb mouse model of Down syndrome (DS), validating it against a landmark-based approach. We identify cranial dysmorphologies in Dp1Tyb mice, especially smaller size and brachycephaly (front-back shortening) homologous to the human phenotype. The landmark-free phenotyping was less labour-intensive and required less user training than the landmark-based method. It also enabled mapping of local differences as planar expansion or shrinkage. This higher resolution and local mapping pinpointed reductions in interior mid-snout structures and occipital bones in this DS model that were not as apparent using a traditional landmark-based method. This approach could make morphometrics widely-accessible beyond traditional niches in zoology and palaeontology, especially in characterising mutant phenotypes.
The apical annuli ofToxoplasma gondiiare composed of coiled-coil and signaling proteins embedded in the IMC sutures
Authors: Engelberg Klemens , Chen Chun-Ti , Bechtel Tyler , Guzmán Victoria Sánchez , Drozda Allison A. , Chavan Suyog , Weerapana Eranthie , Gubbels Marc-Jan
Published: July 2019

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasiteToxoplasma gondii. We mapped the proteome of the annuli inToxoplasmaby reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2 and a methyltransferase (AAMT). Moreover, Inner Membrane Complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy (SR-SIM) identified a concentric organization comprising four rings with diameters ranging from 200-400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggest that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed the AAPs are conserved narrowly in Coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signaling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitnessin vitrobut had no significant impact on acute virulencein vivo. In conclusion, the apical annuli are composed of coiled-coil and signaling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.
A comparison of resting state EEG and structural MRI for classifying Alzheimer’s disease and mild cognitive impairment
Authors: Farina FR , Emek-Savaş DD , Rueda-Delgado L , Boyle R , Kiiski H , Yener G , Whelan R
Published: July 2019

Alzheimer’s disease (AD) is a neurodegenerative disorder characterised by severe cognitive decline and loss of autonomy. AD is the leading cause of dementia. AD is preceded by mild cognitive impairment (MCI). By 2050, 68% of new dementia cases will occur in low- and middle-income countries. In the absence of objective biomarkers, psychological assessments are typically used to diagnose MCI and AD. However, these require specialist training and rely on subjective judgements. The need for low-cost, accessible and objective tools to aid AD and MCI diagnosis is therefore crucial. Electroencephalography (EEG) has potential as one such tool: it is relatively inexpensive (cf. magnetic resonance imaging; MRI) and is portable. In this study, we collected resting state EEG, structural MRI and rich neuropsychological data from older adults (55+ years) with AD, with MCI and from healthy controls (n~60 per group). Our goal was to evaluate the utility of EEG, relative to MRI, for the classification of MCI and AD. We also assessed the performance of combined EEG and behavioural (Mini-Mental State Examination; MMSE) and structural MRI classification models. Resting state EEG classified AD and HC participants with moderate accuracy (AROC=0.76), with lower accuracy when distinguishing MCI from HC participants (AROC=0.67). The addition of EEG data to MMSE scores had no additional value compared to MMSE alone. Structural MRI out-performed EEG (AD vs HC, AD vs MCI: AROCs=1.00; HC vs MCI: AROC=0.73). Resting state EEG does not appear to be a suitable tool for classifying AD. However, EEG classification accuracy was comparable to structural MRI when distinguishing MCI from healthy aging, although neither were sufficiently accurate to have clinical utility. This is the first direct comparison of EEG and MRI as classification tools in AD and MCI participants.
DEET and other repellents are inhibitors of mosquito odorant receptors for oviposition attractants
Authors: Xu Pingxi , Zeng Fangfang , Bedoukian Robert H. , Leal Walter S.
Published: July 2019

In addition to its primary function as an insect repellent, DEET has many “off-label” properties, including a deterrent effect on attraction of gravid female mosquitoes. DEET negatively affects oviposition sites. While deorphanizing odorant receptors (ORs) using the Xenopus oocyte recording system, we have previously observed that DEET generated outward (inhibitory) currents on ORs sensitive to oviposition attractants. Here, we systematically investigated these inhibitory currents. We recorded dose-dependent outward currents elicited by DEET and other repellents on ORs from Culex quinquefasciatus, Aedes aegypti, and Anopheles gambiae. Similar responses were observed with other plant-derived and plant-inspired compounds, including methyl jasmonate and methyl dihydrojasmolate. Inward (regular) currents elicited by skatole upon activation of CquiOR21 were modulated when this oviposition attractant was coapplied with a repellent. Compounds that generate outward currents in ORs sensitive to oviposition attractants elicited inward currents in a DEET-sensitive receptor, CquiOR136. The best ligand for this receptor, methyl dihydrojasmolate, showed repellency activity, but was not as strong as DEET in our test protocol.