bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative assessment of anti-gravity reflexes to evaluate vestibular dysfunction in rats
Authors: Martins-Lopes Vanessa , Bellmunt Anna , Greguske Erin A. , Maroto Alberto F. , Boadas-Vaello Pere , Llorens Jordi
Published: March 2019

The tail-lift reflex and the air-righting reflex are anti-gravity reflexes in rats that depend on vestibular function. To obtain objective and quantitative measures of performance, we recorded these reflexes with slow motion video in two experiments. In the first experiment, vestibular dysfunction was elicited by acute exposure to 0 (control), 400, 600 or 1000 mg/kg of 3,3’-iminodipropionitrile (IDPN), which causes dose-dependent hair cell degeneration. In the second, rats were exposed to sub-chronic IDPN in the drinking water for 0 (control), 4 or 8 weeks; this causes reversible or irreversible loss of vestibular function depending on exposure time. In the tail-lift test, we obtained the minimum angle defined during the lift and descent maneuver by the nose, the back of the neck and the base of the tail. In the air-righting test, we obtained the time to right the head. We also obtained Vestibular Dysfunction Ratings (VDRs) using a previously validated behavioral test battery. Each measure, VDR, tail-lift angle and air-righting time, demonstrated dose-dependent loss of vestibular function after acute IDPN, and time-dependent loss of vestibular function after sub-chronic IDPN. All measures showed high correlations between each other, and maximal correlation coefficients were found between VDRs and tail-lift angles. In scanning electron microscopy evaluation of the vestibular sensory epithelia, the utricle and the saccule showed diverse pathological outcomes, suggesting that they have a different role in these reflexes. We conclude that these anti-gravity reflexes provide useful objective and quantitative measures of vestibular function in rats that are open to further development.
The structure of an injectisome export gate demonstrates conservation of architecture in the core export gate between flagellar and virulence type three secretion systems
Authors: Johnson Steven , Kuhlen Lucas , Deme Justin C. , Abrusci Patrizia , Lea Susan M.
Published: March 2019

Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.
High-throughput Phenotyping with Temporal Sequences
Authors: Estiri Hossein , Strasser Zachary H , Murphy Shawn N.
Published: March 2019

Objective

High-throughput electronic phenotyping algorithms can accelerate translational research using data from electronic health record (EHR) systems. The temporal information buried in EHRs are often underutilized in developing computational phenotypic definitions. The objective of this study is to develop a high-throughput phenotyping method, leveraging temporal sequential patterns of discrete events from electronic health records.

Materials and Methods

We develop a representation mining algorithm to extract five classes of representations from EHR diagnosis and medication records: the aggregated vector of the records (AVR), the traditional immediate sequential patterns (SPM), the transitive sequential patterns (tSPM), as well as two hybrid classes of SPM+AVR and tSPM+AVR. A final small set of representations were selected from each class using the MSMR dimensionality reduction algorithm. Using EHR data on 10 phenotypes from Mass General Brigham Biobank, we trained regularized logistic regression algorithms, which we validated using labeled data.

Results

Phenotyping with temporal sequences resulted in a superior classification performance across all 10 phenotypes compared with the AVR representations that are conventionally used in electronic phenotyping. Although this study only utilizes the diagnosis and medication records, the high-throughput algorithm’s classification performance was superior or similar to the performance of previously published electronic phenotyping algorithms. We characterize and evaluate the top transitive sequences of diagnosis records paired with the records of risk factors, symptoms, complications, medications, or vaccinations.

Discussion

The proposed high-throughput phenotyping approach enables seamless discovery of sequential record combinations that may be difficult to assume from raw EHR data. A transitive sequence can offer a more accurate characterization of the phenotype, compared with its individual components. Additionally, the identified transitive sequences of a given phenotype reflect the actual lived experiences of the patients with that particular disease.

Conclusion

Sequential data representations provide a precise mechanism for incorporating raw EHR records into downstream Machine Learning.
NatB modulates Rb mutant cell death and tumor growth by regulating EGFR/MAPK signaling through the N-end rule pathways
Authors: Sheng Zhentao , Du Wei
Published: March 2019

Despite the prevalence of N-terminal acetylation (Nt-acetylation), little is known of its biological functions. In this study, we show that NatB regulates Rb mutant cell survival, EGFR/MAPK signaling activity, and EGFR signaling-dependent tumor growth. We identify Grb2/Drk, MAPK, and PP2AC as the key NatB targets of EGFR pathway. Surprisingly, NatB activity increases the levels of positive pathway components Grb2/Drk and MAPK while decreases the levels of negative pathway component PP2AC despite these proteins have the same first two amino acids that are recognized by NatB and N-end rule pathways. Mechanistically, we show that NatB regulates Grb2/Drk protein stability through its N-terminal sequences and that Grb2/Drk and MAPK are selectively degraded by the Arg/N-end rule E3 ubiquitin ligase Ubr4, which targets proteins with free N-terminus. In contrast, PP2AC is selectively degraded by the Ac/N-end rule pathway E3 ubiquitin ligase Cnot4 that targets proteins with acetylated N-terminus. These results reveal a novel mechanism by which NatB-mediated Nt-acetylation and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of positive and negative components. Since mutation or overexpression that deregulate the EGFR/Ras signaling pathway are common in human cancers and NatB subunits are significant unfavorable prognostic markers, this study can potentially lead to the development of novel therapeutic approaches.

Significance Statement

Nt-acetylation is often regarded as a constitutive, irreversible, and static modification that is not suited to serve regulatory functions. Our observation that Nt-acetylation by NatB coordinately regulate the levels of positive and negative components of the EGFR/MAPK pathway show that Nt-acetylation and N-end rule pathways can play important roles regulating important signaling pathways. As Acetyl-CoA level, which is influenced by cell metabolism, can be rate limiting for Nt-acetylation, our results also suggest a potentially new mechanism by which cellular metabolic status can regulate growth factor signaling.
Unique Trophoblast Chromatin Environment Mediated by the PcG Protein SFMBT2
Authors: Tang Priscilla , Miri Kamelia , Varmuza Susannah
Published: March 2019

Stem/progenitor cells are maintained by a chromatin environment, mediated in part by Polycomb group (PcG) proteins, that depresses differentiation. The trophoblast-specific PcG protein SFMBT2 is known to be required for maintenance of trophoblast progenitors. Rather than binding to trophoblast specific genes repressed in TSC, SFMBT2 is concentrated at chromocenters and regions rich in repetitive elements, specifically LINE sequences and major satellites, suggesting that it is involved in higher order organization of the trophoblast genome. It is also found enriched at a subset of ncRNAs. Comparison of ChIP-seq data sets for other chromatin proteins reveals several stereotypical distribution patterns, suggesting that SFMBT2 interacts with several different types of chromatin complexes specific to the trophoblast lineage.
Online sensory feedback during active search improves tactile localization
Authors: Fuchs Xaver , Wulff Dirk U. , Heed Tobias
Published: March 2019

Many natural behaviors involve closed feedback loops in which ongoing sensory input refines motor behavior. Previous research on tactile localization, however, has implemented localization as open-loop behavior. For instance, participants indicate a touched position on a silhouette shape of the body or on an occluding board mounted above the hand. Such studies have suggested that humans often make large errors when localizing touch on the skin, or that “perceptual body representations” are distorted. However, by artificially preventing tactile feedback from the target body area, the natural action-perception loop is interrupted. Therefore, these localization approaches may underestimate individuals’ localization ability and draw erroneous conclusions about the role and precision of body representations. Here, we tested tactile localization in a natural setting, in which participants first received brief touches on their left forearm and then searched for the target location by moving the right index finger across the skin. Tactile search reduced localization error when the searching finger was far from, but not when it was near the target, resulting in a remaining error of 1-2 cm. Error reduction was absent when participants searched on an acrylic barrier mounted above the arm, suggesting that availability of tactile feedback on the target arm but not proprioceptive and motor signals of the searching arm determined precision, thus confirming the pivotal role of closed-loop sensory feedback for tactile localization. We suggest that actively produced online tactile feedback routinely refines coarse spatial body representations, similar to the refinement of sparse spatial representations in visual memory through consecutive saccades.
Orchestrating Single-Cell Analysis with Bioconductor
Authors: Amezquita Robert A. , Carey Vince J. , Carpp Lindsay N. , Geistlinger Ludwig , Lun Aaron T. L. , Marini Federico , Rue-Albrecht Kevin , Risso Davide , Soneson Charlotte , Waldron Levi , Pagès Hervé , Smith Mike , Huber Wolfgang , Morgan Martin , Gottardo Raphael , Hicks Stephanie C.
Published: March 2019

Recent developments in experimental technologies such as single-cell RNA sequencing have enabled the profiling a high-dimensional number of genome-wide features in individual cells, inspiring the formation of large-scale data generation projects quantifying unprecedented levels of biological variation at the single-cell level. The data generated in such projects exhibits unique characteristics, including increased sparsity and scale, in terms of both the number of features and the number of samples. Due to these unique characteristics, specialized statistical methods are required along with fast and efficient software implementations in order to successfully derive biological insights. Bioconductor - an open-source, open-development software project based on the R programming language - has pioneered the analysis of such high-throughput, high-dimensional biological data, leveraging a rich history of software and methods development that has spanned the era of sequencing. Featuring state-of-the-art computational methods, standardized data infrastructure, and interactive data visualization tools that are all easily accessible as software packages, Bioconductor has made it possible for a diverse audience to analyze data derived from cutting-edge single-cell assays. Here, we present an overview of single-cell RNA sequencing analysis for prospective users and contributors, highlighting the contributions towards this effort made by Bioconductor.
A triple-hybrid cross reveals a new hybrid incompatibility locus betweenD. melanogaster and D. sechellia
Authors: Cooper Jacob C. , Guo Ping , Bladen Jackson , Phadnis Nitin
Published: March 2019

Hybrid incompatibilities are the result of deleterious interactions between diverged genes in the progeny of two species. InDrosophila, crosses between femaleD. melanogasterand males from theD. simulansclade (D. simulans,D. mauritiana,D. sechellia) fail to produce hybrid F1 males. When attempting to rescue hybrid F1 males by depleting the incompatible allele of a previously identified hybrid incompatibility gene, we observed robust rescue in crosses ofD. melanogastertoD. simulansorD. mauritiana, but no rescue in crosses toD. sechellia. To investigate the genetic basis ofD. sechelliaresistance to hybrid rescue, we designed a triple-hybrid cross to generate recombinantD. sechellia/D. simulansgenotypes. We tested the ability of those genotypes to rescue hybrid males withD. melanogaster, and used whole genome sequencing to measure theD. sechellia/D. simulansallele frequency of viable F1 males. We found that recombinant genotypes were rescued when they contained two specific loci fromD. simulans– a region containing previously identifiedLethal hybrid rescue (Lhr), and an unknown region of chromosome 3L which we nameSechellia aversion to hybrid rescue (Satyr). Our results show that the genetic basis for the recent evolution of this hybrid incompatibility is simple rather than a highly dispersed effect. Further, these data suggest that fixation of differences atLhrafter the split of theD. simulansclade strengthened the hybrid incompatibility betweenD. sechelliaandD. melanogaster.
A conserved acidic cluster motif in SERINC5 confers resistance to antagonism by HIV-1 Nef
Authors: Stoneham Charlotte A. , Ramirez Peter W. , Singh Rajendra , Suarez Marissa , Debray Andrew , Lim Christopher , Jia Xiaofei , Xiong Yong , Guatelli John
Published: March 2019

The cellular protein SERINC5 inhibits the infectivity of diverse retroviruses and is counteracted by the glycoGag protein of MLV, the S2 protein of EIAV, and the Nef protein of HIV-1. Determining regions within SERINC5 that provide restrictive activity or Nef-sensitivity should inform mechanistic models of the SERINC5/HIV-1 relationship. Here, we report that deletion of the highly conserved sequence EDTEE, which is located within a cytoplasmic loop of SERINC5 and is reminiscent of an acidic cluster membrane trafficking signal, increases the sensitivity of SERINC5 to antagonism by Nef while having no effect on the intrinsic activity of the protein as an inhibitor of infectivity. The effects on infectivity correlated with enhanced removal of the ΔEDTEE mutant relative to wild type SERINC5 from the cell surface and with enhanced exclusion of the mutant protein from virions by Nef. Mutational analysis revealed that the acidic residues, but not the threonine, within the EDTEE motif are important for the relative resistance to Nef. Deletion of the EDTEE sequence did not increase the sensitivity of SERINC5 to antagonism by the glycoGag protein of MLV, suggesting that its virologic role is Nef-specific. These results are consistent with the reported mapping of the cytoplasmic loop that contains the EDTEE sequence as a general determinant of Nef-responsiveness, but they further indicate that sequences inhibitory to as well as supportive of Nef-activity reside in this region. We speculate that the EDTEE motif might have evolved to mediate resistance against retroviruses that use Nef-like proteins to antagonize SERINC5.

Importance

Cellular membrane proteins in the SERINC family, especially SERINC5, inhibit the infectivity of retroviral virions. This inhibition is counteracted by retroviral proteins, specifically HIV-1 Nef, MLV glycoGag, and EIAV S2. One consequence of such a host-pathogen “arms race” is compensatory change in the host antiviral protein as it evolves to escape the effects of the viral antagonist. This is often reflected in a genetic signature, positive selection, which is conspicuously missing inSERINC5. Here we show that despite this lack of genetic evidence, a sequence in SERINC5 nonetheless provides relative resistance to antagonism by HIV-1 Nef.
CoBATCH for high-throughput single-cell epigenomic profiling
Authors: Wang Qianhao , Xiong Haiqing , Ai Shanshan , Yu Xianhong , Liu Yaxi , Zhang Jiejie , He Aibin
Published: March 2019

An efficient, generalizable method for genome-wide mapping of single-cell histone modifications or chromatin-binding proteins is so far lacking. Here we develop CoBATCH,combinatorialbarcodingandtargetedchromatin release, for single-cell profiling of genomic distribution of chromatin-binding proteins in cell culture and tissue. Protein A in fusion to Tn5 transposase is enriched through specific antibodies to genomic regions and Tn5 generates indexed chromatin fragments ready for the library preparation and sequencing. Importantly, through a combinatorial barcoding strategy, we are able to measure epigenomic features up to tens of thousands single cells per experiment. CoBATCH produces not only high signal-to-noise features, but also ~10,000 reads per cells, allowing for efficiently deciphering epigenetic heterogeneity of cell populations and subtypes and inferring developmental histories. Thus, obviating specialized device, CoBATCH is easily deployable for any laboratories in life science and medicine.
Integrating detection of copy neutral chromosomal losses in a clinical setting in leukemia and lymphoma by means of allelic imbalance and read depth ratio comparison
Authors: Hansen Marcus C. , Cédile Oriane , Ludvigsen Maja , Kjeldsen Eigil , Møller Peter L. , Abildgaard Niels , Hokland Peter , Nyvold Charlotte G.
Published: March 2019

Chromosomal aberrations are common features of hematological malignancies, with several recurrent aberrations recognized as important diagnostic or prognostic molecular markers. While genome-wide genotyping and genomic hybridization microarray analyses have been implemented in clinical laboratories for several years the usage of next generation sequencing for detection of acquired copy number alterations has not yet reached full clinical integration. One evident problem is the identification of copy neutral loss of heterozygosity (CN-LoH), which is not detectable by sequencing read depth correlation or the analogous microarray CGH. We selected 23 paired samples of hematological disorders from 14 individuals, focusing on leukemia and lymphomas, and tested whether a low complexity approach, relying on Fisher’s exact test or χ2, is efficient for the analysis of variant allele frequencies with read-depth ratio correlations in order to resolve both copy-altering and neutral chromosomal aberrations. This combination helped to identify 69 altered chromosomes and offered mutual confirmation. Moreover, six CN-LoHs (>1% AF shifts, p<0.01) were found with one additional suspected low frequency deletion (~10-15% burden) in a case of CLL. We conclude that this simple method is directly clinically applicable for the detection of copy neutral chromosomal loss of single genes from WES with intermediate coverage.

Availability

Mentioned plot software is available at Harvard Dataverse http://doi.org/10.7910/DVN/KFMGNY

Contact

Marcus.celik.hansen@rsyd.dk or marcus@clin.au.dk

Supplementary information

Supplementary plots are available
Information Integration and Collective Motility in Phototactic Cyanobacteria
Authors: Menon S. N. , Varuni P. , Menon G I.
Published: March 2019

Cells in microbial colonies integrate information across multiple spatial and temporal scales while sensing environmental cues. A number of photosynthetic cyanobacteria respond in a directional manner to incident light, resulting in the phototaxis of individual cells. Colonies of such bacteria exhibit large-scale changes in morphology, arising from cell-cell interactions, during phototaxis. These interactions occur through type IV pili-mediated physical contacts between cells, as well as through the secretion of complex polysaccharides (‘slime’) that facilitates cell motion. Here, we describe a computational model for such collective behaviour in colonies of the cyanobacteriumSynechocystis. The model is designed to replicate observations from recent experiments on the emergent response of the colonies to varied light regimes. It predicts the complex colony morphologies that arise as a result. We ask if changes in colony morphology during phototaxis can be used to infer if cells integrate information from multiple light sources simultaneously, or respond to these light sources separately at each instant of time. We find that these two scenarios cannot be distinguished from the shapes of colonies alone. However, we show that tracking the trajectories of individual cyanobacteria provides a way of determining their mode of response. Our model allows us to address the emergent nature of this class of collective bacterial motion, linking individual cell response to the dynamics of colony shape.

Statement of Significance

Microbial colonies in the wild often consist of large groups of heterogeneous cells that coordi-nate and integrate information across multiple spatio-temporal scales. We describe a computational model for one such collective behaviour, phototaxis, in colonies of the cyanobacteriumSynechocystisthat move in response to light. The model replicates experimental observations the response of cyanobacterial colonies to varied light regimes, and predicts the complex colony morphologies that arise as a result. The results suggest that tracking the trajectories of individual cyanobacteria may provide a way of determining their mode of information integration. Our model allows us to address the emergent nature of this class of collective bacterial motion, linking individual cell response to the large scale dynamics of the colony.
Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress
Authors: Hanna N. , Burdet F. , Melotti A. , Bosmani C. , Kicka S. , Hilbi H. , Cosson P. , Pagni M. , Soldati T.
Published: March 2019

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple drug-resistant strains emphasizes the need for more efficient drug treatments. The experimentally versatile Dictyostelium discoideum – Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. In this study, a time-resolved transcriptomic analysis of the amoeba D. discoideum was performed to decipher the different host pathways impacted during infection. We investigated how D. discoideum fine-tunes its gene expression in response to M. marinum infection by assessing the transcriptomic profile covering the critical stages of entry, establishment of a permissive niche, proliferation and dissemination (1, 3, 6, 12, 24 and 48 hours post infection). Differential gene expression provided a fingerprint of the transcriptome of the host cell in the presence of mycobacteria, and helped identify specific markers and molecular signatures of infection. Enrichment pathway analysis showed that most of the Biological Processes (BP) of upregulated genes at early time point of infection hinted towards damage response and cellular defence, especially in specific pathways involved in membrane repair (ESCRT) and bacteria elimination (autophagy). Whereas at late time points of infection, BP related to starvation were upregulated. Some other signatures were more unexpected, such as cell cycle (downregulation of cytosolic large & small ribosomal subunits) and upregulation of metabolic adaptations (lipids transport).
The WT1-like transcription factor Klumpfuss maintains lineage commitment in the intestine
Authors: Korzelius Jerome , Ronnen-Oron Tal , Baldauf Maik , Meier Elke , Sousa-Victor Pedro , Jasper Heinrich
Published: March 2019

Stem cell (SC) lineages in barrier epithelia exhibit a high degree of plasticity. Mechanisms that govern the precise specification of SC daughter cells during regenerative episodes are therefore critical to maintain homeostasis. One such common mechanism is the transient activation of the Notch (N) signaling pathway. N controls the choice between absorptive and entero-endocrine cell fates in both the mammalian small intestine and theDrosophilamidgut, yet how precisely N signaling promotes lineage restriction in progenitor cells remains unclear. Here, we describe a role for the WT1-like transcription factor Klumpfuss (Klu) in restricting the fate ofDrosophilaenteroblasts (EBs) downstream of N activation. Klu is transiently induced in Notch-positive EBs and its transient activity restricts cell fate towards the enterocyte (EC) lineage. Transcriptomics and DamID profiling show that Klu suppresses enteroendocrine (EE) cell fates by repressing E(Spl)m8-HLH and Phyllopod, both negative regulators of the proneural gene Scute, which is essential for EE differentiation. At the same time, Klu suppresses cell cycle genes, committing EBs to differentiation. Klu-mediated repression of its own transcription further sets up a negative feedback loop that ensures temporal restriction of Klu-mediated gene regulation, and is essential for subsequent differentiation of ECs. Our findings define a transient cell state in which EC lineage restriction is cemented, and establish a hierarchy of transcriptional programs critical in executing a differentiation program downstream of initial induction events governed by N signaling.
Mammillothalamic disconnection alters hippocampo-cortical oscillatory activity and microstructure: Implications for diencephalic amnesia
Authors: Dillingham CM , Milczarek MM , Perry JC , Frost BE , Parker GD , Assaf Y , Sengpiel F , O’Mara SM , Vann SD
Published: March 2019

Diencephalic amnesia can be as disruptive as the more commonly known temporal lobe amnesia, yet the precise contribution of diencephalic structures to memory processes remains elusive. We used discrete lesions of the mammillothalamic tract to model aspects of diencephalic amnesia and assessed the impact of these lesions on multiple measures of activity and plasticity within the hippocampus and retrosplenial cortex. Lesions of the mammillothalamic tract had widespread indirect effects on hippocampo-cortical oscillatory activity within both theta and gamma bands. Both within-region oscillatory activity and cross-regional synchrony were altered. The network changes were state-dependent, displaying different profiles during locomotion and paradoxical sleep. Consistent with the associations between oscillatory activity and plasticity, complementary analyses using several convergent approaches revealed microstructural changes, which appeared to reflect a suppression of learning-induced plasticity in lesioned animals. Together, these combined findings suggest a mechanism by which damage to the medial diencephalon can impact upon learning and memory processes, highlighting important role for the mammillary bodies in the co-ordination of hippocampo-cortical activity.
Space-independent community and hub structure of functional brain networks
Authors: Esfahlani Farnaz Zamani , Bertolero Maxwell A. , Bassett Danielle S. , Betzel Richard F.
Published: March 2019

Coordinated brain activity reflects underlying cognitive processes and can be modeled as a network of inter-regional functional connections. The most costly connections in the network are long-distance correlations that, in the absence of underlying structural connections, are maintained by sustained energetic inputs. Here, we present a spatial modeling approach that amplifies contributions made by long-distance functional connections to whole-brain network architecture, while simultaneously suppressing contributions made by short-range connections. We use this method to characterize the long-distance architecture of functional networks and to identify aspects of community and hub structure that are driven by long-distance correlations and that, we argue, are of greater functional significance. We find that based only on patterns of long-distance connectivity, primary sensory cortices occupy increasingly central positions and appear more “hub-like”. Additionally, we show that the community structure of long-distance connections spans multiple topological levels and differs from the community structure detected in networks that include both short-range and long-distance connections. In summary, these findings highlight the complex relationship between the brain’s physical layout and its functional architecture. The results presented here inform future analyses of community structure and network hubs in health, across development, and in the case of neuropsychiatric disorders.
Unintended inhibition of protein function using GFP nanobodies in human cells
Authors: Küey Cansu , Larocque Gabrielle , Clarke Nicholas I. , Royle Stephen J.
Published: March 2019

Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed “dongles”, that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further.
The MITF-SOX10 regulated long non-coding RNA DIRC3 is a melanoma tumour suppressor
Authors: Coe Elizabeth A , Tan Jennifer Y , Shapiro Michael , Louphrasitthiphol Pakavarin , Bassett Andrew R , Marques Ana C , Goding Colin R , Vance Keith W
Published: March 2019

The MITF and SOX10 transcription factors regulate the expression of genes important for melanoma proliferation, invasion and metastasis. Despite growing evidence of the contribution of long noncoding RNAs (lncRNAs) in cancer, including melanoma, their functions within MITF-SOX10 transcriptional programmes remain poorly investigated. Here we identified 245 candidate melanoma associated lncRNAs whose loci are co-occupied by MITF-SOX10 and that are enriched at active enhancer-like regions. We characterise the function and molecular mechanism of action of one of these lncRNAs,Disrupted In Renal Carcinoma 3(DIRC3), and show that it operates as a MITF-SOX10 regulated tumour suppressor.DIRC3depletion in human melanoma cells leads to increased anchorage-independent growth, a hallmark of malignant transformation, whilst melanoma patients classified by lowDIRC3expression have decreased survival.DIRC3is a nuclear lncRNA that functions locally to activate expression of its neighbouringIGFBP5tumour suppressor through modulating chromatin structure and suppressing SOX10 binding to putative regulatory elements within theDIRC3locus. In turn,DIRC3dependent regulation ofIGFBP5impacts the expression of genes involved in multiple cancer associated processes. Our work indicates that lncRNA components of the MITF-SOX10 networks are an important new class of melanoma regulators and candidate therapeutic targets.
A Chemical Probe of CARM1 Alters Epigenetic Plasticity against Breast Cancer Cell Invasion
Authors: Cai Xiao-Chuan , Zhang Tuo , Kim Eui-jun , Jiang Ming , Wang Ke , Wang Junyi , Chen Shi , Zhang Nawei , Wu Hong , Li Fengling , dela Seña Carlo C. , Zheng Hong , Vivcharuk Victor , Niu Xiang , Zheng Weihong , Lee Jonghan P. , Chen Yuling , Barsyte Dalia , Szewczyk Magda , Hajian Taraneh , Ibáñez Glorymar , Dong Aiping , Dombrovsky Ludmila , Zhang Zhenyu , Deng Haiteng , Min Jinrong , Arrowsmith Cheryl H. , Mazutis Linas , Shi Lei , Vedadi Masoud , Brown Peter J. , Xiang Jenny , Qin Li-Xuan , Xu Wei , Luo Minkui
Published: March 2019

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. HereSKI-73is presented as a CARM1 chemical probe with pro-drug properties.SKI-73can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement.SKI-73recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that theSKI-73-associated reduction of invasiveness act via altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly,SKI-73and CARM1 knockout alter the epigenetic plasticity with remarkable difference, arguing distinct modes of action between the small-molecule and genetic perturbation. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.
Ethanolic extract of Ocimum sanctum leaves reduced invasion and matrix metalloproteinase activity of head and neck cancer cell lines
Authors: Utispan Kusumawadee , Niyomtham Nattisa , Yingyongnarongkul Boon-ek , Koontongkaew Sittichai
Published: March 2019

Head and neck squamous cell carcinoma (HNSCC) has a yearly incidence of 600,000 cases worldwide with a low survival rate. Ocimum sanctum L. or Ocimum tenuiflorum L. (Holy basil; Tulsi in Hindi), is a traditional medicine herb that demonstrates numerous effects, including anti-oxidant, anti-microbial, and anti-tumor effects. The aim of this study was to evaluate the anti-invasive effect of O. sanctum leaf extract on HNSCC cell lines. Ethanolic extract of O. sanctum leaf (EEOS) was prepared and the phenolic compounds were identified using high-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry. Genetically-matched HNSCC cell lines derived from primary (HN30 and HN4) and metastatic sites (HN31 and HN12) from the same patient were used in this study. The EEOS cytotoxicity to the cell lines was determined using an MTT assay. The invasion and matrix metalloproteinase (MMP)-2 and -9 activity of EEOS-treated cells were tested using a modified Boyden chamber assay and zymography, respectively. We found that EEOS significantly inhibited the invasion and MMP-2 and MMP-9 activity of HN4 and HN12 cells, but not HN30 and HN31 cells. Rosmarinic acid, caffeic acid, and apigenin were detected in EEOS. Moreover, rosmarinic acid was found as the major phenolic compound. Therefore, EEOS exerted its anti-invasive effect on HNSCC cells by attenuating MMP activity.
Rapidly increasing prevalence of overweight and obesity in older Ghanaian adults from 2007-2015: evidence from WHO-SAGE Waves 1 & 2
Authors: Lartey Stella T. , Magnussen Costan G. , Si Lei , Boateng Godfred O. , Graaff Barbara de , Biritwum Richard Berko , Minicuci Nadia , Kowal Paul , Blizzard Leigh , Palmer Andrew J.
Published: March 2019

Background

Studies on changes in the prevalence and determinants of obesity in older adults living in sub-Saharan Africa are limited. We examined recent changes in obesity prevalence and associated factors for older adults in Ghana between 2007/08 and 2014/15.

Methods

Data on adults aged 50 years and older in Ghana were collected in the WHO SAGE Wave 1 (2007/08; n=4158) and Wave 2 (2014/15; n=1663). The weighted prevalence of obesity, overweight, normal weight and underweight, and of high central adiposity was compared in Waves 1 and 2. Multinomial and binomial logistic regressions were used to examine whether the determinants of weight status based on objectively measured body mass index and waist circumference changed between the two survey periods.

Results

The prevalence of obesity (Wave 1=10.2%, 95% CI: 8.9-11.7%; Wave 2=15.0%, 95% CI: 12.6-17.7%) and overweight (Wave 1=19.6%, 95% CI: 18.0-21.4%; Wave 2=24.5%, 95% CI: 21.7-27.5%) was higher in Wave 2 than Wave 1 and more than half of the population had high central adiposity (Wave 1=57.7%, 95% CI: 55.4-60.1%; Wave 2=66.9%, 95% CI: 63.7-70.0%) in both study periods. Obesity prevalence was 16% lower in males and 55% higher in females comparing Wave 1 to Wave 2. Female sex, urban residence, and high household wealth were associated with higher odds of overweight/obesity and high central adiposity. Those aged 70+ years had lower odds of obesity in both study waves. In Wave 2, females with physical activity level were more likely to be obese.

Conclusion

Over the 7-year period between survey waves, the population prevalence of overweight and obesity increased by 25% and 47%, respectively, while underweight reduced by 43%. These findings differed considerably by sex, which points to differential impacts of past initiatives to reduce overweight/obesity, potential high-risk groups in Ghana, and the need to increase surveillance.
Dietary acculturation of Asian and the Middle East and North African region immigrants to Saudi Arabia: healthy or unhealthy acquired nutritional behavior?
Authors: Al Zeidan Rasmieh , Tharkar Shabana , Hersi Ahmed , Ullah Anhar
Published: March 2019

Travel and migration influences food behavior. This study assessed the dietary acculturation of immigrants in Saudi Arabia, with regard to length of stay and health status of immigrants. This cross-sectional study included 880 university immigrant employees and their family members from Asian and Middle East and North African regions. Dietary acculturation was assessed based on knowledge and practice of methods of food preparation, type of food consumption, and nutrition label reading behavior, using a measurement tool on a 5-point Likert scale. Furthermore, a scoring system was adapted for healthy practices. Anthropometric, biochemical, and blood pressure measurements were performed as per the World Health Organization guidelines, to determine participants’ health and comorbid status. In addition, scores were calculated for healthy options. Factors influencing better awareness were determined by logistic regression analysis. The most adopted methods of food preparation after migration were barbeque (p=0.018), microwave cooking (p=0.002), and raw food consumption (salads) (p<0.001). Consumption of carbonated drinks (p=0.025), fried fatty and processed food (p=0.037), and sweets and candies (p=0.008) were significantly higher among recent immigrants of <5 years of residency. Label reading behavior of nutritional contents and low-fat options was higher among immigrants with ≥5 years duration of residency (63%; p<0.001). Although female gender, longer duration of residency in Saudi Arabia and presence of comorbidity significantly improved the overall awareness and practice scores in the binary analysis, they failed to show significance in regression model except for the presence of diabetes which improved only awareness. None of the other independent factors seem to influence healthy practices. Chronic diseases like obesity, diabetes and hypertension increased with longer duration of migration(p<0.001).

New immigrants are at risk of acquiring negative dietary habits compromising health, necessitating follow-up studies to establish causation. Interventional policy measures are recommended to formulate dietary guidelines.
Francisella novicidaCas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing
Authors: Acharya Sundaram , Mishra Arpit , Paul Deepanjan , Ansari Asgar Hussain , Azhar Mohd. , Kumar Manoj , Rauthan Riya , Sharma Namrata , Aich Meghali , Sinha Dipanjali , Sharma Saumya , Jain Shivani , Ray Arjun , Jain Suman , Ramalingam Sivaprakash , Maiti Souvik , Chakraborty Debojyoti
Published: March 2019

Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely usedStreptococcus pyogenesCas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report thatFrancisella novicidaCas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher HDR rates and very low non-specific genome editing compared to SpCas9. We demonstrate FnCas9 mediated correction of the sickle cell mutation in patient derived iPSCs and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

SIGNIFICANCE STATEMENT

Therapeutic genome editing has been significantly accentuated by the advent of CRISPR based gene correction. However, genomic off-targeting has been a major setback for clinical translation. Although high fidelity versions of Cas9 have been rationally designed, they recognize and bind to off-targets. In this study, we characterize a naturally occurring Cas9 fromFrancisella novicida(FnCas9) that shows negligible binding affinity to off targets differing by one or more mismatches, rendering it highly specific in target recognition and editing. We show that FnCas9 can direct both HDR and NHEJ mediated DNA repair, generates higher rate of HDR and negligible off-target editing. Finally we show its potential in therapeutic genome editing by correcting the sickle cell anemia mutation in patient derived iPSCs.
Discrete and Continuous Cell Identities of the Adult Murine Striatum
Authors: Stanley Geoffrey , Gokce Ozgun , Malenka Robert C. , Südhof Thomas C. , Quake Stephen R.
Published: March 2019

The striatum is a large brain region containing two major cell types: D1 (dopamine receptor 1) and D2 (dopamine receptor 2) expressing spiny projection neurons (SPNs). We generated a cell type atlas of the adult murine striatum using single-cell RNA-seq of SPNs combined with quantitative RNAin situhybridization (ISH). We developed a novel computational pipeline that distinguishes discrete versus continuous cell identities in scRNA-seq data, and used it to show that SPNs in the striatum can be classified into four discrete types that reside in discrete anatomical clusters or are spatially intermingled. Within each discrete type, we find multiple independent axes of continuous cell identity that map to spatial gradients and whose genes are conserved between discrete types. These gradients correlate well to previously-mapped gradients of connectivity. Using these insights, we discovered multiple novel spatially localized region of the striatum, one of which contains patch-D2 SPNs that expressTac1, Htr7, andTh. Intriguingly, we found one subtype that strongly co-expresses both D1 and D2 dopamine receptors, and uniquely expresses a rare D2 receptor splice variant. These results collectively suggest an organizational principal of neuron identity in which major neuron types can be separated into discrete classes with little overlap and no implied spatial relationship. However these discrete classes are then continuously subdivided by multiple spatial gradients of expression defining anatomical location via a combinatorial mechanism. Finally, they suggest that neuronal circuitry has a substructure at far higher resolution than is typically interrogated which is defined by the precise identity and location of a neuron.
Identification of QTL and Underlying Genes for Root System Architecture associated with Nitrate Nutrition in Hexaploid Wheat
Authors: Griffiths Marcus , Atkinson Jonathan A. , Gardiner Laura-Jayne , Swarup Ranjan , Pound Michael P. , Wilson Michael H. , Bennett Malcolm J. , Wells Darren M.
Published: March 2019

The root system architecture (RSA) of a crop has a profound effect on the uptake of nutrients and consequently the potential yield. However, little is known about the genetic basis of RSA and resource adaptive responses in wheat (Triticum aestivumL.). Here, a high-throughput germination paper plant phenotyping system was used to identify seedling traits in a wheat doubled haploid mapping population, Savannah × Rialto. Significant genotypic and nitrate-N treatment variation was found across the population for seedling traits with distinct trait grouping for root size-related traits and root distribution-related traits. Quantitative trait locus (QTL) analysis identified a total of 59 seedling trait QTLs. Across two nitrate treatments, 27 root QTLs were specific to the nitrate treatment. Transcriptomic analyses for one of the QTLs on chromosome 2D found under low nitrate conditions was pursued revealing gene enrichment in N-related biological processes and 17 candidate up-regulated genes with possible involvement in a root angle response. Together, these findings provide genetic insight into root system architecture and plant adaptive responses to nitrate and provide targets that could help improve N capture in wheat.
Limits to Genomic Divergence Under Sexually Antagonistic Selection
Authors: Kasimatis Katja R. , Ralph Peter L. , Phillips Patrick C.
Published: March 2019

Since the autosomal genome is shared between the sexes, sex-specific fitness optima present an evolutionary challenge. While sexually antagonistic selection might favor different alleles within females and males, segregation randomly reassorts alleles at autosomal loci between sexes each generation. This process of homogenization during transmission thus prevents between-sex allelic divergence generated by sexually antagonistic selection from accumulating across multiple generations. However, recent empirical studies have reported high male-femaleFSTstatistics. Here, we use a population genetic model to evaluate whether these observations could plausibly be produced by sexually antagonistic selection. To do this, we use both a single-locus model with nonrandom mate choice, and individual-based simulations to study the relationship between strength of selection, degree of between-sex divergence, and the associated genetic load. We show that selection must be exceptionally strong to create measurable divergence between the sexes and that the decrease in population fitness due to this process is correspondingly high. Individual-based simulations with selection genome-wide recapitulate these patterns and indicate that small sample sizes and sampling variance can easily generate substantial male-female divergence. We therefore conclude that caution should be taken when interpreting autosomal allelic differentiation between the sexes.