bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers
Authors: Patrick Eric M. , Slivka Joseph , Payne Bramyn , Comstock Matthew J. , Schmidt Jens C.
Published: July 2019

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. After release of multiple telomeric repeats from telomerase, we observed folding of product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.
Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein
Authors: Landeck Natalie , Strathearn Katherine E. , Ysselstein Daniel , Buck Kerstin , Dutta Sayan , Banerjee Siddhartha , Lv Zhengjian , Hulleman John D. , Hindupur Jagadish , Lin Li-Kai , Padalkar Sonal , McCabe George P. , Stanciu Lia A. , Lyubchenko Yuri L. , Kirik Deniz , Rochet Jean-Christophess
Published: July 2019

α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson’s disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models as well as their biochemical properties relevant to aSyn pathobiology. We report that mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.
Gclc deletion in surface-ectoderm tissues induces microphthalmia
Authors: Thompson Brian , Chen Ying , Philippe Julien , Anderson David , Golla Jaya Prakash , Davidson Emily , Apostolopoulos Nicholas , Schey Kevin , Katsanis Nicholas , Orlicky David J. , Thompson David , Vasiliou Vasilis
Published: July 2019

Glutamate cysteine ligase catalytic subunit (Gclc) is the catalytic subunit for the glutamate-cysteine ligase (Gcl) enzyme. Gcl catalyzes the rate limiting step in glutathione (GSH) synthesis. Gclc is highly expressed in the developing eye. To define the regulatory role of Gclc in eye development, we developed a novel, Le-Cre transgene-driven, Gclc knockout mouse model. Gclcf/f/Le-CreTg/− mice present with deformation of the retina, cornea, iris, and lens, consistent with a microphthalmia phenotype. Controlling for the microphthalmia phenotype of Gclcwt/wt/Le-CreTg/− mice revealed that Gclcf/f/Le-CreTg/− mice have a more severe microphthalmia phenotype. Thus, the loss of Gclc expression exacerbates the microphthalmia phenotype in Le-Cre mice. Gclcf/f/Le-CreTg/− eyes present with reduced retinal and lens epithelium proliferation and increased lens cell death. Imaging mass spectrometry of ocular tissues revealed changes in the intensity and distribution of several lipid species and proteins in the retina and corneas of Gclcf/f/Le-CreTg/− eyes. Lastly, using splice-blocking morpholinos and CRISPR/Cas9, we created two gclc knockdown zebrafish models, both of which display a microphthalmia phenotype. Combined, the mouse and zebrafish results indicate that, in chordates, Gclc has a conserved role in regulating eye development. In summary, these novel animal models are useful tools for elucidating the mechanisms involved in microphthalmia development.
BRCA1 Mediated Homologous Recombination and S Phase DNA Repair Pathways Restrict LINE-1 Retrotransposition in Human Cells
Authors: Sun Xiaoji , Mita Paolo , Kahler David J. , Li Donghui , Wudzinska Aleksandra , Yun Chi , Bader Joel S. , Fenyö David , Boeke Jef D.
Published: July 2019

Long interspersed element-1 (LINE-1 or L1) is the only autonomous retrotransposon active in human cells. L1s DNA makes about 17% of the human genome and retrotransposition of a few active L1 copies has been detected in various tumors, underscoring the potential role of L1 in mediating or increasing genome instability during tumorigenic development. Different host factors have been shown to influence L1 mobility through several mechanisms. However, systematic analyses of host factors affecting L1 retrotransposition are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay and coupled it to a genome-wide siRNA knockdown screen to study the cellular regulators of L1 retrotransposition in human cells. We showed that L1 insertion frequency was stimulated by knockdown of Double-Stranded Break (DSB) repair factors that are active in the S/G2 phase of the cell cycle including Homologous Recombination (HR), Fanconi Anemia (FA) and, to a less extent, microhomology-mediated end-joining (MMEJ) factors. In particular, we show that BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, plays multiple roles in regulating L1; BRCA1 knockdown directly affects L1 retrotransposition frequency and structure and also plays a role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a “battle” between HR factors and L1 retrotransposons, revealing a potential role for L1 in development of tumors characterized by BRCA1 and HR repair deficiencies.
Cyclin B1-Cdk1 binding to MAD1 links nuclear pore disassembly to chromosomal stability
Authors: Jackman Mark , Marcozzi Chiara , Pardo Mercedes , Yu Lu , Tyson Adam L. , Choudhary Jyoti S. , Pines Jonathon
Published: July 2019

How the cell completely reorganises its architecture when it divides is a problem that has fascinated researchers for almost 150 years. We now know that the core regulatory machinery is highly conserved in eukaryotes but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated to remodel the cell in a matter of minutes remains a major question. Cyclin B-CDK is the primary kinase that drives mitotic remodelling and here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the spindle assembly checkpoint protein, MAD1. This localised Cyclin B1-CDK1 activity coordinates NPC disassembly with kinetochore assembly: it is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore, which enables MAD1 to be recruited to kinetochores before nuclear envelope breakdown, thereby strengthening the spindle assembly checkpoint to maintain genomic stability.
A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex
Authors: Partlow Edward A. , Baker Richard W. , Beacham Gwendolyn M. , Chappie Joshua S. , Leschziner Andres E. , Hollopeter Gunther
Published: July 2019

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.
Giotto, a toolbox for integrative analysis and visualization of spatial expression data
Authors: Dries Ruben , Zhu Qian , Dong Rui , Linus Eng Chee-Huat , Li Huipeng , Liu Kan , Fu Yuntian , Zhao Tianxiao , Sarkar Arpan , Bao Feng , George Rani E , Pierson Nico , Cai Long , Yuan Guo-Cheng
Published: July 2019

The rapid development of novel spatial transcriptomic and proteomic technologies has provided new opportunities to investigate the interactions between cells and their native microenvironment. However, effective use of such technologies requires the development of innovative computational tools that are easily accessible and intuitive to use. Here we present Giotto, a comprehensive, flexible, robust, and open-source toolbox for spatial transcriptomic and proteomic data analysis and visualization. The data analysis module provides end-to-end analysis by implementing a wide range of algorithms for characterizing cell-type distribution, spatially coherent gene expression patterns, and interactions between each cell and its surrounding neighbors. Furthermore, Giotto can also be used in conjunction with external single-cell RNAseq data to infer the spatial enrichment of cell types from data that do not have single-cell resolution. The data visualization module allows users to interactively visualize the gene expression data, analysis outputs, and additional imaging features, thereby providing a user-friendly workspace to explore multiple modalities of information for biological investigation. These two modules can be used iteratively for refined analysis and hypothesis development. We applied Giotto to a wide range of public datasets encompassing diverse technologies and platforms, thereby demonstrating its general applicability for spatial transcriptomic and proteomic data analysis and visualization.
Insulin potentiates JAK/STAT signaling to broadly inhibit flavivirus replication in insect vectors
Authors: Ahlers Laura R.H. , Trammell Chasity E. , Carrell Grace F. , Mackinnon Sophie , Torrevillas Brandi K. , Chow Clement Y. , Luckhart Shirley , Goodman Alan G.
Published: July 2019

The World Health Organization estimates that over half of the world’s population is at risk for vector-borne diseases, such as those caused by arboviral infection. Because many arboviruses are mosquito-borne, investigation of the insect immune response will help identify targets that could reduce the spread of these viruses by the mosquito. In this study, we used a genetic screening approach to identifyinsulin-like receptoras a novel component of the immune response to arboviral infection. We determined that vertebrate insulin reduces West Nile virus (WNV) replication inDrosophila melanogasteras well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, we showed that insulin signaling activates the JAK/STAT, but not RNAi, pathway to control infection. Finally, we validated that insulin priming of adult femaleCulexmosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to emerging human pathogens.
Parameter-dependent shift from rational to irrational decisions in mice
Authors: Schneider Nathan A. , Ballintyn Benjamin , Katz Donald , Lisman John , Pi Hyun-Jae
Published: July 2019

In the classical view of economic choices, subjects make rational decisions evaluating the costs and benefits of options in order to maximize their overall income. Nonetheless, subjects often fail to reach optimal outcomes. The overt value of an option drives the direction of decisions, but covert factors such as emotion and sunk cost are thought to drive the observed deviations from optimality. Many questions remain to be answered as to 1) which contexts contribute the most to deviation from an optimal solution; and 2) the extent of these effects. In order to tackle these questions, we devised a decision-making task for mice, in which cost and benefit parameters could be independently and flexibly adjusted and for which a tractable optimal solution was known. Comparing mouse behavior with this optimal solution across parameter settings revealed that the factor most strongly contributing to suboptimality was the cost parameter. The quantification of sunk cost, a covert factor implicated in our task design, revealed it as another contributor to suboptimality. In one condition where the large reward option was particularly unattractive and the small reward cost was low, the sunk cost effect and the cost-led suboptimality almost vanished. In this regime and this regime only, mice could be viewed as close to rational. Taken together, our findings support a model whereby parallel neural circuits independently activate and modulate multiple valuation algorithms, and suggest that “rationality” is a task-specific construct even in mice.

Significant Statement

Irrational factors in economic decision-making often cause significant deviation from optimal outcomes. By devising a flexible economic choice behavior for mice and comparing their behavior with an optimal solution, we investigated overt and covert factors that contributed to suboptimal outcomes and quantified the deviation from optimality. This investigation identified regimes where mice could be viewed as rational or irrational depending upon the parameters in the same task. These findings may provide a platform to investigate biological substrates underlying rational and irrational decision factors.
Phospholamban and sarcolipin share similar transmembrane zipper motifs that control self-association affinity and oligomer stoichiometry
Authors: DesLauriers Nicholas R. , Svensson Bengt , Thomas David D. , Autry Joseph M.
Published: July 2019

We have characterized the structural determinants of phospholamban (PLB) and sarcolipin (SLN) self-association using site-directed mutagenesis, SDS-PAGE, and fluorescence resonance energy transfer (FRET) microscopy. PLB and SLN are single-pass transmembrane (TM) peptides that are critically involved in regulation of contractility in cardiac and skeletal muscle via reversible inhibition of calcium (Ca) transport by SERCA. PLB and SLN also exhibit ion channel activityin vitro, yet the physiological significance of these functions is unknown. Here we have determined that structural insights offered by the tetrameric PLB Cys41 to Leu (C41L) mutation, a mutant with four possible leucine/isoleucine zipper interactions for stabilizing PLB tetramers. Using scanning alanine mutagenesis and SDS-PAGE, we have determined the C41L-PLB tetramer is destabilized by mutation of Leu37 to Ala (L37A) or Ile40 to Ala (I40A), which are the samea- andd-arm residues stabilizing the PLB pentamer via leucine/isoleucine zippers, highlighting the importance of these two zippers in PLB higher-order oligomerization. The new possible zipper arm in C41L-PLB (N34, C41L, I48) did not contribute to tetramerization. On the other hand, we determined that tetramer conversion back to pentamer was induced by alanine mutation of Ile48, a residue located on thee-arm below C41L, implicating steric interaction and restriction are the stabilizing and destabilizing forces that control the distribution between pentamer and tetramer populations. We propose that thee-arm and hydrophobic residues in the adjacentb-arm act as secondary structural motifs that help control the stoichiometry of PLB oligomerization. FRET microscopy and alanine mutagenesis of SLN residues Val14 (V14A) or Leu21 (L21A) decreased the binding affinity of the SLN‒SLN complex, demonstrating the importance of each residue in mediating self-association. Helical wheel analysis supports a heptad-repeat TM zipper mechanism of SLN oligomerization, similar to the 3.5 residue/turn Leu and Ile zippers found in PLB pentamers. Collectively, our studies add new insights on the conservation of homologous hydrophobic 3-4 pattern of residues in zipper motifs that mediate PLB and SLN self-assembly. We propose that the importance of these apolar, steric interactions in the TM domain are widespread in stabilizing higher-order oligomerization of membrane proteins.
A missense mutation dissociates triglyceride and phospholipid transfer activities in zebrafish and human microsomal triglyceride transfer protein
Authors: Wilson Meredith H. , Rajan Sujith , Danoff Aidan , White Richard J. , Hensley Monica R. , Quinlivan Vanessa H. , Thierer James H. , Busch-Nentwich Elisabeth M. , Hussain M. Mahmood , Farber Steven A.
Published: July 2019

Microsomal triglyceride transfer protein (MTP) transfers triglycerides and phospholipids and is essential for the assembly of Apolipoprotein B (ApoB)-containing lipoproteins in the endoplasmic reticulum. We have discovered a zebrafish mutant (mttpc655) expressing a C-terminal missense mutation (G863V) in Mttp, one of the two subunits of MTP, that is defective at transferring triglycerides, but retains phospholipid transfer activity. Mutagenesis of the conserved glycine in the human MTTP protein (G865V) also eliminates triglyceride but not phospholipid transfer activity. The G863V mutation reduces the production and size of ApoB-containing lipoproteins in zebrafish embryos and results in the accumulation of cytoplasmic lipid droplets in the yolk syncytial layer. However,mttpc655mutants exhibit only mild intestinal lipid malabsorption and normal growth as adults. In contrast, zebrafish mutants bearing the previously identifiedmttpstlmutation (L475P) are deficient in transferring both triglycerides and phospholipids and exhibit gross intestinal lipid accumulation and defective growth. Thus, the G863V point mutation provides the first evidence that the triglyceride and phospholipid transfer functions of a vertebrate MTP protein can be separated, arguing that selective inhibition of the triglyceride transfer activity of MTP may be a feasible therapeutic approach for dyslipidemia.
Regional heterogeneity of developing GABAergic interneuron excitation in vivo
Authors: Murata Yasunobu , Colonnese Matthew T.
Published: July 2019

GABAergic interneurons are proposed to be critical for early activity and synapse formation by directly exciting, rather than inhibiting, neurons in developing hippocampus and neocortex. However, the role of GABAergic neurons in the generation of neonatal network activity has not been tested in vivo, and recent studies have challenged the excitatory nature of early GABA. By locally manipulating interneuron activity in unanesthetized neonatal mice, we show that GABAergic neurons are indeed excitatory in hippocampus at postnatal-day 3 (P3), and responsible for most of the spontaneous firing of pyramidal cells at that age. Hippocampal interneurons become inhibitory by P7, whereas cortical interneurons are inhibitory at P3 and remain so throughout development. This regional and age heterogeneity is the result of a change in chloride reversal potential as activation of light-gated anion channels expressed in glutamatergic neurons causes firing in hippocampus at P3, but silences it at P7. This study in the intact brain reveals a critical role for GABAergic interneuron excitation in neonatal hippocampus, and a surprising heterogeneity of interneuron function in cortical circuits that was not predicted from in vitro studies.
Reconstructing contact network structure and cross-immunity patterns from multiple infection histories
Authors: Christian Selinger , Samuel Alizon
Published: July 2019

Interactions within a population shape the spread of infectious diseases but contact patterns between individuals are difficult to access. We hypothesised that key properties of these patterns can be inferred from multiple infection data in longitudinal follow-ups. We developed a simulator for epidemics with multiple infections on networks and analysed the resulting individual infection time series by introducing the concept of infection barcodes. We find that, depending on infection multiplicity and network sampling, infection barcode summary statistics can recover network properties such as degree distribution. Furthermore, we show that by mining infection barcodes for multiple infection patterns, one can detect immunological interference between pathogens (i.e. the fact that past infections in a host condition future probability of infection). The combination of individual-based simulations and barcode analysis of infection histories opens promising perspectives to infer and validate transmission networks and immunological interference for infectious diseases from longitudinal cohort data.

Author summary

Infectious disease dynamics are constrained both by between-host contacts and pathogen interactions within a host. Furthermore, multiple parasites circulate such that hosts are infected (sometimes simultaneously) by a variety of strains or species. We hypothesise that multiple infection history can inform us about the networks on which parasites are transmitted, but also on within-host interactions such as immunological interference. We developed a simulator for multiple infections on networks. By combining intuitive novel metrics for multiple infection events and established tools from computational data analysis, we show that similarity in infection history between two hosts correlates with their proximity in the contact network. By analysing pathogens co-occurrence patterns within hosts, we also recover immunological interference at the population level. The demonstrated robustness of our results in terms of observability, network clustering, and pathogen diversity opens new perspectives to extract host contact and between-pathogen immunity information from longitudinal cohort data.
Spinocerebellar Ataxia Type 1 protein Ataxin-1 is signalled to DNA damage by Ataxia Telangiectasia Mutated kinase
Authors: Suart Celeste E. , Perez Alma M. , Al-Ramahi Ismael , Maiuri Tamara , Botas Juan , Truant Ray
Published: July 2019

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the ataxin-1 protein. Recent genetic correlational studies have implicated DNA damage repair pathways in modifying the age at onset of disease symptoms in SCA1 and Huntington’s Disease, another polyglutamine expansion disease. We demonstrate that both endogenous and transfected ataxin-1 localizes to sites of DNA damage, which is impaired by polyglutamine expansion. This response is dependent on ataxia telangiectasia mutated (ATM) kinase activity. Further, we characterize an ATM phosphorylation motif within ataxin-1 at serine 188. We show reduction of the Drosophila ATM homolog levels in a ATXN1[82Q] Drosophila model through shRNA or genetic cross ameliorates motor symptoms. These findings offer a possible explanation as to why DNA repair was implicated in SCA1 pathogenesis by past studies. The similarities between the ataxin-1 and the huntingtin responses to DNA damage provide further support for a shared pathogenic mechanism for polyglutamine expansion diseases.
Treatment with commonly used antiretroviral drugs induces a type I/III interferon signature in the gut in the absence of HIV infection
Authors: Hughes Sean M. , Levy Claire N. , Calienes Fernanda L. , Stekler Joanne D. , Pandey Urvashi , Vojtech Lucia , Berard Alicia R. , Birse Kenzie , Noël-Romas Laura , Richardson Brian , Golden Jackelyn B. , Cartwright Michael , Collier Ann C. , Stevens Claire E. , Curlin Marcel E. , Holtz Timothy H. , Mugo Nelly , Irungu Elizabeth , Katabira Elly , Muwonge Timothy , Lama Javier R. , Baeten Jared M. , Burgener Adam , Lingappa Jairam R. , McElrath M. Juliana , Mackelprang Romel , McGowan Ian , Cranston Ross D. , Cameron Mark J. , Hladik Florian
Published: July 2019

Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are used for HIV treatment and prevention. Previously, we found that topical rectal tenofovir gel caused immunological changes in the mucosa. Here we assessed the effect of oral TDF/FTC in three HIV pre-exposure prophylaxis trials, two with gastrointestinal and one with cervicovaginal biopsies. TDF/FTC induced type I/III interferon-related (IFN I/III) genes in the gastrointestinal tract, but not blood, with strong correlations between the two independent rectal biopsy groups (Spearman r=0.91) and between the rectum and duodenum (r=0.81). Gene set testing also indicated stimulation of type I/III pathways in the ectocervix, as well as of cellular proliferation in the duodenum. mRNA sequencing, digital droplet PCR, proteomics, and immunofluorescence staining confirmed IFN I/III pathway stimulation in the gastrointestinal tract. Thus, oral TDF/FTC stimulates an IFN-I/III signature throughout the gut, which could increase antiviral efficacy but also cause chronic immune activation in HIV prevention and treatment settings.
Predicted genetic gains from introgressing chromosome segments from exotic germplasm into an elite soybean cultivar
Authors: Ru Sushan , Bernardo Rex
Published: July 2019

Broadening the diversity of cultivated soybean [Glycine max(L.) Merrill] through introgression of exotic germplasm has been difficult. Our objectives were to 1) determine if introgressing specific chromosome segments (instead of quantitative trait locus alleles) from exotic soybean germplasm has potential for improving an elite cultivar, and 2) identify strategies to introgress and pyramid exotic chromosome segments into an elite cultivar. We estimated genomewide marker effects for yield and other traits in seven crosses between the elite line IA3023 and seven soybean plant introductions (PIs). We then predicted genetic gains from having ≤2 targeted recombinations per linkage group. When introgression was modeled for yield while controlling maturity in the seven PI × IA3023 populations, the predicted yield was 8 to 25% over the yield of IA3023. Correlated changes in maturity, seed traits, lodging, and plant height were generally small but were in the favorable direction. In contrast, selecting the best recombinant inbred (without targeted recombination) in each of the PI × IA3023 populations led to negative or minimal yield gains over IA3023. In one PI × IA3023 population, introgressing and pyramiding only two linkage groups from recombinant inbreds into IA3023 was predicted to achieve an 8% yield gain over IA3023 without sacrificing the performance of other traits. The probability of inheriting intact chromosomes was high enough to allow introgression and pyramiding of chromosome segments in 5-6 generations. Overall, our study suggested that introgressing specific chromosome segments is an effective way to introduce exotic soybean germplasm into an elite cultivar.

Key message

To improve an elite soybean line, introgress longer chromosome segments instead of QTL alleles from exotic germplasm.
SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans
Authors: Bhatia Sonya , Hunter Craig P.
Published: July 2019

RNA interference (RNAi) is sequence-specific gene silencing triggered by double-stranded (ds)RNA. When dsRNA is expressed or introduced into one cell and is transported to and initiates RNAi in other cells, it is called systemic RNAi. Systemic RNAi is very efficient in C. elegans and genetic screens for systemic RNAi defective (Sid) mutants have identified RNA transporters (SID-1, SID-2 and SID-5) and a signaling protein (SID-3). Here we report that SID-4 is nck-1, a C. elegans NCK-like adaptor protein. sid-4 null mutations cause a weak, dosesensitive, systemic RNAi defect and can be effectively rescued by SID-4 expression in target tissues only, implying a role in dsRNA import. SID-4 and SID-3 (ACK-1 kinase) homologs interact in mammals and insects, suggesting they may function in a common signaling pathway, however, a sid-3; sid-4 double mutants showed additive resistance to RNAi, suggesting that these proteins likely interact with other signaling pathways as well. A bioinformatic screen coupled to RNAi sensitivity tests identified 23 additional signaling components with weak RNAi defective phenotypes. These observations suggest that environmental conditions may modulate systemic RNAi efficacy, and indeed, sid-3 and sid-4 are required for growth temperature effects on systemic RNAi silencing efficiency.
Genetic risks of schizophrenia identified in a matched case-control study
Authors: Oishi Kengo , Niitsu Tomihisa , Kanahara Nobuhisa , Sato Yasunori , Iwayama Yoshimio , Toyota Tomoko , Hashimoto Tasuku , Sasaki Tsuyoshi , Takase Masayuki , Yoshikawa Takeo , Iyo Masaomi
Published: July 2019

Background

Genetic association studies of schizophrenia may be confounded by the pathological heterogeneity and multifactorial nature of this disease. We demonstrated previously that combinations of the three functional single nucleotide polymorphisms (SNPs) rs10770141 of tyrosine hydroxylase (TH) gene, rs4680 of catechol-O-methyltransferase (COMT) gene, and rs1800497 of dopamine D2 receptor (DRD2) gene may be associated with schizophrenia onset, and we tested those associations herein. Methods: We conducted a secondary study of 2,542 individuals in age- and sex-matched case-control populations. The schizophrenia diagnosis was based on the DSM-IV. To reduce the influence of confounders (age and sex), we performed a propensity score matching analysis. Genotyping and associative analyses of rs10770141, rs4680, and rs1800497 with schizophrenia were performed. Results: We analyzed 1,271 schizophrenics (male/female: 574/698; age 47.4±13.9 years) and 1,271 matched controls (male/female: 603/669; age 46.5±13.4 years). The estimated odds ratios (ORs) were 1.245 (p<0.001) for rs4680, 1.727 (p<0.0001) for rs1800497, and 1.788 (p<0.0001) for rs10770141. Double SNP analyses revealed the ORs of 2.010 (p<0.0001) for the combination of rs4680*rs1800497, 1.871 (p<0.001) for rs1800497*rs10770141, and 1.428 (p=0.068) for rs4680*rs1800497. Among the individuals with any of the three double SNP risk combinations (which accounted for 35.8% of the involved patients), the estimated OR was 2.224 (p<0.0001). Conclusions: In this validation study, the combinations of functional polymorphisms related to dopaminergic genes were associated with the development of schizophrenia. Analyzing combinations of functional polymorphisms with the control of possible confounders may provide new insights for association research.
New interpretable machine learning method for single-cell data reveals correlates of clinical response to cancer immunotherapy
Authors: Greene Evan , Finak Greg , D’Amico Leonard A. , Bhardwaj Nina , Church Candice D. , Morishima Chihiro , Ramchurren Nirasha , Taube Janis M. , Nghiem Paul T. , Cheever Martin A. , Fling Steven P. , Gottardo Raphael
Published: July 2019

High-dimensional single-cell cytometry is routinely used to characterize patient responses to cancer immunotherapy and other treatments. This has produced a wealth of datasets ripe for exploration but whose biological and technical heterogeneity make them difficult to analyze with current tools. We introduce a new interpretable machine learning method for single-cell mass and flow cytometry studies, FAUST, that robustly performs unbiased cell population discovery and annotation. FAUST processes data on a per-sample basis and returns biologically interpretable cell phenotypes that can be compared across studies, making it well-suited for the analysis and integration of complex datasets. We demonstrate how FAUST can be used for candidate biomarker discovery and validation by applying it to a flow cytometry dataset from a Merkel cell carcinoma anti-PD-1 trial and discover new CD4+ and CD8+ effector-memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. We then use FAUST to validate these correlates in an independent CyTOF dataset from a published metastatic melanoma trial. Importantly, existing state-of-the-art computational discovery approaches as well as prior manual analysis did not detect these or any other statistically significant T cell sub-populations associated with anti-PD-1 treatment in either data set. We further validate our methodology by using FAUST to replicate the discovery of a previously reported myeloid correlate in a different published melanoma trial, and validate the correlate by identifying itde novoin two additional independent trials. FAUST’s phenotypic annotations can be used to perform cross-study data integration in the presence of heterogeneous data and diverse immunophenotyping staining panels, enabling hypothesis-driven inference about cell sub-population abundance through a multivariate modeling framework we callPhenotypic andFunctionalDifferentialAbundance (PFDA). We demonstrate this approach on data from myeloid and T cell panels across multiple trials. Together, these results establish FAUST as a powerful and versatile new approach for unbiased discovery in single-cell cytometry.
Liquid-liquid phase separation and fibrillization of tau are independent processes with overlapping conditions
Authors: Lin Yanxian , Fichou Yann , Zeng Zhikai , Hu Nicole Y. , Han Songi
Published: July 2019

Amyloid aggregation of the microtubule binding protein tau is a hallmark of Alzheimer’s disease and many other neurodegenerative diseases. Recently, tau has been found to undergo liquid-liquid phase separation (LLPS) near physiological conditions. Although LLPS and aggregation have been shown to simultaneously occur under certain common conditions, it remains to be seen whether tau LLPS promotes aggregation, or if they are two independent processes. In this study, we address this question by combining multiple biochemical and biophysical assays in vitro. We investigated the impacts of LLPS on tau aggregation at three stages: conformation of tau, kinetics of aggregation and fibril quantity. We showed that none of these properties are influenced directly by LLPS, while amyloid aggregation propensity of tau can be altered without affecting its LLPS behavior. LLPS and amyloid aggregation of tau occur under overlapping conditions of enhanced intermolecular interactions and localization, but are two independent processes.
Predictors of arm non-use in chronic stroke: a preliminary investigation
Authors: Buxbaum Laurel J. , Varghese Rini , Stoll Harrison , Winstein Carolee J.
Published: July 2019

Background

The phenomenon of non-use after stroke is characterized by failure to use the contralesional arm despite adequate capacity. It has been suggested that non-use is a consequence of the greater effort and/or attention required to use the affected limb, but such accounts have not been tested, and we have poor understanding of the characteristics of individuals who exhibit non-use.

Objective

We aimed to provide preliminary evidence regarding the demographic, neuropsychological, and psychological factors that may influence non-use in chronic stroke.

Methods

Twenty chronic stroke survivors (10 left and 10 right hemisphere stroke) with mild-to-moderate sensory-motor impairment on the upper extremity Fugl-Meyer (UEFM) were assessed with the Actual Amount of Use Test (AAUT), which measures the disparity between amount of use and quality of movement in “spontaneous” versus “forced” conditions. Participants were als assessed with measures of limb apraxia, spatial neglect, attention/arousal, and self-efficacy (confidence in arm movement). Using stepwise multiple regression, we determined whether demographic information and/or performance on these measures predicted AAUT non-use scores.

Results

Scores on the UEFM as well as attention/arousal and self-efficacy predicted the degree of non-use. Attention/arousal predicted non-use above and beyond UEFM.

Conclusions

Given the complexity of the non-use phenomenon, it follows that a combination of impairment, attention/arousal, and perceived confidence predicted non-use behavior. That a measure of attention/arousal predicted non-use behavior above and beyond sensory-motor functioning highlights the importance of motivated engagement to drive use of the paretic limb. Larger-scale studies incorporating additional measures (e.g., mental health, lesion volume and white matter connectivity, pain, motivation) will be important for future investigations.
Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain
Authors: Kadoya Syun-suke , Urayama Syun-ichi , Nunoura Takuro , Hirai Miho , Takaki Yoshihiro , Kitajima Masaaki , Nakagomi Toyoko , Nakagomi Osamu , Okabe Satoshi , Nishimura Osamu , Sano Daisuke
Published: July 2019

RNA viruses form a dynamic distribution of mutant swarm (termed “quasispecies”) due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission ex-emplifies a bottleneck effect in that only part of a viral population can reach the next susceptible hosts. In the present study, the rhesus rotavirus (RRV) strain of Rotavirus A was serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001 in duplicate (the 1st and 2nd lineages), and three phenotypes (infectious titer, cell binding ability and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and non-synonymous substitutions on rotaviral genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate that a stronger bottleneck effect can create more sequence spaces for minor mutants originally existing in a hidden layer of mutant swarm.

IMPORTANCE

In this study, we investigated a bottleneck effect on an RRV population, which may drastically impact a viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified a human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck could create a new sequence space in a population for minor mutants originally existing in a hidden layer of a mutant swarm of the double-stranded RNA virus. The results of this study suggest that the genetic drift caused by a bottleneck in a human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected by the molecular epidemiology using next generation sequencing in which the viral genetic diversity within a viral population is investigated.
Antiviral and virucidal activities against herpes simplex viruses of umesu phenolics extracted from Japanese apricot
Authors: Nishide Mitsunori , Ikeda Keiko , Mimura Hisa , Yoshida Minoru , Mitani Takahiko , Hajime Koyama Augustine
Published: July 2019

Umesu phenolics were obtained from the salt extracts of Japanese apricot (Nanko‐mume cultivar of Prunus mume Sieb. et Zucc.) as purified phenolics. The antiviral activities of umesu phenolics obtained were then examined against herpes simplex virus type 1 (HSV‐1) and type 2 (HSV‐2), enveloped DNA viruses. The phenolics inhibited the multiplication of these viruses when added to the culture media of the infected cells. This inhibition occurred at phenolic concentrations at which they showed no severe cytotoxicity. One‐step growth experiments showed that the eclipse period in the HSV‐1 multiplication process was extended in the presence of umesu phenolics and that the addition of phenolics after the completion of viral DNA replication did not affect their multiplication. More drastic effects were observed on virucidal activities against HSV‐1 and HSV‐2; the infectivity decreased to 0.0001 when infected cells were incubated with 3 mg/ml phenolics at 30°C for 5 min. These results demonstrate the antiviral and virucidal activities of umesu phenolics and suggest a potential pharmacological use for these phenolics as a sanitizing or preventive medicine against superficial HSV infections.
HprKXcc is a serine kinase that regulates virulence in the Gram‐negative phytopathogen Xanthomonas campestris
Authors: Li Rui‐Fang , Cui Ping , Wei Ping‐Zhen , Liu Xing‐Yan , Tang Ji‐Liang , Lu Guang‐Tao
Published: July 2019

The HprK serine kinase is a component of the phosphoenolpyruvate phosphotransferase system (PTS) of bacteria that generally regulates catabolite repression through phosphorylation/dephosphorylation of the PTS protein PtsH at a conserved serine residue. However, many bacteria do not encode a complete PTS or even have an HprK homologue. Xanthomonas campestris pv. campestris (Xcc) is a pathogen that cause black rot disease in crucifer plants and one of the few Gram‐negative bacteria that encodes a homologue of HprK protein (herein HprKXcc). To gain insight into the role of HprKXcc and other PTS‐related components in Xcc we individually mutated and phenotypically assessed the resulting strains. Deletion of hprK
Xcc demonstrated its requirement for virulence and other diverse cellular processes associated including extracellular enzyme activity, extracellular‐polysaccharide production and cell motility. Global transcriptome analyses revealed the HprKXcc had a broad regulatory role in Xcc. Additionally, through overexpression, double gene deletion and transcriptome analysis we demonstrated that hprK
Xcc shares an epistatic relationship with ptsH. Furthermore, we demonstrate that HprKXcc is a functional serine kinase, which has the ability to phosphorylate PtsH. Taken together, the data illustrates the previously unappreciated global regulatory role of HprKXcc and previously uncharacterized PTS components that control virulence in this pathogen.
Diagnostic yield of computed tomography‐guided biopsy and aspiration for vertebral osteomyelitis
Authors: Ang Megan T , Wong Geoffrey R , Wong Davy R , Clements Warren , Joseph Tim
Published: July 2019

Introduction

The diagnostic yield of and best approaches for imaging‐guided percutaneous biopsy for vertebral osteomyelitis is controversial. Early studies suggest yields of up to 90%; however, recent evidence shows lower yields of 30–40%. We aim to determine yield and predictors of yield in percutaneous CT‐guided biopsies in vertebral osteomyelitis.

Methods

We conducted a retrospective observational single‐centre study cohort study of all patients presenting for vertebral biopsy or aspiration between 2014 and 2018. Only patients undergoing biopsy for suspected infection were included. Patients with malignant indications were excluded. Comprehensive review of medical records was performed for clinical presentation, comorbidities, imaging, biomarkers, microbiology and treatment.

Results

Overall, 40 out of 88 biopsies were performed for suspected infection, in 36 patients. Mean age was 59 ± 18 years; 29 (81%) were male. Of the 40 samples, an organism was identified in 14 samples (35%). Gram‐positive organisms were most commonly identified; Staphylococcus aureus was cultured in 7 (50%) of samples. Mean admission CRP was significantly higher in patients with identified organisms compared to those without (137 ± 106 vs 54 ± 78, P = 0.008). Aspiration was a strong independent predictor of positive microbiological growth on multivariate analysis (OR 6.52 [1.25–34.02], P = 0.026). Biopsy or aspiration aided clinical decision‐making in half of cases.

Conclusions

Percutaneous CT‐guided biopsy has a modest yield for identifying the culprit organism in suspected cases of vertebral osteomyelitis. Elevated CRP and aspiration of fluid collections are associated with improved microbiological yield and should be considered in deciding when and where to biopsy.
Cohesive framework for modelling plant cover class data
Authors: Irvine Kathryn M. , Wright Wilson J. , Shanahan Erin K. , Rodhouse Thomas J.
Published: July 2019

The study of plant distribution and abundance is a fundamental pursuit in ecology and conservation biology. Measuring plant abundance by visually assessing percent cover and recording a cover class is a common field method that yields ordinal data. Statistical models for ordinal data exist but entail cumbersome interpretations and sometimes restrictive assumptions.

We propose a Bayesian hierarchical framework for analysing cover class data that allows for linking ordinal observations to a latent beta distribution and accounts for zero inflation. Harnessing a latent beta distribution supports interpreting changes in abundance in terms of mean percent cover rather than odds ratios of cumulative cover classes as for cumulative link models. The zero augmentation allows for simultaneous inferences on both occurrence (distribution) and abundance. We show how our model can account for true and false zeros, misclassification of cover classes, multiple species and hierarchical sampling designs, using empirical examples and simulations.

Simulated observation errors, when ignored, led to models overestimating abundance and underestimating occurrence. Based on simulations, we found no substantial difference between mean percent cover estimates when analyzing ordinal cover classes versus continuous percent cover as the response. Our empirical datasets displayed high probability of detection (>0.85 on average for all species), likely due to the sampling design used and training of observers. Probability of occurrence was slightly underestimated for bare ground, Artemisia tridentata, Elycap medusae, and Poa secunda using a model that ignored imperfect detection. Estimated mean percent cover was not substantially impacted by ignoring measurement error for five plant species and bare ground.

Our modelling framework for cover class data allows for an explicit separation of distribution from abundance and, importantly, allows for interpreting species–environment relationships in terms of variation in mean percent cover as compared to cumulative odds ratios. The beta distribution inherently accommodates heteroscedasticity and skewness, statistical properties that are a consequence of spatially aggregated patterns common to plant survey data. Recording cover classes provides a reliable, efficient way to measure plants and our simulations suggest little loss of information compared to assuming continuous percent cover. We provide JAGS and Stan model code for implementation.