bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly
Authors: Li Wenqiang , Li Shuntang , Qiao Jie , Wang Fei , Liu Yang , He Ruyi , Liu Yi , Ma Lixin
Published: May 2018

CRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.
Hydrogen-based metabolism – an ancestral trait in lineages sibling to the Cyanobacteria
Authors: Matheus Carnevali Paula B. , Schulz Frederik , Castelle Cindy J. , Kantor Rose , Shih Patrick , Sharon Itai , Santini Joanne M. , Olm Matthew , Amano Yuki , Thomas Brian C. , Anantharaman Karthik , Burstein David , Becraft Eric D. , Stepanauskas Ramunas , Woyke Tanja , Banfield Jillian F.
Published: May 2018

The metabolic machinery from which microbial aerobic respiration evolved is tightly linked to the origins of oxygenic Cyanobacteria (Oxyphotobacteria). Even though the majority of Oxyphotobacteria are photoautotrophs and can use carbohydrates with oxygen (O2) as the electron acceptor, all are fermenters under dark anoxic conditions. Studies suggest that the ancestor of Oxyphotobacteria may have used hydrogen (H2) as an electron donor and that two types of NiFe hydrogenases are essential for its oxidation. Melainabacteria and Sericytochromatia, close phylogenetic neighbors to Oxyphotobacteria comprise fermentative and aerobic representatives, or organisms capable of both. Margulisbacteria (candidate divisions RBX-1 and ZB3) and Saganbacteria (candidate division WOR-1), a novel cluster of bacteria phylogenetically related to Melainabacteria, Sericytochromatia and Oxyphotobacteria may further constrain the metabolic platform in which oxygenic photosynthesis and aerobic respiration arose. Here, we predict the metabolisms of Margulisbacteria and Saganbacteria from new and published metagenome-assembled genomes (MAGs) and single amplified genomes (SAGs), and compare them to their phylogenetic neighbors. Sediment-associated Margulisbacteria are predicted to have a fermentation-based metabolism featuring a variety of hydrogenases, a nitrogenase for nitrogen (N2) fixation, and electron bifurcating complexes involved in cycling of ferredoxin and NAD(P)H. Overall, the genomic features suggest the capacity for metabolic fine-tuning under strictly anoxic conditions. In contrast, the genomes of Margulisbacteria from the ocean ecosystem encode an electron transport chain that supports aerobic growth. Similarly, some Saganbacteria genomes encode various hydrogenases, and others may have the ability to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Like Melainabacteria and Sericytochromatia, Margulisbacteria and Saganbacteria have diverse energy metabolisms capable of fermentation, and aerobic or anaerobic respiration. In summary, our findings support the hypothesis that the ancestor of these groups was an anaerobe in which fermentation and H2 metabolism were central metabolic features. Our genomic data also suggests that contemporary lineages sibling to the Oxyphotobacteria may have acquired the ability to use O2 as a terminal electron acceptor under certain environmental conditions.
The marine goby – alpheid shrimp symbiosis doesnotcorrelate with larger fish eye size
Authors: Stiefel Klaus M. , Reyes Rodolfo B.
Published: May 2018

The symbiosis between marine gobies and Alpheid shrimp is based on an exchange of sensory performance (look-out for predators) by the goby versus muscular performance (burrow digging) by the shrimp. Using a comparative approach, we estimate the excess investment by the goby into its visual system as a consequence of the symbiosis. When correlating eye size with fish length for both shrimp-associated and solitary gobies, we find that the shrimp-associated gobies donothave larger eyes than size-matched solitary gobies.

We do find a trend, however, in that the shrimp-associated gobies live at shallower depths than the solitary gobies, indicative of the visual nature of the symbiosis. We discuss the implications of symbiosis based on large and small energy investments, and the evolutionary modifications likely necessary to include shrimp-goby communication into the behavior of the goby.
Accounting for taste: A multi-attribute neurocomputational model explains the neural dynamics of choices for self and others
Authors: Harris Alison , Clithero John A. , Hutcherson Cendri A.
Published: May 2018

How do we make choices for others with different preferences from our own? Although neuroimaging studies implicate similar circuits in representing preferences for oneself and others, some models propose that additional corrective mechanisms come online when choices for others diverge from one’s own preferences. Here we used event-related potentials (ERP) in humans, in combination with computational modeling, to examine how social information is integrated in the time leading up to choices for oneself and others. Hungry male and female participants with unrestricted diets selected foods for themselves, a similar unrestricted eater, and a dissimilar, self-identified healthy eater. Across choices for both oneself and others, ERP value signals emerged within the same time window but differentially reflected taste and health attributes based on the recipient’s preferences. Choices for the dissimilar recipient were associated with earlier activity localized to brain regions implicated in social cognition including temporoparietal junction (TPJ). Finally, response-locked analysis revealed a late ERP component specific to choices for the similar recipient, localized to the parietal lobe, that appeared to reflect differences in the response threshold based on uncertainty. A multi-attribute computational model supported the link between specific ERP components and distinct model parameters, and was not significantly improved by adding time-dependent dual processes. Model simulations suggested that longer response times (RTs) previously associated with effortful correction may alternatively arise from higher choice uncertainty. Together these results provide a parsimonious neurocomputational mechanism for social decision-making, additionally explaining divergent patterns of choice and RT data in decisions for oneself and others.

Significance Statement

How do we choose for others, particularly when they have different preferences? Whereas some studies suggest that similar neural circuits underlie decision-making for oneself and others, others argue for additional, slower perspective-taking mechanisms. Combining event-related potentials with computational modeling, we found that integration of others’ preferences occurs over the same timescale as for oneself, while differentially tracking recipient-relevant attributes. Although choosing for others took longer and produced differences in late-emerging neural responses, computational modeling attributed these patterns to greater response caution rather than egocentric bias correction. Computational simulations also correctly predicted when and why choosing differently for others takes longer, suggesting that a model incorporating value integration and evidence accumulation can parsimoniously account for complex patterns in social decision-making.
Base-pair Ambiguity and the Kinetics of RNA Folding
Authors: Zhou Guangyao , Loper Jackson , Geman Stuart
Published: May 2018

Background

A folding RNA molecule encounters multiple opportunities to form non-native yet energetically favorable pairings of nucleotide sequences. Given this forbidding free-energy landscape, mechanisms have evolved that contribute to a directed and efficient folding process, including catalytic proteins and error-detecting chaperones. Among structural RNA molecules we make a distinction between “bound” molecules, which are active as part of ribonucleoprotein (RNP) complexes, and “unbound,” with physiological functions performed without necessarily being bound in RNP complexes. We hypothesized that unbound molecules, lacking the partnering structure of a protein, would be more vulnerable than bound molecules to kinetic traps that compete with native stem structures. We defined an “ambiguity index”—a normalized function of the primary and secondary structure of an individual molecule that measures the number of kinetic traps available to nucleotide sequences that are paired in the native structure, presuming that unbound molecules would have lower indexes. The ambiguity index depends on the purported secondary structure, and was computed under both the comparative (“gold standard”) and an equilibrium-based prediction which approximates the minimum free energy (MFE) structure. Arguing that kinetically accessible metastable structures might be more biologically relevant than thermodynamic equilibrium structures, we also hypothesized that MFE-derived ambiguities would be less effective in separating bound and unbound molecules.

Results

We have introduced an intuitive and easily computed function of primary and secondary structures that measures the availability of complementary sequences that could disrupt the formation of native stems on a given molecule—an ambiguity index. Using comparative secondary structures, the ambiguity index is systematically smaller among unbound than bound molecules, as expected. Furthermore, the effect is lost when the presumably more accurate comparative structure is replaced instead by the MFE structure.

Conclusions

A statistical analysis of the relationship between the primary and secondary structures of non-coding RNA molecules suggests that stem-disrupting kinetic traps are substantially less prevalent in molecules not participating in RNP complexes. In that this distinction is apparent under the comparative but not the MFE secondary structure, the results highlight a possible deficiency in structure predictions when based upon assumptions of thermodynamic equilibrium.
The QNOAEL vs. BMD for Point of Departure
Authors: Dickerson Cynthia , Lodder Robert A.
Published: May 2018

Quantile bootstrap (QB) methods can be applied to the problem of estimating the No Observed Adverse Effect Level (NOAEL) of a New Molecular Entity (NME) to anticipate a safe starting dose for beginning clinical trials. An estimate of the NOAEL from the extended QB method (called the QNOAEL) can be calculated using multiple disparate studies in the literature and/or from laboratory experiments. The QNOAEL is similar in some ways to the Benchmark Dose (BMD) and is superior to the BMD in others. The Benchmark Dose method is currently widely used in toxicological research.

Results are used in a simulation based on nonparametric cluster analysis methods to calculate confidence levels on the difference between the Effect and the No Effect studies. The QNOAEL simulation generates an intuitive curve that is comparable to the dose-response curve.

The QNOAEL of ellagic acid (EA) will be calculated for clinical trials of its use as a component therapeutic agent (in BSN476) for treating Chikungunya infections. This will be the first application of QB to the problem of NOAEL estimation for a drug. The specific aims of the proposed study are to evaluate the accuracy and precision of the QB Simulation and QNOAEL compared to the Benchmark Dose Method, and to calculate the QNOAEL of EA for BSN476 Drug Development.
Netrin G1 promotes pancreatic tumorigenesis through cancer associated fibroblast driven nutritional support and immunosuppression
Authors: Francescone Ralph , Vendramini-Costa Débora Barbosa , Franco-Barraza Janusz , Wagner Jessica , Muir Alexander , Lau Allison N. , Gabitova Linara , Pazina Tatiana , Gupta Sapna , Luong Tiffany , Shah Neelima , Rollins Dustin , Malik Ruchi , Thapa Roshan , Restifo Diana , Zhou Yan , Cai Kathy Q. , Hensley Harvey H. , Tan Yinfei , Kruger Warren D. , Devarajan Karthik , Balachandran Siddharth , Klein-Szanto Andres J. , Wang Huamin , El-Deiry Wafik S. , Vander Heiden Matthew G. , Peri Suraj , Campbell Kerry S. , Astsaturov Igor , Cukierman Edna
Published: May 2018

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturingin vitroassays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. NetG1+cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. NetG1+CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These pro-tumor functions were controlled by a signaling circuit downstream to NetG1, which was comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally blocking NetG1 with a neutralizing antibody stuntedin vivotumorigenesis, suggesting NetG1 as potential target in PDAC.

Significance

PDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered roles for NetG1 in CAFs to promote tumorigenesis. NetG1 was important for two major CAF functions: the metabolic support of PDAC cells and the intrinsic immunosuppressive capacity of CAFs. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Moreover, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity, and identified a signaling circuit that governs NetG1 functions. Finally, we demonstrated the therapeutic potential of inhibiting NetG1in vivoby limiting tumorigenesis in mice with a neutralizing antibody, illustrating that targeting stromal NetG1 could be an attractive therapeutic approach.
Let-7i-5p regulation of cell morphology and migration through distinct signaling pathways in normal and pathogenic fibroblasts from urethra
Authors: Zhang Kaile , Chen Jun , Zhou Shukui , Yang Ranxin , Wang Ying , Fu Qiang , Chen Rong , Fang Xiaolan
Published: May 2018

Pelvic fracture urethral distraction defects (PFUDD) is a common disease that could severely affect patients’ life quality, yet little is known about the molecular mechanism associated with pathogenic fibrosis in PFUDD. In this study, we found that let-7i-5p could regulate different cellular events in normal and pathogenic fibroblasts through three distinct signaling pathways. Interestingly, those regulations are compromised during the translation from mRNA to protein, and partially based on pathogenic status of the fibroblasts. By analyzing the molecular mechanism associated with its function, we conclude that let-7i-5p plays an essential role in regulating cell shape and tissue elasticity, cell migration, cell morphology and cytoskeleton, and could serve as a potential target for clinical treatment of urethral stricture patients.
A competence-regulated toxin-antitoxin system in Haemophilus influenzae
Authors: Black Hailey Findlay , Mastromatteo Scott , Sinha Sunita , Ehrlich Rachel L. , Nislow Corey , Mell Joshua Chang , Redfield Rosemary J.
Published: May 2018

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenae, the DNA uptake machinery is induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that HI0659—which is absolutely required for DNA uptake— encodes the antitoxin of a competence-regulated toxin-antitoxin operon (‘toxTA’), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the toxin restores uptake to the antitoxin mutant. In addition to the expected Sxy-and CRP-dependent-competence promoter, transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We present evidence that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture, and we show that the toxin gene has been inactivated by deletion in many H. influenzae strains. We suggest that this competence-regulated toxin-antitoxin system may facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions, or alternatively be a simple genetic parasite.
crossword: A data-driven simulation language for the design of genetic-mapping experiments and breeding strategies
Authors: Walid Korani , Vaughn Justin N
Published: May 2018

The simulation of genetic systems can save time and resources by optimizing the logistics of an experiment. Current tools are difficult to use by those unfamiliar with programming, and these tools rarely address the actual genetic structure of the population under study. Here, we introduce crossword, which utilizes the widely available results of re-sequencing and genomics data to create more realistic simulations and to simplify user input. The software was written in R, making installation and implementation straightforward. Because crossword is a domain-specific language, it allows complex and unique simulations to be performed, but the language is supported by a graphical interface that guides users through functions and options. We first show crossword’s utility in QTL-seq design, where its output accurately reflects empirical data. By introducing the concept of levels to reflect family relatedness, crossword is suitable to a broad range of breeding programs and crops. Using levels, we further illustrate crossword’s capabilities by examining the effect of family size and number of selfing generations on phenotyping accuracy and genomic selection. Additionally, we explore the ramifications of effect polarity among parents in a mapping cross, a scenario that is common in crop genetics but often difficult to simulate. Given the ease of use and apparent realism, we anticipate crossword will quickly become a “bicycle for the [geneticist’s] mind”.
Allostery in oligomeric receptor models
Authors: Douglas Conradi Smith Gregory
Published: May 2018

We show how equilibrium binding curves of receptor heterodimers and homodimers can be expressed as rational polynomial functions of the equilibrium binding curves of the constituent monomers, without approximation and without assuming independence of receptor monomers. Using a distinguished spanning tree construction for reduced graph powers, the method properly accounts for thermodynamic constraints and allosteric coupling between receptor monomers.
Revealing subthreshold motor contributions to perceptual confidence
Authors: Gajdos Thibault , Fleming Stephen M. , Garcia Marta Saez , Weindel Gabriel , Davranche Karen
Published: May 2018

Established models of perceptual metacognition, the ability to evaluate our perceptual judgments, posit that perceptual confidence depends on the strength or quality of feedforward sensory evidence. However, alternative theoretical accounts suggest the entire perception-action cycle, and not only variation in sensory evidence, is monitored when evaluating confidence in one’s percepts. Such models lead to the counterintuitive prediction that perceptual confidence should be directly modulated by features of motor output. To evaluate this proposal here we recorded electromyographic (EMG) activity of motor effectors while subjects performed a near-threshold perceptual discrimination task and reported their confidence in each response. A subset of trials exhibited sub-threshold EMG activity in response effectors before a decision was made. Strikingly, trial-by-trial analysis showed that confidence, but not accuracy, was significantly higher on trials with subthreshold motor activation. These findings support a hypothesis that preparatory motor activity impacts upon confidence over and above performance, consistent with models in which perceptual metacognition integrates information across the perception-action cycle.
Multi-Tissue Transcriptome-Wide Association Study Identifies 26 Novel Candidate Susceptibility Genes for High Grade Serous Epithelial Ovarian Cancer
Authors: Gusev Alexander , Lawrenson Kate , Segato Felipe , Fonseca Marcos A.S. , Kar Siddhartha , Vavra Kevin C. , Lee Janet M , Pejovic Tanya ,   Ovarian Cancer Association Consortium, Karlan Beth Y. , Freedman Matthew L. , Noushmehr Houtan , Pharoah Paul D.P. , Pasaniuc Bogdan , Gayther Simon A.
Published: May 2018

Genome-wide association studies (GWASs) have identified about 30 different susceptibility loci associated with high grade serous ovarian cancer (HGSOC) risk. We sought to identify potential susceptibility genes by integrating the risk variants at these regions with genetic variants impacting gene expression and splicing of nearby genes. We compiled gene expression and genotyping data from 2,169 samples for 6 different HGSOC-relevant tissue types. We integrated these data with GWAS data from 13,037 HGSOC cases and 40,941 controls, and performed a transcriptome-wide association study (TWAS) across >70,000 significantly heritable gene/exon features. We identified 24 transcriptome-wide significant associations for 14 unique genes, plus 90 significant exon-level associations in 20 unique genes. We implicated multiple novel genes at risk loci, e.g.LRRC46at 19q21.32 (TWASP=1×10−9) and aPRC1splicing event (TWASP=9×10−8) which was splice-variant specific and exhibited no eQTL signal. Functional analyses in HGSOC cell lines found evidence of essentiality forGOSR2, INTS1, KANSL1andPRC1; with the latter gene showing levels of essentiality comparable to that ofMYC. Overall, gene expression and splicing events explained 41% of SNP-heritability for HGSOC (s.e. 11%,P=2.5×10−4), implicated at least one target gene for 6/13 distinct genome-wide significant regions and revealed 2 known and 26 novel candidate susceptibility genes for HGSOC.

STATEMENT OF SIGNIFICANCE

For many ovarian cancer risk regions, the target genes regulated by germline genetic variants are unknown. Using expression data from >2,100 individuals, this study identified novel associations of genes and splicing variants with ovarian cancer risk; with transcriptional variation now explaining over one-third of the SNP-heritability for this disease.
Individual behavioral type captured by a Bayesian model comparison of cap making by sponge crabs
Authors: Harada Keita , Hayashi Naoki , Kagaya Katsushi
Published: May 2018

‘Animal personality’ is considered to be developed through complex interactions of an individual with its surrounding environment. How can we quantify the ‘personality’ of an individual? Quantifying intra- and inter-individual variability of behavior, or individual behavioral type, appears to be a prerequisite in the study of animal personality. We propose a statistical method from a predictive point of view to measure the appropriateness of our assumption of ‘individual’ behavior in repeatedly measured behavioral data from several individuals. For a model case, we studied the sponge crab Lauridromia dehaani known to make and carry a ‘cap’ from a natural sponge for camouflage. Because a cap is most likely to be rebuilt and replaced repeatedly, we hypothesized that each individual crab would grow a unique behavioral type and it would be observed under an experimentally controlled environmental condition. To test the hypothesis, we conducted behavioral experiments and employed a new Bayesian model-based comparison method to examine whether crabs have individual behavioral types in the cap making behavior. Crabs were given behavioral choices by using artificial sponges of three different sizes. We modeled the choice of sponges, size of the trimmed part of a cap, size of the cavity of a cap, and the latency to produce a cap, as random variables in 26 models, including hierarchical models specifying the behavioral types. In addition, we calculated the marginal-level widely applicable information criterion (mWAIC) values for hierarchical models to evaluate and compared them with the non-hierarchical models from the predictive point of view. As a result, the crabs of less than about 9 cm in size were found to make caps from the sponges. The body size explained the behavioral variables namely, choice, trimmed cap characteristics, and cavity size, but not latency. Furthermore, we captured the behavioral type as a probabilistic distribution structure of the behavioral data by comparing WAIC. Our statistical approach is not limited to behavioral data but is also applicable to physiological or morphological data when examining whether some group structure exists behind fluctuating empirical data.
Thermal plasticity of the circadian clock is under nuclear and cytoplasmic control in wild barley
Authors: Bdolach Eyal , Prusty Manas Ranjan , Faigenboim-Doron Adi , Filichkin Tanya , Helgerson Laura , Schmid Karl J , Greiner Stephan , Fridman Eyal
Published: May 2018

Temperature compensation, expressed as the ability to maintain clock characteristics (mainly period) in face of temperature changes, is considered a key feature of circadian clock systems. In this study, we explore the genetic basis for circadian clock plasticity under high temperatures by utilizing a new doubled haploid (DH) population derived from two reciprocalHordeum vulgaresps.spontaneumhybrids genotypes (crosses between B1K-50-04 and B1K-09-07). Genotyping by sequencing of DH lines indicated a rich recombination landscape, with minor fixation (less than 8%), for one of the parental alleles, yet with prevalent and varied segregation distortion across seven barley chromosomes. Phenotyping was conducted with a high-throughput platform under optimal and high temperature environments. Genetic analysis, which included QxE and binary-threshold models, identified a significant influence of the maternal organelle genome (the plasmotype), as well as several nuclear quantitative trait loci (QTL), on clock phenotypes (free-running period and amplitude). Moreover, it showed the differential contribution of cytoplasmic genome clock rhythm buffering against high temperature. Resequencing of the parental chloroplast indicated the presence of several candidate genes underlying these significant effects. This first reported plasmotype-driven clock plasticity paves the way for identifying an hitherto unknown impact of nuclear and plasmotype variations on clock robustness and on plant adaptation to changing environments.

Highlight

Circadian clock robustness to high temperature is controlled by nuclear and plasmotype quantitative trait loci in a wild barley(Hordeum vulgaressp.spontaneum)reciprocal doubled haploid population.
Genetic determinants of resistance and virulence among carbapenemase-producing Klebsiella pneumoniae from Sri Lanka
Authors: Zhu Chendi , Liyanapathirana Veranja , Li Carmen , Pinto V. , Hui Mamie , Lo Norman , Wong Kam-Tak , Dissanayake N. , Ip Margaret
Published: May 2018

Whole genome sequencing of carbapenem-resistant Enterobacteriaceae from the intensive care units of a Sri Lankan teaching hospital revealed the presence of carbapenemase gene, blaOXA-181 among isolates of carbapenase-producing Klebsiella pneumoniae belonging to ST437 (2 strains) and ST147 (8 strains) in 2015. blaOXA-181 genes were carried in three variants of ColE-type plasmids. Elevated carbapemen resistance were observed in ompK36 mutant strains. ESBL genes, plasmid–mediated quinolone resistance (PMQR) determinants (qnr, aac(6’)-Ib-cr, oqxAB) and mutations on chromosomal quinolone resistance-determining regions (QRDRs) with substitutions at ser83→I of gyrA and ser80→I of parC were observed. All strains possessed yersiniabactin on the mobile element ICEkp and other virulence determinants. Strict infection control and judicious use of antibiotics are warranted to prevent further spread of multidrug-resistant Klebsiella pneumoniae in the intensive care units.
Pathways of pathogenicity: The transcriptional stages of germination in the fatal fungal pathogen Rhizopus delemar
Authors: Sephton Clark Poppy C. S. , Muñoz Jose F. , Ballou Elizabeth R. , Cuomo Christina A. , Voelz Kerstin
Published: May 2018

Rhizopus delemar is an invasive fungal pathogen, responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which spores of Rhizopus delemar infect and cause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. Understanding the molecular mechanisms which underpin this transformation may be key to controlling mucormycosis, however the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes which take place over the course of germination. This process is characterised by four distinct stages: dormancy, isotropic swelling, germ tube emergence and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transforming Rhizopus delemar from a single to a multicellular organism.

Importance

Germination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.
Analysis of Transcripts and splice isoforms in Red Clover (Trifolium pratense L.) by single-molecule long-read sequencing
Authors: Chao Yuehui , Yuan Jianbo , Li Sifeng , Jia Siqiao , Han Liebao , Xu Lixin
Published: May 2018

Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover. In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2,194 novel isoforms from novel genes were identified. A total of 5,492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8,719 including 1,86,517 transcripts have at least one poly(A) site. Furthermore, we identified 4,333 long non-coding RNAs and 3,762 fusion transcripts. Our results show the feasibility of deep sequencing full-length RNA from red clover transcriptome on a single-molecule level.
Distinct Patterns of the Autofluorescence of Body Surface: Potential Novel Diagnostic Biomarkers for Stable Coronary Artery Disease and Myocardial Infarction
Authors: Qu Xinkai , Li Yujia , Tao Yue , Zhang Mingchao , Wu Danhong , Guan Shaofeng , Han Wenzheng , Ying Weihai
Published: May 2018

Searches for new biomarkers of stable coronary artery disease (SCAD) and myocardial infarction (MI) are critical for therapeutic efficacy of the diseases. In this study we tested our hypothesis that distinct patterns of autofluorescence (AF) of skin and fingernails may become novel diagnostic biomarkers for MI and SCAD. Our study has indicated that SCAD and MI have distinct patterns of AF of their body surface: First, the AF intensity of the MI patients is significantly higher than that of the Healthy and Low-Risk group in their right and left Centremetacarpus, Ventroforefinger, Dorsal Index Finger and Ventribrachium, while the AF intensity of the SCAD patients is significantly higher than that of the Healthy and Low-Risk group in their right and left Index Fingernails and Dorsal Antebrachium; and second, the AF asymmetry of the MI patients is significantly higher than that of the Healthy and Low-Risk group in their Centremetacarpus, Ventroforefinger, Index Fingernails and Dorsal Antebrachium, while the AF asymmetry of the SCAD patients is significantly higher than that of the Healthy and Low-Risk group in their Ventroforefinger, Dorsal Index Finger, Dorsal Centremetacarpus and Index Fingernails. Moreover, the AF pattern of acute ischemic stroke is markedly different from those of SCAD and MI. The oxidative stress in the plasma of the MI and SCAD patients may cause the increased AF by altering the AF of keratins. Collectively, our study has indicated that SCAD and MI patients have distinct patterns of AF changes, which may become novel diagnostic biomarkers for SCAD and MI.
Heavy metal sensitivities of gene deletion strains forITT1andRPS1Aconnect their activities to the expression ofURE2, a key gene involved in metal detoxification in yeast
Authors: Moteshareie Houman , Hajikarimlou Maryam , Indrayanti Alex Mulet , Burnside Daniel , Dias Ana Paula , Lettl Clara , Ahmed Duale , Omidi Katayoun , Kazmirchuk Tom , Puchacz Nathalie , Zare Narges , Takallou Sarah , Naing Thet , Hernández Raúl Bonne , Willmore William G. , Babu Mohan , McKay Bruce , Samanfar Bahram , Holcik Martin , Golshani Ashkan
Published: May 2018

Heavy metal and metalloid contaminations are among the most concerning types of pollutant in the environment. Consequently, it is important to investigate the molecular mechanisms of cellular responses and detoxification pathways for these compounds in living organisms. To date, a number of genes have been linked to the detoxification process. The expression of these genes can be controlled at both transcriptional and translational levels. In baker’s yeast,Saccharomyces cerevisiae, resistance to a wide range of toxic metals is regulated by glutathione S-transferases. YeastURE2encodes for a protein that has glutathione peroxidase activity and is homologous to mammalian glutathione S-transferases. TheURE2expression is critical to cell survival under heavy metal stress. Here, we report on the finding of two genes,ITT1, an inhibitor of translation termination, andRPS1A, a small ribosomal protein, that when deleted yeast cells exhibit similar metal sensitivity phenotypes to gene deletion strain forURE2. Neither of these genes were previously linked to metal toxicity. Our gene expression analysis illustrates that these two genes affectURE2mRNA expression at the level of translation.

Summary statement

We identified two yeast genes,ITT1andRPS1A, that when deleted, results in yeast cells sensitivity to heavy metals and metalloids. Further investigation indicated that they influence the expression ofURE2gene, a key player in metal detoxification, by upregulating its translation. Our findings suggest thatITT1andRPS1Aplay an indirect role in responding to toxic metal stress.
Effects of gap junction misexpression on synapses between auditory sensory neurons and the giant fiber ofDrosophila melanogaster
Authors: Jezzini Sami H. , Merced Amelia , Blagburn Jonathan M.
Published: May 2018

The synapse between auditory Johnston’s Organ neurons (JONs) and the giant fiber (GF) ofDrosophilais structurally mixed, being composed of cholinergic chemical synapses and Neurobiotin-(NB) permeable gap junctions, which consist of the innexin Shaking-B (ShakB). Misexpression of one ShakB isoform, ShakB(N+16), in a subset of JONs that do not normally form gap junctions, results in theirde novodye coupling to the GF. This is similar to the effect of misexpression of the transcription factor Engrailed (En) in these same neurons, which also causes the formation of additional chemical synapses. In order to test the hypothesis that ShakB misexpression would similarly affect the distribution of chemical synapses, fluorescently-labeled presynaptic active zone protein (Brp) was expressed in JONs and the changes in its distribution were assayed with confocal microscopy. Both ShakB(N+16) and En increased the dye-coupling of JONs with the GF, indicating the formation of ectopic gap junctions. Conversely, expression of the ‘incorrect’ isoform, ShakB(N) abolishes dye coupling. However, while En misexpression increased the chemical contacts with the GF and the amount of GF medial branching, ShakB misexpression did not. ShakB immunocytochemistry showed that misexpression of ShakB(N+16) increases gap junctional plaques in JON axons but ShakB(N) does not. We conclude that both subsets of JON form chemical synapses onto the GF dendrites but only one population forms gap junctions, comprised of ShakB(N+16). Misexpression of this isoform in all JONs does not result in the formation of new mixed synapses but in the insertion of gap junctions, presumably at the sites of existing chemical synaptic contacts with the GF.
Fast animal pose estimation using deep neural networks
Authors: Pereira T.D. , Aldarondo D. E. , Willmore L. , Kislin M. , Wang S. S.-H. , Murthy M. , Shaevitz J. W.
Published: May 2018

Recent work quantifying postural dynamics has attempted to define the repertoire of behaviors performed by an animal. However, a major drawback to these techniques has been their reliance on dimensionality reduction of images which destroys information about which parts of the body are used in each behavior. To address this issue, we introduce a deep learning-based method for pose estimation, LEAP (LEAP Estimates Animal Pose). LEAP automatically predicts the positions of animal body parts using a deep convolutional neural network with as little as 10 frames of labeled data for training. This framework consists of a graphical interface for interactive labeling of body parts and software for training the network and fast prediction on new data (1 hr to train, 185 Hz predictions). We validate LEAP using videos of freely behaving fruit flies (Drosophila melanogaster) and track 32 distinct points on the body to fully describe the pose of the head, body, wings, and legs with an error rate of <3% of the animal’s body length. We recapitulate a number of reported findings on insect gait dynamics and show LEAP’s applicability as the first step in unsupervised behavioral classification. Finally, we extend the method to more challenging imaging situations (pairs of flies moving on a mesh-like background) and movies from freely moving mice (Mus musculus) where we track the full conformation of the head, body, and limbs.
Transmembrane exchange of fluorosugars: characterization of red cell GLUT1 kinetics using 19F NMR
Authors: Shishmarev D , Fontenelle CQ , Kuprov I , Linclau B , Kuchel PW
Published: May 2018

We developed a novel approach for quantifying the equilibrium-exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. Our method is based on an adapted kinetic theory describing the concentration dependence of the transmembrane-exchange rates of two simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogues in human erythrocytes (red blood cells: RBCs) using 19F nuclear magnetic resonance (NMR) exchange spectroscopy (EXSY). An inosine-based glucose-free medium was shown to promote survival and stable metabolism of RBCs over the duration of the experiments (a few hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we were able to categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 towards them. Differences in the membrane permeability of the three glucose analogues were qualitatively interpreted in terms of local perturbations in the bonding of substrates to amino-acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results will apply to the design of probes of glucose transport, or inhibitors of glucose metabolism in cells including those exhibiting the Warburg effect.

ABBREVIATIONS

EXSY

exchange spectroscopy

FDG

fluoro-deoxy-glucose

FDG-n

n-fluoro-n- deoxy-D-glucose (n = 2, 3, 4)

FID

free induction decay

Glc

D-glucose

NMR

nuclear magnetic resonance

RBC

red blood cell
pTSara-NatB, an improved N-terminal acetylation system for recombinant protein expression in E. coli
Authors: Rovere Matteo , Powers Alex E. , Patel Dushyant S. , Bartels Tim
Published: May 2018

N-terminal acetylation is one of the most common post-translational modifications of the eukaryotic proteome and regulates numerous aspects of cellular physiology, such as protein folding, localization and turnover. In particular α-synuclein, whose dyshomeostasis has been tied to the pathogenesis of several neurodegenerative disorders, is completely Nα-acetylated in nervous tissue. In this work, building on previous reports, we develop and characterize a bacterial N-terminal acetylation system based on the expression of the yeast N-terminal acetyltransferase B (NatB) complex under the control of the PBAD (L-arabinose-inducible) promoter. We show its functionality and the ability to completely Nα-acetylate our model substrate α-synuclein both upon induction of the construct with L-arabinose and also by only relying on the constitutive expression of the NatB genes.
Reconstruction of the cell entry pathway of an extinct virus
Authors: Robinson-McCarthy Lindsey R. , McCarthy Kevin R. , Raaben Matthijs , Piccinotti Silvia , Nieuwenhuis Joppe , Stubbs Sarah H. , Bakkers Mark J.G. , Whelan Sean P. J.
Published: May 2018

Endogenous retroviruses (ERVs), remnants of ancient germline infections, comprise 8% of the human genome. The most recently integrated includes human ERV-K (HERV-K) where several envelope (env) sequences remain intact. Viral pseudotypes decorated with one of those Envs are infectious. Using a recombinant vesicular stomatitis virus encoding HERV-K Env as its sole attachment and fusion protein (VSV-HERVK) we conducted a genome-wide haploid genetic screen to interrogate the host requirements for infection. This screen identified 11 genes involved in heparan sulfate biosynthesis. Genetic inhibition or chemical removal of heparan sulfate and addition of excess soluble heparan sulfate inhibit infection. Direct binding of heparin to soluble HERVK Env and purified VSV-HERVK defines it as critical for viral attachment. Cell surface bound VSV-HERVK particles are triggered to infect on exposure to acidic-pH, whereas acid pH pretreatment of virions blocks infection. Testing of additional endogenous HERV-Kenvsequences reveals they bind heparin and mediate acid pH triggered fusion. This work reconstructs and defines key steps in the infectious entry pathway of an extinct virus.

Author Summary

The genomes of all vertebrates are littered with the remains of once exogenous retroviruses. The properties of these ancient viruses that fostered germline colonization and their subsequent inheritance as genetic elements are largely unknown. The viral envelope protein (Env) dictates the cell entry pathway. Here we define host factors involved in the cell-entry of the youngest human ERV, HERV-K. Using a forward genetic screen, we identified heparan sulfate as a critical mediator of productive cell-entry. The abundance of this carbohydrate on almost all cells in the body suggests that HERV-K endogenization was a consequence of a broad tropism and not a specific targeting of germ cells. We demonstrate that multiple HERV-K Env protein encoded in the genome bind heparin. As HERV-K Envs are expressed in some transformed and virus-infected cells as well as during inflammation, it is tempting to speculate that this heparan sulfate binding property could be physiologically relevant during disease.
A congenital pain insensitivity mutation in the nerve growth factor gene uncouples nociception from affective pain in heterozygous humans and mice
Authors: Testa Giovanna , Perini Irene , Mainardi Marco , Morelli Chiara , Olimpico Francesco , Pancrazi Laura , Petrella Carla , Severini Cinzia , Florio Rita , Malerba Francesca , Heppenstall Paul , Costa Mario , Morrison India , Capsoni Simona , Cattaneo Antonino
Published: May 2018

Pain is an unpleasant but necessary sensory experience, which facilitates adaptive behaviours, such as fear. Despite recent advances, the question of how the pain experience influences learning of the fear response is still debated1,2. Genetic disorders rendering patients congenitally unable to feel pain have been described, and are usually explained by defects in peripheral nociceptors3-5. It is not known how growing up without pain affects central emotional and motivational responses to aversive stimuli. The rare autosomal recessive Hereditary Sensory and Autonomic Neuropathy type V (HSAN V) is caused by a mutation in the nerve growth factor (NGF) gene (R100W). HSAN V homozygous patients display a congenital indifference to painful events, with deficits in peripheral nociceptors but without overt cognitive impairment6. In contrast, heterozygous carriers do not present with pain-related deficits and have been identified only through pedigree and genetic screening7,8. We exploited this clinically silent population to dissociate nociceptive from affective features of pain. To address this we investigated both heterozygous knock-in mice bearing the R100W mutation (NGFR100W/m mice) and a cohort of HSAN V heterozygous human carriers. Surprisingly, we found that NGFR100W/m mice, despite normal responses to a noxious conditioning stimulus, show a deficit in learned fear while their social and innate fear responses are normal. The lack of pain-related fear response was linked to a reduced activation of anterior cingulate and motor cortices and of striatum, but not in primary somatosensory cortex. Likewise, human heterozygous R100W carriers, despite perceiving noxious stimuli and reporting subjective pain thresholds, show increased reaction latencies in response to painful stimulation, alongside a decreased subjective urgency to react. Functional magnetic resonance imaging (fMRI) revealed, comparably to the mouse data, an altered processing of painful stimuli in rostral anterior cingulate, medial premotor cortical regions and striatum. These findings from both human and mouse HSAN V carriers uncover behavioural and motivational consequences of a mild genetic pain insensitivity on the establishment of pain-dependent affective responses and memories.