bluuebunny/crossref_metadata_2025 huggingface dataset:
Periphilin self-association underpins epigenetic silencing by the HUSH complex
Authors: Prigozhin Daniil M. , Albecka Anna , Douse Christopher H. , Tchasovnikarova Iva A. , Timms Richard T. , Farleigh Laura E. , Oda Shun , Freund Stefan M. V. , Maslen Sarah , Lehner Paul J. , Modis Yorgo
Published: December 2019

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin formsα-helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.
An exhaustive analysis of single amino acid variants in helical transmembrane proteins
Authors: Llorian-Salvador Oscar , Bernhofer Michael , Mahlich Yannick , Rost Burkhard
Published: December 2019

Single nucleotide variants (SNVs) have been widely studied in the past due to being the main source of human genetic variation. Less is known about the effect of single amino acid variants (SAVs) due to the immense resources required for comprehensive experimental studies. In contrast, in silico methods predicting the effects of sequence variants upon molecular function and upon the organism are readily available and have contributed unexpected suggestions, e.g. that SAVs common to a human population (shared by >5% of the population) have, on average, more significant impact on the molecular function of proteins than do rare SAVs (shared by <1% of the population). Here, we investigated the impact of variants in a human population upon helical transmembrane proteins (TMPs). Three main results stood out. Firstly, common SAVs, on average, have stronger effects than rare SAVs for TMPs, and are enriched, in particular, in the membrane helices. Secondly, proteins with seven transmembrane helices (7TM, including GPCRs, i.e. G protein-coupled receptors) are depleted of SAVs in comparison to other proteins, possibly due to increased evolutionary constraints in these important proteins. Thirdly, rare SAVs with strong effect are significantly absent (over common SAVs) in signal peptide regions.
Lost and found: re-searching and re-scoring proteomics data aids the discovery of bacterial proteins and improves proteome coverage
Authors: Willems Patrick , Fijalkowski Igor , Van Damme Petra
Published: December 2019

Prokaryotic genome annotation is heavily dependent on automated gene annotation pipelines that are prone to propagate errors and underestimate genome complexity. We describe an optimized proteogenomic workflow that uses ribo-seq and proteomic data ofSalmonella Typhiumuriumto identify unannotated proteins or alternative protein forms raised upon alternative translation initiation (i.e. N-terminal proteoforms). This data analysis encompasses the searching of co-fragmenting peptides and post-processing with extended peptide-to-spectrum quality features including comparison to predicted fragment ion intensities. When applying this strategy, an enhanced proteome-depth is achieved as well as greater confidence for unannotated peptide hits. We demonstrate the general applicability of our pipeline by re-analyzing publicDeinococcus radioduransdatasets. Taken together, systematic re-analysis using available prokaryotic (proteome) datasets holds great promise to assist in experimentally-based genome annotation.
Methods for combining multiple correlated biomarkers with application to the study of low-grade inflammation and muscle mass in senior horses
Authors: Vsevolozhskaya Olga A. , Herbst Alisa C. , Adams Amanda A. , Burns Cailey , Cantu Bertsie , Barker Virginia D. , Zaykin Dmitri V.
Published: December 2019

The simplest analysis of biomarker data is based on a series of single biomarker hypothesis tests, followed by correction for multiple testing. However, it is intuitively plausible that a joint analysis of multiple biomarkers will have higher statistical power and promise improved discrimination over tests based on single markers. In this article, we study analytical properties of the approach for joint analysis of correlated summary statistics based on the test for quadratic forms (TQ). Based on the derivation of the TQ-distribution, we proposed a scale-location approximation of the TQ statistic, which we call approximate TQ. We show that the approximate TQ has very similar power to the traditional TQ test under varying correlation structures among biomarkers. Our application of both the TQ and the approximate TQ test to data on biomarkers for inflamm-aging – an age-related low-grade chronic inflammation – reveals an association between the percentage of IFNγ positive lymphocytes and overall muscle condition in senior horses.
Tuning Extracellular Electron Transfer byShewanella oneidensisUsing Transcriptional Logic Gates
Authors: Dundas Christopher M. , Keitz Benjamin K.
Published: December 2019

Extracellular electron transfer pathways, such as those in the bacteriumShewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux inS. oneidensishas proven challenging since a functional understanding of its EET pathway proteins and their effect on engineering parameterizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway ofS. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriateS. oneidensisknockout strains, we pinpointed construct/strain pairings that expressedcymA, mtrA, andmtrCwith maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures, that predictably turn on and turn off EET flux, respectively, in response to IPTG. Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs. inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.
Resolving the spatial profile of figure enhancement in human V1 through population receptive field modeling
Authors: Poltoratski Sonia , Tong Frank
Published: December 2019

The detection and segmentation of meaningful figures from their background is a core function of vision. While work in non-human primates has implicated early visual mechanisms in this figure-ground modulation, neuroimaging in humans has instead largely ascribed the processing of figures and objects to higher stages of the visual hierarchy. Here, we used high-field fMRI at 7Tesla to measure BOLD responses to task-irrelevant orientation-defined figures in human early visual cortex, and employed a novel population receptive field (pRF) mapping-based approach to resolve the spatial profiles of two constituent mechanisms of figure-ground modulation: a local boundary response, and a further enhancement spanning the full extent of the figure region that is driven by global differences in features. Reconstructing the distinct spatial profiles of these effects reveals that figure enhancement modulates responses in human early visual cortex in a manner consistent with a mechanism of automatic, contextually-driven feedback from higher visual areas.

Significance Statement

A core function of the visual system is to parse complex 2D input into meaningful figures. We do so constantly and seamlessly, both by processing information about visible edges and by analyzing large-scale differences between figures and background. While influential neurophysiology work has characterized an intriguing mechanism that enhances V1 responses to perceptual figures, we have a poor understanding of how the early visual system contributes to figure-ground processing in humans. Here, we use advanced computational analysis methods and high-field human fMRI data to resolve the distinct spatial profiles of local edge and global figure enhancement in the early visual system (V1 and LGN); the latter is distinct and consistent a mechanism of automatic, stimulus-driven feedback from higher-level visual areas.
Visualizing the metazoan proliferation-terminal differentiation decisionin vivo
Authors: Adikes Rebecca C. , Kohrman Abraham Q. , Martinez Michael A. Q. , Palmisano Nicholas J. , Smith Jayson J. , Medwig-Kinney Taylor N. , Min Mingwei , Sallee Maria D. , Ahmed Ononnah B. , Kim Nuri , Liu Simeiyun , Morabito Robert D. , Weeks Nicholas , Zhao Qinyun , Zhang Wan , Feldman Jessica L. , Barkoulas Michalis , Pani Ariel M. , Spencer Sabrina L. , Martin Benjamin L. , Matus David Q.
Published: December 2019

Cell proliferation and terminal differentiation are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these cell cycle-associated events live inC. elegansand zebrafish. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from terminally differentiated cells in G0, revealing a commitment point and a cryptic stochasticity in an otherwise invariantC. eleganscell lineage. We also derive a predictive model of future proliferation behavior inC. elegansand zebrafish based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitatein vivostudies of cell cycle control in a wide-range of developmental contexts.
Fully Human Monoclonal Antibodies Effectively Neutralizing Botulinum Neurotoxin Serotype B
Authors: Matsumura Takuhiro , Amatsu Sho , Misaki Ryo , Yutani Masahiro , Du Anariwa , Kohda Tomoko , Fujiyama Kazuhito , Ikuta Kazuyoshi , Fujinaga Yukako
Published: December 2019

Botulinum neurotoxin (BoNT) is the most potent natural toxin known. Of the seven BoNT serotypes (A to G), types A, B, E, and F cause human botulism. Treatment of human botulism requires the development of effective toxin-neutralizing antibodies without side effects such as serum sickness and anaphylaxis. In this study, we generated fully human monoclonal antibodies (HuMAbs) against serotype B BoNT (BoNT/B1) using a murine–human chimera fusion partner cell line named SPYMEG. Of these HuMAbs, M2, which specifically binds to the light chain of BoNT/B1, showed neutralization activity in a mouse bioassay (approximately ≥ 100 i.p. LD50/mg of antibody), and M4, which binds to the C-terminal of heavy chain, showed partial protection. The combination of M2 and M4 was able to completely neutralize BoNT/B1 with a potency greater than 10,000 i.p. LD50/mg of antibodies, and was effective both prophylactically and therapeutically in the mouse model of botulism. Moreover, this combination showed broad neutralization activity against three type B subtypes, namely BoNT/B1, BoNT/B2, and BoNT/B6. These data demonstrate that the combination of M2 and M4 is promising in terms of a foundation for new human therapeutics for BoNT/B intoxication.

IMPORTANCE

Botulinum neurotoxins (BoNTs) produced byClostridium botulinumand related species cause human botulism. Immunotherapy is the most effective treatment for botulism and equine immune serum formulations are used in cases of human botulism. However, these antisera may cause serum sickness or anaphylaxis. Additionally, the production of immune sera involves complicated and time-consuming manufacturing processes and quality management. Therefore, the development of safe, effective, and higher productive antibodies is required. Here we generated fully human monoclonal antibodies against serotype B BoNT (BoNT/B). We found that the combination of these antibodies (M2+M4) had potent and broad neutralization activity against BoNT/B, and showed therapeutic and preventive effects against botulism in mouse models. These data indicate that M2+M4 are promising candidates for the development of human therapeutics and prophylactics for BoNT/B intoxication.
A clinically and genomically annotated nerve sheath tumor biospecimen repository
Authors: Pollard Kai , Banerjee Jineta , Doan Xengie , Wang Jiawan , Guo Xindi , Allaway Robert , Langmead Shannon , Slobogean Bronwyn , Meyer Christian F. , Loeb David M. , Morris Carol D. , Belzberg Allan J. , Blakeley Jaishri O. , Rodriguez Fausto J. , Guinney Justin , Gosline Sara J.C. , Pratilas Christine A.
Published: December 2019

Nerve sheath tumors occur as a heterogeneous group of neoplasms in patients with neurofibromatosis type 1 (NF1). The malignant form represents the most common cause of death in people with NF1, and even when benign, these tumors can result in significant disfigurement, neurologic dysfunction, and a range of profound symptoms. Lack of human tissue across the peripheral nerve tumors common in NF1 has been a major limitation in the development of new therapies. To address this unmet need, we have created an annotated collection of patient tumor samples, patient-derived cell lines, and patient-derived xenografts, and carried out high-throughput genomic and transcriptomic characterization to serve as a resource for further biologic and preclinical therapeutic studies. In this work, we release genomic and transcriptomic datasets comprised of 55 tumor samples derived from 23 individuals, complete with clinical annotation. All data are publicly available through the NF Data Portal and athttp://synapse.org/jhubiobank.
Neural evidence of cognitive conflict during binocular rivalry
Authors: Drew Alice , Torralba Mireia , Ruzzoli Manuela , Fernández Luis Morís , Sabaté Alba , Pápai Márta Szabina , Soto-Faraco Salvador
Published: December 2019

To make sense of ambiguous and, at times, incomplete sensory input, the brain relies on a process of active interpretation. At any given moment, only one of several possible perceptual outcomes prevails in our conscious experience. Our hypothesis is that the competition between alternative representations induces cognitive conflict, eventually leading to fluctuations between different perceptual interpretations. We used binocular rivalry, a popular protocol to probe changes in perceptual awareness [1–3] and drew on the conflict monitoring theory, which holds that cognitive control is invoked by the detection of conflict during information processing. We looked for an increase in power of fronto-medial theta oscillations (5-7 Hz), an established EEG marker of conflict detection [4–7]. Our results show that fm-theta power increases right before perceptual alternations and decreases thereafter, suggesting that conflict monitoring is related to perceptual competition. Furthermore, to investigate conflict resolution via attentional engagement, as held by the conflict monitoring theory [8], we also looked for changes in parieto-occipital alpha oscillations (8-12 Hz) associated to perceptual switches. These oscillations have been associated to attention allocation via functional inhibition in sensory cortices [9–12]. The power of parieto-occipital alpha was inversely related to that of fm-theta, reflecting periods of high inter-ocular inhibition during stable perception, and low inhibition around moments of perceptual change. Our findings validate a prediction made 20 years ago in the seminal paper formulating the conflict monitoring theory, and establish a previously unknown link between conflict mechanisms and the processes leading to perceptual awareness.

Highlights

Binocular rivalry induces conflict between competing perceptual representations

Mid-frontal theta power increases around the resolution of perceptual conflict

Posterior alpha decreases reflect neural excitability around perceptual switches

The results link cognitive conflict theory with perceptual inference
Genomic analysis of carbapenemase-encoding plasmids fromKlebsiella pneumoniaeacross Europe highlights three major patterns of dissemination
Authors: David Sophia , Cohen Victoria , Reuter Sandra , Sheppard Anna E. , Giani Tommaso , Parkhill Julian ,   the European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) Working Group,   the ESCMID Study Group for Epidemiological Markers (ESGEM), Rossolini Gian Maria , Feil Edward J. , Grundmann Hajo , Aanensen David M.
Published: December 2019

The incidence ofKlebsiella pneumoniaeinfections that are resistant to carbapenems, a last-line class of antibiotics, has been rapidly increasing. The primary mechanism of carbapenem resistance is production of carbapenemase enzymes, which are most frequently encoded on plasmids byblaOXA-48-like,blaVIM,blaNDMandblaKPCgenes. Using short-read sequence data, we previously analysed genomes of 1717 isolates from theK. pneumoniaespecies complex submitted during the European survey of carbapenemase-producingEnterobacteriaceae(EuSCAPE). Here, we investigated the diversity, prevalence and transmission dynamics of carbapenemase-encoding plasmids using long-read sequencing of representative isolates (n=79) from this collection in combination with short-read data from all isolates. We highlight three major patterns by which carbapenemase genes have disseminated via plasmids. First,blaOXA-48-likegenes have spread across diverse lineages primarily via a highly conserved, epidemic pOXA-48-like plasmid. Second,blaVIMandblaNDMgenes have spread via transient associations of diverse plasmids with numerous lineages. Third,blaKPCgenes have transmitted predominantly by stable association with one clonal lineage (ST258/512) despite frequent mobilisation between pre-existing yet diverse plasmids within the lineage. Despite contrasts in these three modes of carbapenemase gene spread, which can be summarised as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage, all are underpinned by significant propagation along high-risk clonal lineages.
UMAP does not preserve global structure any better than t-SNE when using the same initialization
Authors: Kobak Dmitry , Linderman George C.
Published: December 2019

One of the most ubiquitous analysis tools employed in single-cell transcriptomics and cytometry is t-distributed stochastic neighbor embedding (t-SNE) [1], used to visualize individual cells as points on a 2D scatter plot such that similar cells are positioned close together. Recently, a related algorithm, called uniform manifold approximation and projection (UMAP) [2] has attracted substantial attention in the single-cell community. InNature Biotechnology, Becht et al. [3] argued that UMAP is preferable to t-SNE because it better preserves the global structure of the data and is more consistent across runs. Here we show that this alleged superiority of UMAP can be entirely attributed to different choices of initialization in the implementations used by Becht et al.: t-SNE implementations by default used random initialization, while the UMAP implementation used a technique called Laplacian eigenmaps [4] to initialize the embedding. We show that UMAP with random initialization preserves global structure as poorly as t-SNE with random initialization, while t-SNE with informative initialization performs as well as UMAP with informative initialization. Hence, contrary to the claims of Becht et al., their experiments do not demonstrate any advantage of the UMAP algorithmper se, but rather warn against using random initialization.
Identification of a Novel Retrieval-Dependent Memory Process in the CrabNeohelice granulata
Authors: Merlo Santiago A. , Santos Maria J. , Pedreira Maria E. , Merlo Emiliano
Published: December 2019

Fully consolidated associative memories may be altered by alternative retrieval dependent memory processes. While a brief exposure to the conditioned stimulus (CS) can trigger reconsolidation of the original memory, a prolonged CS exposure will trigger memory extinction. The conditioned response is maintained after reconsolidation, but is inhibited after extinction, presumably by the formation of a new inhibitory memory trace. In rats and humans, it has been shown that CS exposure of intermediate duration leave the memory in an insensitive or limbo state. Limbo is characterised by the absence of reconsolidation or extinction. Here we investigated the evolutionary conserved nature of limbo using a contextual Pavlovian conditioning (CPC) memory paradigm in the crabNeohelice granulata. In animals with fully consolidated CPC memory, systemic administration of the protein synthesis inhibitor cycloheximide after 1 CS presentation disrupted the memory, presumably by interfering with memory reconsolidation. The same intervention given after 320 CSs prevented CPC memory extinction. Cycloheximide had no behavioural effect when administered after 80 CS presentations, a protocol that failed to extinguish CPC memory. Also, we observed that a stronger CPC memory engaged reconsolidation after 80 CS instead of limbo, indicating that memory strength affects the parametrical conditions to engage either reconsolidation or limbo. Altogether, these results indicate that limbo is an evolutionary conserved memory process segregating reconsolidation from extinction in the number of CSs space. Limbo appears as an intrinsic component of retrieval dependent memory processing, with a key function in the transition from memory maintenance to inhibition.

Author statement (CRediT Roles)

Santiago A. Merlo:Conceptualization, Data curation, Formal Analysis, Investigation, Software, Validation, Visualization, Writing – original draft, Writing – review & editing.

Jimena Santos:Investigation, Writing – review & editing.

Maria Eugenia Pedreira:Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Emiliano Merlo:Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.
Vitamin D-binding protein is required for the maintenance of α-cell function and glucagon secretion
Authors: Viloria Katrina , Nasteska Daniela , Briant Linford J.B. , Heising Silke , Larner Dean , Fine Nicholas H.F. , Ashford Fiona B. , Silva Xavier Gabriela da , Ramos Maria Jiménez , Manning Fox Jocelyn E. , MacDonald Patrick E. , Akerman Ildem , Lavery Gareth G. , Flaxman Christine , Morgan Noel G. , Richardson Sarah J. , Hewison Martin , Hodson David J.
Published: December 2019

Vitamin D-binding protein (DBP) or GC-globulin carries vitamin D metabolites from the circulation to target tissues. DBP expression is highly-localized to the liver and pancreatic α-cells. While DBP serum levels, gene polymorphisms and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α-cell morphology, α-cell function and glucagon secretion. Deletion of DBP led to smaller and hyperplastic α-cells, altered Na+channel conductance, impaired α-cell activation by low glucose, and reduced rates of glucagon secretion. Mechanistically, this involved reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects were also seen in β-cell and δ-cell function. Immunostaining of human pancreata revealed generalized loss of DBP expression as a feature of late-onset and longstanding, but not early-onset type 1 diabetes. Thus, DBP is a critical regulator of α-cell phenotype, with implications for diabetes pathogenesis.

HIGHLIGHTS

DBP expression is highly-localized to mouse and human α-cells

Loss of DBP increases α-cell number, but decreases α-cell size

α-cells in DBP knockout islets are dysfunctional and secrete less glucagon

DBP expression is decreased in α-cells of donors with late-onset or longstanding type 1 diabetes
Investigating β-lactam drug targets inMycobacterium tuberculosisusing chemical probes
Authors: Levine Samantha R. , Beatty Kimberly E.
Published: December 2019

Tuberculosis is a deadly disease that requires a flexible arsenal of drugs to treat it. Although β-lactam antibiotics are rarely used to treatMycobacterium tuberculosis(Mtb) infections today, the targets of these drugs are present in the bacterium. Moreover, the cell wall peptidoglycan ofMtbcontains an abundance of unusual (3→3) cross-links. These cross-links are formed by enzymes called L,D-transpeptidases, which are susceptible to inhibition by the carbapenem class of antibiotics. We developed new small molecule probes to investigate the L,D-transpeptidases and other β-lactam drug targets inMtb. We synthesized probes based on three classes of antibiotics, a monobactam, cephalosporin, and carbapenem. For the carbapenem, we synthesized a meropenem analogue conjugated to a far-red fluorophore. This probe was particularly useful in identifying active L,D-transpeptidases in protein gel-resolved lysates. Next we analyzed β-lactam targets in lysates from both hypoxic and actively-replicating cultures ofMtb. We identified numerous targets, including transpeptidases, carboxypeptidases, and the β-lactamase BlaC. Overall, we provide evidence thatMtbdynamically regulates the enzymes responsible for maintaining cell wall peptidoglycan and that meropenem is a good inhibitor of those enzymes.
Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins inBacillus subtilis
Authors: Sharifzadeh Shabnam , Dempwolff Felix , Kearns Daniel B. , Carlson Erin E.
Published: December 2019

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis inBacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs inB. subtiliswere discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in resistance inB. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.
FluidFM as a Tool to Study Adhesion Forces of Bacteria - Optimization of Parameters and Comparison to Conventional Bacterial Probe Scanning Force Spectroscopy
Authors: Hofherr Linda , Müller-Renno Christine , Ziegler Christiane
Published: December 2019

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells, we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell.
Ser-653-Asn substitution in the acetohydroxyacid synthase gene confers resistance in weedy rice to imidazolinone herbicides imazapic and imazapyr in Malaysia
Authors: Ruzmi Rabiatuladawiyah , Ahmad-Hamdani M. S. , Mazlan Norida
Published: December 2019

The IMI-herbicides rice package has been recognized by all means among the most efficient chemical approaches for weedy rice control nowadays. Inevitably, the continuous and sole dependence, as well as ignorance on the appropriate use of imidazolinone herbicides in the IMI-herbicides rice package by rice growers has caused the development of herbicide resistance in weedy rice populations across many IMI-herbicides rice package adopted countries, inclusive of Malaysia. Hence, a comprehensive study was conducted to elucidate the occurrence, level, and mechanisms endowing resistance to IMI-herbicides on field-reported resistant (R) weedy rice populations collected from IMI-rice fields in Kampung Simpang Sanglang, Perlis (A), Kampung Behor Mentalon, Perlis (B), and Kampung Sungai Kering, Kedah (C). The collected weedy rice populations were compared with a susceptible weedy rice population (S), an imidazolinone-resistant rice cultivar (IMI-rice), and a susceptible local rice cultivar (MR219). Dose-response experiments were carried out using commercial IMI-herbicides (premix of imazapic and imazapyr) available in the IMI-herbicides rice package, in the seed bioassay and whole-plant dose-response. Based on the Resistance Index (RI) quantification in both experiments, the cross-resistance pattern of weedy rice populations and rice varieties to imazapic and imazapyr was determined. Molecular investigation was carried out by comparing acetohydroxyacid synthase (AHAS) gene sequences between resistant (R) weedy rice populations (A, B, and C), S population, IMI-rice, and MR219. Evidently, the AHAS gene sequences of R weedy rice were identical to the IMI-rice, revealing that amino acid substitution of Ser-653-Asn occurs in both R populations and IMI-rice, but neither in MR219 nor S plants.In vitroassays were conducted using analytical grade imidazolinone herbicides of imazapic (99.3%) and imazapyr (99.6%) with seven concentrations. The results demonstrated that the AHAS enzyme extracted from R populations and IMI-rice were less sensitive to IMI-herbicides in comparison to S and MR219, further supporting the IMI-herbicides resistance was conferred by target site mutation. In conclusion, the basis of imidazolinone resistance in selected populations of Malaysia weedy rice was due to a Ser-653-Asn mutation that reduced sensitivity of the target site to IMI-herbicides. The current study presents the first report of resistance mechanism in weedy rice in Malaysian rice fields.
A novel IKK- and proteasome-independent mechanism of RelA activation triggers senescence associated secretome via transcriptional repression of NFKBIA
Authors: Kolesnichenko Marina , Mikuda Nadine , Höpken Uta E. , Milanovic Maja , Tufan A. Bugra , Uyar Bora , Sun Wei , Schleich Kolja , von Hoff Linda , Willenbrock Michael , Krahn Inge , Jungmann Sabine , Hinz Michael , Akalin Altuna , Lee Soyoung , Schmidt-Ullrich Ruth , Schmitt Clemens A. , Scheidereit Claus
Published: December 2019

The IκB kinase (IKK) - NF-κB pathway is activated as part of the DNA damage response and controls both resistance to apoptosis and inflammation. How these different functions are achieved remained unknown. We demonstrate here that DNA double strand breaks elicit two subsequent phases of NF-κB activationin vivoandin vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The first, rapidly activated phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of IκBα and is terminated through IκBα (NFKBIA) re-expression. The second phase is activated days later in senescent cells but is independent of IKK and the proteasome. An altered phosphorylation status of p65, in part driven by GSK3β, results in transcriptional silencing ofNFKBIAand IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.
Loss of the Reissner Fiber and increased URP neuropeptide signaling underlie scoliosis in a zebrafish ciliopathy mutant
Authors: Vesque Christine , Anselme Isabelle , Pezeron Guillaume , Cantaut-Belarif Yasmine , Eschstruth Alexis , Djebar Morgane , Santos Diego López , Ribeuz Hélène Le , Jenett Arnim , Khoury Hanane , Véziers Joëlle , Parmentier Caroline , Schneider-Maunoury Sylvie
Published: December 2019

Cilia-driven movements of the cerebrospinal fluid (CSF) are involved in zebrafish axis straightness, both in embryos and juveniles [1, 2]. In embryos, axis straightness requires cilia-dependent assembly of the Reissner fiber (RF), a SCO-spondin polymer running down the brain and spinal cord CSF-filled cavities [3]. Reduced expression levels of theurp1andurp2genes encoding neuropeptides of the Urotensin II family in CSF-contacting neurons (CSF-cNs) also underlie embryonic ventral curvature of several cilia motility mutants [4]. Moreover, mutants forscospondinanduts2r3(a Urotensin II peptide family receptor gene) develop scoliosis at juvenile stages [3, 4]. However, whether RF maintenance and URP signaling are perturbed in juvenile scoliotic ciliary mutants and how these perturbations are linked to scoliosis is unknown. Here we produced mutants in the zebrafish ortholog of the humanRPGRIP1Lciliopathy gene encoding a transition zone protein [5–7].rpgrip1l-/-zebrafish had normal embryogenesis and developed 3D spine torsions in juveniles. Cilia lining the CNS cavities were normal inrpgrip1l-/-embryos but sparse and malformed in juveniles and adults. Hindbrain ventricle dilations were present at scoliosis onset, suggesting defects in CSF flow. Immunostaining showed a secondary loss of RF correlating with juvenile scoliosis. Surprisingly, transcriptome analysis ofrgprip1lmutants at scoliosis onset uncovered increased levels ofurp1andurp2expression. Overexpressingurp2infoxj1-expressing cells triggered scoliosis inrpgrip1lheterozygotes. Thus, our results demonstrate that increased URP signaling drives scoliosis onset in a ciliopathy mutant. We propose that imbalanced levels of URP neuropeptides in CSF-cNs may be an initial trigger of scoliosis.
Local sleep-like cortical reactivity in the awake brain after focal injury
Authors: Sarasso Simone , D’Ambrosio Sasha , Fecchio Matteo , Casarotto Silvia , Viganò Alessandro , Landi Cristina , Mattavelli Giulia , Gosseries Olivia , Quarenghi Matteo , Laureys Steven , Devalle Guya , Rosanova Mario , Massimini Marcello
Published: December 2019

The functional consequences of brain injury are known to depend on neuronal alterations extending beyond the area of structural damage. Although a lateralized EEG slowing over the injured hemisphere was known since the early days of clinical neurophysiology, its electrophysiological mechanisms were not systematically investigated. In parallel, basic sleep research has thoroughly characterized the neuronal events underlying EEG slow waves in physiological conditions. These EEG events reflect brief interruptions of neuronal firing (OFF-periods) that can occur locally and have prominent consequences on network and behavioral functions. Notably, the EEG slow waves observed following focal brain injury have been never explicitly connected to local sleep-like neuronal events. In previous works, probing cortical circuits with transcranial magnetic stimulation coupled with EEG (TMS/EEG) proved as an effective way to reveal the tendency of cortical circuits to transiently plunge into silent OFF-periods. Here, using this approach, we show that the intact cortex surrounding focal brain injuries engages locally in pathological sleep-like dynamics. Specifically, we employed TMS/EEG in a cohort of thirty conscious awake patients with chronic focal and multifocal brain injuries of various etiologies. TMS systematically evoked prominent slow waves associated with sleep-like OFF-periods in the area surrounding focal cortico-subcortical lesions. These events were associated with a local disruption of signal complexity and were absent when stimulating the contralateral hemisphere. Perilesional sleep-like OFF-periods may represent a valid read-out of the electrophysiological state of discrete cortical circuits following brain injury as well as a potential target of interventions aimed at fostering functional recovery.

One Sentence Summary

Focal cortical injuries are associated with local intrusion of sleep-like dynamics over the perilesional areas which disrupt local signal complexity and coexist with typical wakefulness cortical reactivity patterns within the same brain.
A dual mechanism underlying retroactive shifts of auditory spatial attention: dissociating target- and distractor-related modulations of alpha lateralization
Authors: Klatt Laura-Isabelle , Getzmann Stephan , Begau Alexandra , Schneider Daniel
Published: December 2019

Attention can be allocated to mental representations to select information from working memory. To date, it remains ambiguous whether such retroactive shifts of attention involve the inhibition of irrelevant information or the prioritization of relevant information. Investigating asymmetries in posterior alpha-band oscillations during an auditory retroactive cueing task, we aimed at differentiating those mechanisms. Participants were cued to attend two out of three sounds in an upcoming sound array. Importantly, the resulting working memory representation contained one laterally and one centrally presented item. A centrally presented retro-cue then indicated the lateral, the central, or both items as further relevant for the task (comparing the cued item(s) to a memory probe). Time-frequency analysis revealed opposing patterns of alpha lateralization depending on target eccentricity: A contralateral decrease in alpha power intarget lateraltrials indicated the involvement of target prioritization. A contralateral increase in alpha power when the central item remained relevant (distractor lateraltrials) suggested the de-prioritization of irrelevant information. No lateralization was observed when both items remained relevant, supporting the notion that auditory alpha lateralization is restricted to situations in which spatial information is task-relevant. Altogether, the data demonstrate that retroactive attentional deployment involves excitatory and inhibitory control mechanisms.
Druggability Assessment in TRAPP using Machine Learning Approaches
Authors: Yuan Jui-Hung , Han Sungho Bosco , Richter Stefan , Wade Rebecca C. , Kokh Daria B.
Published: December 2019

Accurate protein druggability predictions are important for the selection of drug targets in the early stages of drug discovery. Due to the flexible nature of proteins, the druggability of a binding pocket may vary due to conformational changes. We have therefore developed two statistical models, a logistic regression model (TRAPP-LR) and a convolutional neural network model (TRAPP-CNN), for predicting druggability and how it varies with changes in the spatial and physicochemical properties of a binding pocket. These models are integrated into TRAPP (TRAnsient Pockets in Proteins), a tool for the analysis of binding pocket variations along a protein motion trajectory. The models, which were trained on publicly available and self-augmented data sets, show equivalent or superior performance to existing methods on test sets of protein crystal structures, and have sufficient sensitivity to identify potentially druggable protein conformations in trajectories from molecular dynamics simulations. Visualization of the evidence for the decisions of the models in TRAPP facilitates identification of the factors affecting the druggability of protein binding pockets.
Microclimate buffering and thermal tolerance across elevations in a tropical butterfly
Authors: Montejo-Kovacevich Gabriela , Martin Simon H. , Meier Joana I. , Bacquet Caroline N. , Monllor Monica , Jiggins Chris D. , Nadeau Nicola J.
Published: December 2019

Microclimatic variability in tropical forests plays a key role in shaping species distributions and their ability to cope with environmental change, especially for ectotherms. Yet, currently available climatic datasets lack data from the forest interior and our knowledge of thermal tolerance among tropical ectotherms is limited. To tackle this, we studied natural variation in the microclimate experienced by a tropical genus of butterflies (Heliconius sp.) along their Andean range across a single year. We found that the forest strongly buffers temperature and humidity in the understory, especially in the lowlands where temperatures are more extreme. There were systematic differences between our yearly records and macroclimate databases (WorldClim2), with lower interpolated minimum temperatures and maximum temperatures higher than expected. We then assessed thermal tolerance of ten Heliconius butterfly species in the wild and showed that populations at high elevations had significantly lower heat tolerance than those at lower elevations. However, when we reared populations of the widespread H. erato from high and low elevations in a common-garden environment, the difference in heat tolerance across elevations was reduced, indicating plasticity in this trait. Microclimate buffering is not currently captured in publicly available datasets but could be crucial for enabling upland shifting of species sensitive to heat such as highland Heliconius. Plasticity in thermal tolerance may alleviate the effects of global warming on some widespread ectotherm species, but more research is needed to understand the long-term consequences of plasticity on populations and species.

Summary statement

Tropical forests along the Andes were found to greatly buffer climate. The butterflies inhabiting high elevations were less thermally tolerant but not when reared in common-garden conditions, indicating plasticity.
Neural correlates of conscious tactile perception: An analysis of BOLD activation patterns and graph metrics
Authors: Grund Martin , Forschack Norman , Nierhaus Till , Villringer Arno
Published: December 2019

Theories of human consciousness substantially vary in the proposed spatial extent of brain activity associated with conscious perception as well as in the assumed functional alterations within the involved brain regions. Here, we investigate which local and global changes in brain activity accompany conscious somatosensory perception following electrical finger nerve stimulation, and whether there are whole-brain functional network alterations by means of graph metrics. Thirty-eight healthy participants performed a somatosensory detection task and reported their decision confidence during fMRI. For conscious tactile perception in contrast to undetected near-threshold trials (misses), we observed increased BOLD activity in the precuneus, the intraparietal sulcus, the insula, the nucleus accumbens, the inferior frontal gyrus and the contralateral secondary somatosensory cortex. For misses compared to correct rejections, bilateral secondary somatosensory cortices, supplementary motor cortex and insula showed greater activations. The analysis of whole-brain functional network topology for hits, misses and correct rejections, did not result in any significant differences in modularity, participation, clustering or path length, which was supported by Bayes factor statistics. In conclusion, for conscious somatosensory perception, our results are consistent with an involvement of (probably) domain-general brain areas (precuneus, insula, inferior frontal gyrus) in addition to somatosensory regions; our data do not support the notion of specific changes in graph metrics associated with conscious experience. For the employed somatosensory submodality of fine electrical current stimulation, this speaks for a global broadcasting of sensory content across the brain without substantial reconfiguration of the whole-brain functional network resulting in an integrative conscious experience.
Role of adenosine in functional recovery following anoxic coma inLocusta migratoria
Authors: Van Dusen Rachel A. , Lanz Christopher , Robertson R. Meldrum
Published: December 2019

When exposed to prolonged anoxia insects enter a reversible coma during which neural and muscular systems temporarily shut down. Nervous system shut down is a result of spreading depolarization throughout neurons and glial cells. Upon return to normoxia, recovery occurs following the restoration of ion gradients. However, there is a delay in the functional recovery of synaptic transmission following membrane repolarization. In mammals, the build-up of extracellular adenosine following spreading depolarization contributes to this delay. Adenosine accumulation is a marker of metabolic stress and it has many downstream effects through the activation of adenosine receptors. Here we demonstrate that adenosine lengthens the time to functional recovery following anoxic coma in locusts. Caffeine, used as an adenosine receptor antagonist, decreased the time to recovery in intact animals and lengthened the time to recovery in semi-intact animals. Our results show that the rate of recovery in insect systems is affected by the presence of adenosine.
A machine learning approach to define antimalarial drug action from heterogeneous cell-based screens
Authors: Ashdown George W. , Dimon Michelle , Fan Minjie , Terán Fernando Sánchez-Román , Witmer Katrin , Gaboriau David C. A. , Armstrong Zan , Hazard Jon , Ando D. Michael , Baum Jake
Published: December 2019

Drug resistance threatens the effective prevention and treatment of an ever-increasing range of human infections. This highlights an urgent need for new and improved drugs with novel mechanisms of action to avoid cross-resistance. Current cell-based drug screens are, however, restricted to binary live/dead readouts with no provision for mechanism of action prediction. Machine learning methods are increasingly being used to improve information extraction from imaging data. Such methods, however, work poorly with heterogeneous cellular phenotypes and generally require time-consuming human-led training. We have developed a semi-supervised machine learning approach, combining human- and machine-labelled training data from mixed human malaria parasite cultures. Designed for high-throughput and high-resolution screening, our semi-supervised approach is robust to natural parasite morphological heterogeneity and correctly orders parasite developmental stages. Our approach also reproducibly detects and clusters drug-induced morphological outliers by mechanism of action, demonstrating the potential power of machine learning for accelerating cell-based drug discovery.

One Sentence Summary

A machine learning approach to classifying normal and aberrant cell morphology from plate-based imaging of mixed malaria parasite cultures, facilitating clustering of drugs by mechanism of action.