bluuebunny/crossref_metadata_2025 huggingface dataset:
High-throughput phenotyping of leaf discs infected with grapevine downy mildew using shallow convolutional neural networks
Authors: Zendler Daniel , Malagol Nagarjun , Schwandner Anna , Töpfer Reinhard , Hausmann Ludger , Zyprian Eva
Published: August 2021

Objective and standardized recording of disease severity in mapping crosses and breeding lines is a crucial step in characterizing resistance traits utilized in breeding programs and to conduct QTL or GWAS studies. Here we report a system for automated high-throughput scoring of disease severity on inoculated leaf discs. As proof of concept, we used leaf discs inoculated with Plasmopara viticola causing grapevine downy mildew (DM). This oomycete is one of the major grapevine pathogens and has the potential to reduce grape yield dramatically if environmental conditions are favorable. Breeding of DM resistant grapevine cultivars is an approach for a novel and more sustainable viticulture. This involves the evaluation of several thousand inoculated leaf discs from mapping crosses and breeding lines every year. Therefore, we trained a shallow convolutional neural-network (SCNN) for efficient detection of leaf disc segments showing P. viticola sporangiophores. We could illustrate a high and significant correlation with manually scored disease severity used as ground truth data for evaluation of the SCNN performance. Combined with an automated imaging system, this leaf disc-scoring pipeline has the potential to reduce the amount of time during leaf disc phenotyping considerably. The pipeline with all necessary documentation for adaptation to other pathogens is freely available.
CRISPR-based kinome-screening revealed MINK1 as a druggable player to rewire 5FU-resistance in OSCC through AKT/MDM2/p53 axis
Authors: Mohanty Sibasish , Mohapatra Pallavi , Shriwas Omprakash , Ansari Shamima Azma , Priyadarshini Manashi , Priyadarsini Swatismita , Rath Rachna , Sultania Mahesh , Das Majumdar Saroj Kumar , Swain Rajeeb Kumar , Dash Rupesh
Published: August 2021

Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responder as compared to chemotherapy responders. The nude mice and zebrafish xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. The patient derived TPF resistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC.
A novel lineage-tracing mouse model for MmuPV1 infection enables in vivo studies in the absence of cytopathic effects
Authors: Yilmaz Vural , Louka Panayiota , Strati Katerina
Published: August 2021

Human papillomaviruses (HPVs) are DNA viruses that ubiquitously infect humans and have been associated with hyperproliferative lesions. The recently discovered mouse specific papillomavirus, MmuPV1, provides the opportunity to study papillomavirus infections in vivo in the context of a common laboratory mouse model (Mus musculus). To date, a major challenge in the field has been the lack of tools to identify, observe and characterize individually the papillomavirus hosting cells and also trace the progeny of these cells over time.

Here, we present the successful generation of an in vivo lineage-tracing model of MmuPV1-harboring cells and their progeny by means of genetic reporter activation. Following the validation of the system both in vitro and in vivo, we used it to provide a proof-of-concept of its utility. Using flow-cytometry analysis, we observed increased proliferation dynamics and decreased MHC-I cell surface expression in MmuPV1-treated tissues which could have implications in tissue regenerative capacity and ability to clear the virus. This model is a novel tool to study the biology of the MmuPV1 host-pathogen interactions.
Audiovisual integration in the Mauthner cell enhances escape probability and reduces response latency
Authors: Martorell Nicolás , Medan Violeta
Published: August 2021

Fast and accurate threat detection is critically important for animal survival. Reducing perceptual ambiguity by integrating multiple sources of sensory information can enhance threat detection and reduce response latency. However, studies showing a direct link between behavioral correlates of multisensory integration and its underlying neural basis are rare. In fish, an explosive escape behavior known as C-start is driven by an identified neural circuit centered on the Mauthner cell. The Mauthner cell can trigger C-starts in response to visual and auditory stimuli allowing to investigate how multisensory integration in a single neuron affects behavioral outcome after threat detection. Here we demonstrate that in goldfish visual looms and brief auditory stimuli can be integrated to increase C-start probability and that this enhancement is inversely correlated to the saliency of the cues with weaker auditory cues producing a proportionally stronger multisensory effect. We also show that multisensory stimuli reduce response latency locked to the presentation of the auditory cue. Finally, we make a direct link between behavioral data and its underlying neural mechanism by reproducing empirical data with an integrate-and-fire computational model of the Mauthner cell.
Oculomotor freezing indicates conscious detection free of decision bias
Authors: White Alex L. , Moreland James C. , Rolfs Martin
Published: August 2021

The appearance of a salient stimulus rapidly inhibits saccadic eye movements. Curiously, this “oculomotor freezing” reflex is triggered only by stimuli that the observer reports seeing. It remains unknown, however, if oculomotor freezing is linked to the observer’ssensory experience, or theirdecisionthat a stimulus was present. To dissociate between these possibilities, we manipulated decision criterion via monetary payoffs and stimulus probability in a detection task. These manipulations greatly shifted observers’ decision criteria but did not affect the degree to which microsaccades were inhibited by stimulus presence. Moreover, the link between oculomotor freezing and explicit reports of stimulus presence was stronger when the criterion was conservative rather than liberal. We conclude that the sensory threshold for oculomotor freezing is independent of decision bias. Provided that conscious experience is also unaffected by such bias, oculomotor freezing is an implicit indicator of sensory awareness.

New & Noteworthy

Sometimes a visual stimulus reaches awareness, and sometimes it does not. To understand why, we need objective, bias-free measures of awareness. We discovered that a reflexive freezing of small eye movements indicates when an observer detects a stimulus. Furthermore, when we biased observers’ decisions to report seeing the stimulus, the oculomotor reflex was unaltered. This suggests that the threshold for conscious perception is independent of the decision criterion and is revealed by oculomotor freezing.
Dynamic representation of space in the hippocampus: spatial novelty detection and consolidation in CA1 and CA2
Authors: Bhasin Guncha
Published: August 2021

Hippocampal place cells are the functional units of spatial navigation and are present in all subregions-CA1, CA2, CA3 and CA4. Recent studies on CA2 have indicated its role in social and contextual memory, but its contribution towards spatial novelty detection and consolidation remains largely unknown. The current study aims to uncover how CA1 and CA2 detect, process, assimilate and consolidate spatial novelty. Accordingly, a novel 3-day paradigm was designed where the animal was introduced to a completely new environment on the first day and to varying degrees of familiarity and novelty on subsequent days, as the track was extended in length and modified in shape, keeping other environmental constraints fixed. Detection of spatial novelty was found to be a dynamic and complex phenomenon, characterized by different responses from hippocampal place cells, depending on when novelty was introduced. Therefore, the study concludes that early novelty detection (the first time a novel space is introduced in a relatively familiar environment) and subsequent novelty detection are not processed in the same way. Additionally, while neuronal responses to spatial novelty detection (early and subsequent) were found to be the same in CA1 and CA2 ensembles, their responses differed in spatial consolidation mechanisms during subsequent sleep replays. For CA1, spatial coverage of prior behaviour was found to be closely reflected in subsequent sleep for that particular day, but CA2 showed no such ‘coherent response’, highlighting mnemonic processing differences between CA2 and CA1 with respect to spatial novelty.
Diverse Genotype-by-Weather Interactions in Switchgrass
Authors: MacQueen Alice H. , Zhang Li , Smith Samuel Pattillo , Bonnette Jason E. , Boe Arvid R. , Fay Philip A. , Fritschi Felix B. , Lowry David B. , Mitchell Robert B. , Rouquette Francis M. , Wu Yanqi , Harpak Arbel , Juenger Thomas E.
Published: August 2021

The timing of vegetative and reproductive growth in plants (“phenological timings”) depend on genetic effects (G), environmental (e.g., weather) cues, and their interaction. Here, we measure phenological timings in two highly divergent switchgrass (Panicum virgatum) subpopulations using repeated plantings of cloned individuals at eight sites across the central United States. The timing of vegetative growth for the two subpopulations reversed between their two natural ranges and had strong negative correlations between these regions; in contrast, the timing of flowering was positively correlated between gardens. We expect that these phenotypic correlations consist of polygenic effects on phenology which have distinct patterns of GxE segregating at different mapped loci. Thus, we infer the mixture of ways genetic effects impact phenological timings, such as across common gardens (GxE) or with weather cues (GxWeather). We demonstrate that we can identify genetic variation with GxWeather and assign genetic loci to specific weather-based cues or other patterns. For example, in the Gulf subpopulation, 65% of genetic effects on the timing of vegetative growth covary with daylength 14 days prior to green-up date, and 33% of genetic effects on the timing of flowering covary with cumulative rainfall in the week prior to flowering. However, most variation in genetic effects cannot be attributed to variation in weather variables. Selective breeding for particular alleles at GxWeather loci could alter flowering responsiveness in a photoperiod or rainfall-specific way. More broadly, our approach refines the characterization of genotype-by-environment interactions and can be implemented in any species phenotyped in multiple environments.
Somatic mutation rates scale with lifespan across mammals
Authors: Cagan Alex , Baez-Ortega Adrian , Brzozowska Natalia , Abascal Federico , Coorens Tim H. H. , Sanders Mathijs A. , Lawson Andrew R. J. , Harvey Luke M. R. , Bhosle Shriram , Jones David , Alcantara Raul E. , Butler Timothy M. , Hooks Yvette , Roberts Kirsty , Anderson Elizabeth , Lunn Sharna , Flach Edmund , Spiro Simon , Januszczak Inez , Wrigglesworth Ethan , Jenkins Hannah , Dallas Tilly , Masters Nic , Perkins Matthew W. , Deaville Robert , Druce Megan , Bogeska Ruzhica , Milsom Michael D. , Neumann Björn , Gorman Frank , Constantino-Casas Fernando , Peachey Laura , Bochynska Diana , John Smith Ewan St. , Gerstung Moritz , Campbell Peter J. , Murchison Elizabeth P. , Stratton Michael R. , Martincorena Iñigo
Published: August 2021

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans. Comparative analyses can shed light on the diversity of mutagenesis across species and on long-standing hypotheses regarding the evolution of somatic mutation rates and their role in cancer and ageing. Here, we used whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found somatic mutagenesis to be dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans, although the relative contribution of each signature varied across species. Remarkably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied displaying a comparable association. Despite widely different life histories among the species surveyed, including ∼30-fold variation in lifespan and ∼40,000-fold variation in body mass, the somatic mutation burden at the end of lifespan varied only by a factor of ∼3. These data unveil common mutational processes across mammals and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing.
A flexible point and variance estimator to assess bird/bat fatality from carcass searches
Authors: Mercker Moritz
Published: August 2021

Estimation of bird and bat fatalities due to collision with anthropogenic structures (such as power lines or wind turbines) is an important ecological issue. However, searching for collision victims usually only detects a proportion of the true number of collided individuals. Various mortality estimators have previously been proposed to correct for this incomplete detection, based on regular carcass searches and additional field experiments. However, each estimator implies specific assumptions/restrictions, which may easily be violated in practice. In this study, we extended previous approaches and developed a versatile algorithm to compute point and variance estimates for true carcass numbers. The presented method allows for maximal flexibility in the data structure. Using simulated data, we showed that our point and variance estimators ensured unbiased estimates under various challenging data conditions. The presented method may improve the estimation of true collision numbers, as an important pre-condition for calculating collision rates and evaluating measures to reduce collision risks, and may thus provide a basis for management decisions and/or compensation actions with regard to planned or existing wind turbines and power lines.
Learning the local landscape of protein structures with convolutional neural networks
Authors: Kulikova Anastasiya V. , Diaz Daniel J. , Loy James M. , Ellington Andrew D. , Wilke Claus O.
Published: August 2021

The fundamental problem of protein biochemistry is to predict protein structure from amino acid sequence. The inverse problem, predicting either entire sequences or individual mutations that are consistent with a given protein structure, has received much less attention even though it has important applications in both protein engineering and evolutionary biology. Here, we ask whether 3D convolutional neural networks (3D CNNs) can learn the local fitness landscape of protein structure to reliably predict either the wild-type amino acid or the consensus in a multiple sequence alignment from the local structural context surrounding a site of interest. We find that the network can predict wild type with good accuracy, and that network confidence is a reliable measure of whether a given prediction is likely going to be correct or not. Predictions of consensus are less accurate, and are primarily driven by whether or not the consensus matches the wild type. Our work suggests that high-confidence mis-predictions of the wild type may identify sites that are primed for mutation and likely targets for protein engineering.
Corticostriatal suppression of appetitive Pavlovian conditioned responding
Authors: Villaruel Franz R. , Martins Melissa , Chaudhri Nadia
Published: August 2021

The capacity to suppress learned responses is essential for animals to adapt in dynamic environments. Extinction is a process by which animals learn to suppress conditioned responding when an expected outcome is omitted. The infralimbic cortex (IL) to nucleus accumbens shell (NAcS) neural circuit is implicated in suppressing conditioned responding after extinction, especially in the context of operant cocaine-seeking behaviour. However, the role of the IL-to-NAcS neural circuit in the extinction of responding to appetitive Pavlovian cues is unknown and the psychological mechanisms involved in response suppression following extinction are unclear. We trained rats to associate a 10 s auditory conditioned stimulus (CS; 14 trials per session) with a sucrose unconditioned stimulus (US; 0.2 mL per CS) in a specific context and then, following extinction in a different context, precipitated a renewal of CS responding by presenting the CS alone in the original Pavlovian conditioning context. Unilateral, optogenetic stimulation of the IL-to-NAcS circuit selectively during CS trials suppressed renewal. In a separate experiment, IL-to-NAcS stimulation suppressed CS responding regardless of prior extinction and impaired extinction retrieval. Finally, IL-to-NAcS stimulation during the CS did not suppress the acquisition of Pavlovian conditioning but was required for the subsequent expression of CS responding. These results are consistent with multiple studies showing that the IL-to-NAcS neural circuit is involved in the suppression of operant cocaine-seeking, extending these findings to appetitive Pavlovian cues. The suppression of appetitive Pavlovian responding following IL-to-NAcS circuit stimulation does not, however, appear to require an extinction-dependent process.

SIGNIFICANCE STATEMENT

Extinction is a form of inhibitory learning through which animals learn to suppress conditioned responding in the face of non-reinforcement. We investigated the role of infralimbic (IL) cortex inputs to the nucleus accumbens shell (NAcS) in the extinction of responding to appetitive Pavlovian cues and the psychological mechanisms involved in response suppression following extinction. Using in vivo optogenetics, we found that stimulating the IL-to-NAcS neural circuit suppressed context-induced renewal of conditioned responding after extinction. In a separate experiment, stimulating the IL-to-NAcS circuit suppressed conditioned responding in an extinction-independent manner. These findings can be leveraged by future research aimed at understanding how corticostriatal circuits contribute to behavioural flexibility and mental disorders that involve the suppression of learned behaviours.
Tools and applications for integrative analysis of DNA methylation in social insects
Authors: Morandin Claire , Brendel Volker P.
Published: August 2021

DNA methylation is a common epigenetic signaling tool and an important biological process which is widely studied in a large array of species. The presence, level, and function of DNA methylation varies greatly across species. In insects, DNA methylation systems are reduced, and methylation rates are often low. Low methylation levels probed by whole genome bisulfite sequencing require great care with respect to data quality control and interpretation. Here we introduce BWASP/R, a complete workflow that allows efficient, scalable, and entirely reproducible analyses of raw DNA methylation sequencing data. Consistent application of quality control filters and analysis parameters provides fair comparisons among different studies and an integrated view of all experiments on one species. We describe the capabilities of the BWASP/R workflow by re-analyzing several publicly available social insect WGBS data sets, comprising 70 samples and cumulatively 147 replicates from four different species. We show that the CpG methylome comprises only about 1.5% of CpG sites in the honeybee genome and that the cumulative data are consistent with genetic signatures of site accessibility and physiological control of methylation levels.

Significance Statement

DNA methylation in the honeybee genome occurs almost entirely at CpG sites. Methylation rates are small compared to rates in mammalian or plant genomes. De novo analysis of all published honeybee methylation studies and statistical modeling suggests that the CpG methylome consists of about only 300,000 sites. The development of a fully reproducible, scalable, portable workflow allows for easy accessible updates of integrative views of all current experiments. The integrated results for the honeybee are consistent with genetic determination of methylation site accessibility by yet uncharacterized sequence features and physiological control of methylation levels at those sites.
The IRE1/XBP1 signaling axis promotes skeletal muscle regeneration through a cell non-autonomous mechanism
Authors: Roy Anirban , da Silva Meiricris Tomaz , Bhat Raksha , Bohnert Kyle R. , Iwawaki Takao , Kumar Ashok
Published: August 2021

Skeletal muscle regeneration is regulated by coordinated activation of multiple signaling pathways activated in both injured myofibers and satellite cells. The unfolded protein response (UPR) is a major mechanism that detects and alleviates protein-folding stresses in ER. However, the role of individual arms of the UPR in skeletal muscle regeneration remains less understood. In the present study, we demonstrate that IRE1α (also known as ERN1) and its downstream target, XBP1, are activated in skeletal muscle of mice upon injury. Myofiber-specific ablation of IRE1 or XBP1 in mice diminishes skeletal muscle regeneration that is accompanied with reduced number of satellite cells and their fusion to injured myofibers. Ex vivo cultures of myofiber explants demonstrate that ablation of IRE1α reduces the proliferative capacity of myofiber- associated satellite cells. Myofiber-specific deletion of IRE1α dampens Notch signaling and canonical NF-κB pathway in skeletal muscle of mice. Our results also demonstrate that targeted ablation of IRE1α reduces skeletal muscle regeneration in the mdx mice, a model of Duchenne muscular dystrophy. Collectively, our results reveal that the IRE1α-mediated signaling promotes muscle regeneration through augmenting the proliferation of satellite cells in a cell non- autonomous manner.
Improving robustness of 3D multi-shot EPI by structured low-rank reconstruction of segmented CAIPI sampling for fMRI at 7T
Authors: Chen Xi , Wu Wenchuan , Chiew Mark
Published: August 2021

Three-dimensional (3D) encoding methods are increasingly being explored as alternatives to multi-slice two-dimensional (2D) acquisitions in fMRI, particularly in cases where high isotropic resolution is needed. 3D multi-shot EPI is the most popular 3D fMRI acquisition method, but is susceptible to physiological fluctuations which can induce inter-shot phase variations, and thus reducing the achievable tSNR, negating some of the benefit of 3D encoding. This issue can be particularly problematic at ultra-high fields like 7T, which have more severe off-resonance effects. In this work, we aim to improve the temporal stability of 3D multi-shot EPI at 7T by improving its robustness to inter-shot phase variations. We presented a 3D segmented CAIPI sampling trajectory (“seg-CAIPI”) and an improved reconstruction method based on Hankel structured low-rank matrix recovery. Simulation and in-vivo results demonstrate that the combination of the seg-CAIPI sampling scheme and the proposed structured low-rank reconstruction is a promising way to effectively reduce the unwanted temporal variance induced by inter-shot physiological fluctuations, and thus improve the robustness of 3D multi-shot EPI for fMRI.

Highlights

A 3D multi-shot EPI sampling trajectory using interleaved ordering along kz and a CAIPI blipping pattern improves robustness to inter-shot phase variations

Reconstruction based on Hankel structured low-rank matrix completion can significantly improve the temporal stability of 3D multi-shot acquisitions at 7T

1.5mm resolution brain fMRI data show that ~60% improvement in mean tSNR can be obtained using the proposed method compared to the conventional method

1.8mm resolution brain fMRI data demonstrate that the proposed method allows for 4-fold acceleration without loss of tSNR compared to conventional 3D EPI

Preliminary brainstem fMRI data show that ~40% improvement in mean tSNR can be obtained using the proposed sampling and reconstruction
Village in a dish: a model system for population-scale hiPSC studies
Authors: Neavin Drew R. , Steinmann Angela M. , Chiu Han Sheng , Daniszewski Maciej S. , Moutinho Cátia , Chan Chia-Ling , Tyebally Mubarika , Gnanasambandapillai Vikkitharan , Lam Chuan E. , Nguyen Uyen , Hernández Damián , Lidgerwood Grace E. , Hewitt Alex W. , Pébay Alice , Palpant Nathan J. , Powell Joseph E.
Published: August 2021

The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and under different conditions. Human induced pluripotent stem cells (hiPSCs) are uniquely suited to study these context-dependent effects, but to do so requires cell lines from hundreds or potentially thousands of individuals. Village cultures, where multiple hiPSC lines are cultured and differentiated together in a single dish, provide an elegant solution for scaling hiPSC experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned back to a donor line using single cell sequencing, and addressing whether line-specific signaling alters the transcriptional profiles of companion lines in a village culture. We generated single cell RNA sequence data from hiPSC lines cultured independently (uni-culture) and in villages at three independent sites. We show that the transcriptional profiles of hiPSC lines are highly consistent between uni- and village cultures for both fresh (0.46 < R < 0.88) and cryopreserved samples (0.46 < R < 0.62). Using a mixed linear model framework, we estimate that the proportion of transcriptional variation across cells is predominantly due to donor effects, with minimal evidence of variation due to culturing in a village system. We demonstrate that the genetic, epigenetic or hiPSC line-specific effects on gene expression are consistent whether the lines are uni- or village-cultured (0.82 < R < 0.94). Finally, we identify the consistency in the landscape of cell states between uni- and village-culture systems. Collectively, we demonstrate that village methods can be effectively used to detect hiPSC line-specific effects including sensitive dynamics of cell states.
A scalable algorithm for clonal reconstruction from sparse time course genomic sequencing data
Authors: Ismail Wazim Mohammed , Tang Haixu
Published: August 2021

Long-term evolution experiments (LTEEs) reveal the dynamics of clonal compositions in an evolving bacterial population over time. Accurately inferring the haplotypes - the set of mutations that identify each clone, as well as the clonal frequencies and evolutionary history in a bacterial population is useful for the characterization of the evolutionary pressure on multiple correlated mutations instead of that on individual mutations. Here, we study the computational problem of reconstructing the haplotypes of bacterial clones from the variant allele frequencies (VAFs) observed during a time course in a LTEE. Previously, we formulated the problem using a maximum likelihood approach under the assumption that mutations occur spontaneously, and thus the likelihood of a mutation occurring in a specific clone is proportional to the frequency of the clone in the population when the mutation occurs. We also developed several heuristic greedy algorithms to solve the problem, which were shown to report accurate results of clonal reconstruction on simulated and real time course genomic sequencing data in LTEE. However, these algorithms are too slow to handle sparse time course data when the number of novel mutations occurring during the time course are much greater than the number of time points sampled. In this paper, we present a novel scalable algorithm for clonal reconstruction from sparse time course data. We employed a statistical method to estimate the sampling variance of VAFs derived from low coverage sequencing data and incorporated it into the maximum likelihood framework for clonal reconstruction on noisy sequencing data. We implemented the algorithm (named ClonalTREE2) and tested it using simulated and real sparse time course genomic sequencing data. The results showed that the algorithm was fast and achieved near-optimal accuracy under the maximum likelihood framework for the time course data involving hundreds of novel mutations at each time point. The source code of ClonalTREE2 is available at https://github.com/COL-IU/ClonalTREE2.
Selective estrogen receptor modulators limit alphavirus infection by targeting the viral capping enzyme nsP1
Authors: Mudgal Rajat , Bharadwaj Chandrima , Dubey Aakriti , Choudhary Shweta , Nagarajan Perumal , Aggarwal Megha , Ratra Yashika , Basak Soumen , Tomar Shailly
Published: August 2021

Alphaviruses cause animal or human diseases that are characterized by febrile illness, debilitating arthralgia, or encephalitis. Selective estrogen receptor modulators (SERMs), a class of FDA-approved drugs, have been shown to possess antiviral activities against multiple viruses, including Hepatitis C virus, Ebola virus, dengue virus, and vesicular stomatitis virus. Here, we evaluated three SERM compounds, namely 4-hydroxytamoxifen, tamoxifen, and clomifene, for plausible antiviral properties against two medically important alphaviruses, chikungunya virus (CHIKV) and Sindbis virus (SINV). In cell culture settings, these SERMs displayed potent activity against CHIKV and SINV at non-toxic concentrations with EC50 values ranging between 400 nM and 3.9 μM. Further studies indicated that these compounds inhibit a post-entry step of the alphavirus life cycle, while enzymatic assays involving purified recombinant proteins confirmed that these SERMs target the enzymatic activity of non-structural protein 1 (nsP1), the capping enzyme of alphaviruses. Finally, tamoxifen treatment restrained CHIKV growth in the infected mice and diminished musculoskeletal pathologies. Combining biochemical, cell culture-based studies, and in vivo analyses, we strongly argue that SERM compounds, or their derivatives, may provide for attractive therapeutic options against alphaviruses.
In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center
Authors: Khara Pratick , Song Liqiang , Christie Peter J. , Hu Bo
Published: August 2021

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1-VirB11 and VirD4. We visualized the pKM101-encoded T4SS in the native cellular context by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but differs strikingly from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers-of-dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially-dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer.

SIGNIFICANCE

Bacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for development of intervention strategies aimed at suppressing the action of T4SS nanomachines.
Unveiling the links between peptide identification and differential analysis FDR controls by means of a practical introduction to knockoff filters
Authors: Etourneau Lucas , Varoquaux Nelle , Burger Thomas
Published: August 2021

In proteomic differential analysis, FDR control is often performed through a multiple test correction (i.e., the adjustment of the original p-values). In this protocol, we apply a recent and alternative method, based on so-called knockoff filters. It shares interesting conceptual similarities with the target-decoy competition procedure, classically used in proteomics for FDR control at peptide identification. To provide practitioners with a unified understanding of FDR control in proteomics, we apply the knockoff procedure on real and simulated quantitative datasets. Leveraging these comparisons, we propose to adapt the knockoff procedure to better fit the specificities of quantitive proteomic data (mainly very few samples). Performances of knockoff procedure are compared with those of the classical Benjamini-Hochberg procedure, hereby shedding a new light on the strengths and weaknesses of target-decoy competition.
An Ancestral Function of Strigolactones as Symbiotic Rhizosphere Signals
Authors: Kodama Kyoichi , Rich Mélanie K. , Yoda Akiyoshi , Shimazaki Shota , Xie Xiaonan , Akiyama Kohki , Mizuno Yohei , Komatsu Aino , Luo Yi , Suzuki Hidemasa , Kameoka Hiromu , Libourel Cyril , Keller Jean , Sakakibara Keiko , Nishiyama Tomoaki , Nakagawa Tomomi , Mashiguchi Kiyoshi , Uchida Kenichi , Yoneyama Kaori , Tanaka Yoshikazu , Yamaguchi Shinjiro , Shimamura Masaki , Delaux Pierre-Marc , Nomura Takahito , Kyozuka Junko
Published: August 2021

In flowering plants, carotenoid-derived strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), a previously unidentified SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating that it is ancestral in land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.
Exact distribution of threshold-crossing times for protein concentrations: Implication for biological timekeeping
Authors: Rijal Krishna , Prasad Ashok , Singh Abhyudai , Das Dibyendu
Published: August 2021

Stochastic transcription and translation dynamics of protein accumulation up to some concentration threshold sets the timing of many cellular physiological processes. Here we obtain the exact distribution of first threshold-crossing times of protein concentration, in either Laplace or time domain, and its associated cumulants: mean, variance and skewness. The distribution is asymmetric and its skewness non-monotonically varies with the threshold. We study lysis times of E-coli cells for holin gene mutants of bacteriophage-λ and find a good match with theory. Mutants requiring higher holin thresholds show more skewed lysis time distributions as predicted.
Nanomechanics combined with HDX reveal allosteric drug binding sites of CFTR NBD1
Authors: Padányi Rita , Farkas Bianka , Tordai Hedvig , Kiss Bálint , Grubmüller Helmut , Soya Naoto , Lukács Gergely L. , Kellermayer Miklós , Hegedűs Tamás
Published: August 2021

Cystic fibrosis is most frequently caused by the deletion of F508 (ΔF508) in CFTR’s nucleotide binding domain 1 (NBD1), compromising CFTR folding, stability and domain assembly. The limitation of developing a successful therapy is due to the lack of molecules that synergistically facilitate folding by targeting distinct structural defects of ΔF508-CFTR. To improve drug efficacy by targeting the ΔF508-NBD1 folding and stability, and to study potential ΔF508-NBD1 allosteric corrector binding sites at the atomic level, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. These methods allowed us to describe unfolding intermediates and forces acting during NBD1 mechanical unfolding and to elucidate the stabilization mechanism of ΔF508-NBD1 by 5-bromoindole-3-acetic acid (BIA). An NBD1 region, including the α-subdomain, was identified as a potentially important participant of the first folding steps, characterized by non-native interactions of F508, thus destabilized in the deletion mutant. The instability was counteracted by the low-potency corrector BIA, increasing the mechanical resistance of the ΔF508-NBD1 α-subdomain, which was confirmed as a binding site by computational modeling and HDX experiments. Our results underline the complementarity of computational and experimental methods and provide a possible strategy to improve folding correctors.
NR4A3 is oxidative stress responsible transcription factor through HMOX1, and also controls cell cycle through CCNE1 and CDK2 in pancreatic islet derived 1.1B4 cells
Authors: Nakayama Motoko , Ueta Etsuko , Yoshida Mitsuru , Shimizu Yuri , Oguchi Reiko , Tokuda Atsuko , Sone Yasuko , Nomi Yuri , Otsuka Yuzuru
Published: August 2021

The mechanism of antioxidant defense system is still controversial. As islet ß-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation.

Graphical abstract

Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase.
Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans
Authors: Roux Indra , Chooi Yit-Heng
Published: August 2021

Building strains of filamentous fungi for stable long-term heterologous expression of large biosynthetic pathways is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase-mediated cassette exchange. We built A. nidulans strains harboring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21 kb DNA fragment in a single step. We further evaluated the integration at two loci by analyzing the expression of a fluorescent reporter and the production of a heterologous polyketide metabolite. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterized aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.
Sequence grammar underlying unfolding and phase separation of globular proteins
Authors: Ruff Kiersten M. , Choi Yoon Hee , Cox Dezerae , Ormsby Angelique R. , Myung Yoochan , Ascher David B. , Radford Sheena E. , Pappu Rohit V. , Hatters Danny M.
Published: August 2021

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). For UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and sequence features of cellular proteins. Overall, we find that the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammar of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and lead to gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.

Highlights

Unfolded states of globular proteins phase separate to form UPODs in cells

The fraction of unfolded molecules and the sticker grammar govern phase separation

Hydrophobic residues act as stickers that engage in intermolecular interactions

Sticker grammar also influences gain-of-function recruitment into aberrant UPODs
Bimodal inference in humans and mice
Authors: Weilnhammer Veith , Stuke Heiner , Standvoss Kai , Sterzer Philipp
Published: August 2021

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Perception is known to cycle through periods of enhanced and reduced sensitivity to external information. Here, we asked whether such infra-slow fluctuations arise as a noise-related epiphenomenon of limited processing capacity or, alternatively, represent a structured mech­anism of perceptual inference. Using two large-scale datasets, we found that humans and mice waver between alternating intervals of externally- and internally-oriented modes of sensory analysis. During external mode, perception aligned more closely with the external sensory information, whereas internal mode was characterized by enhanced biases toward perceptual history. Computational modeling indicated that dynamic changes in mode are enabled by two interlinked factors: (i), the integration of subsequent inputs over time and, (ii), infra-slow anti-phase oscillations in the perceptual impact of external sensory information versus internal predictions that are provided by perceptual history. Simulated data suggested that between-mode fluctuations may benefit perception by generating unambiguous error signals that enable robust learning and metacognition in volatile environments.

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One sentence summary

Humans and mice fluctuate between external and internal modes of sensory processing.
The Hybrid Drive: a chronic implant device combining tetrode arrays with silicon probes for layer-resolved ensemble electrophysiology in freely moving mice
Authors: Guardamagna Matteo , Eichler Ronny , Pedrosa Rafael , Aarts Arno , Meyer Arne F. , Battaglia Francesco P.
Published: August 2021

Understanding the function of brain cortices requires simultaneous investigation at multiple spatial and temporal scales and to link neural activity to an animal’s behavior. A major challenge is to measure within- and across-layer information in actively behaving animals, in particular in mice that have become a major species in neuroscience due to an extensive genetic toolkit. Here we describe the Hybrid Drive, a new chronic implant for mice that combines tetrode arrays to record within-layer information with silicon probes to simultaneously measure across-layer information. The design of our device combines up to 14 tetrodes and 2 silicon probes, that can be arranged in custom arrays to generate unique areas-specific (and multi-area) layouts. We show that large numbers of neurons and layer-resolved local field potentials can be recorded from the same brain region across weeks without loss in electrophysiological signal quality. The drive’s lightweight structure (≈ 3.5 g) leaves animal behavior largely unchanged during a variety of experimental paradigms. We demonstrate how the data collected with the Hybrid Drive allow state-of-the-art analysis in a series of experiments linking the spiking activity of CA1 pyramidal layer neurons to the oscillatory activity across hippocampal layers. Our new device fits a gap in the existing technology and increases the range and precision of questions that can be addressed about neural computations in freely behaving mice.