bluuebunny/crossref_metadata_2025 huggingface dataset:
The polymorphism ofHydramicrosatellite sequences provides strain-specific signatures
Authors: Schenkelaars Quentin , Perez-Cortez Diego , Perruchoud Chrystelle , Galliot Brigitte
Published: March 2020

Hydraare freshwater polyps widely studied for their amazing regenerative capacity, adult stem cell populations, low senescence and value as ecotoxicological marker. Many wild-type strains ofH. vulgarishave been collected worldwide and maintained effectively under laboratory conditions by asexual reproduction, while stable transgenic lines have been continuously produced since 2006. Efforts are now needed to ensure the genetic characterization of all these strains, which despite similar morphologies, show significant variability in their response to gene expression silencing procedures, pharmacological treatments or environmental conditions. Here, we established a rapid and reliable procedure at the single polyp level to produce via PCR amplification of three distinct microsatellite sequences molecular signatures that clearly distinguish betweenHydrastrains and species. The TG-rich region of an uncharacterized gene (ms-c25145) helps to distinguish between EurasianH. vulgarisstrains (Hm-105,Basel1,Basel2andreg-16), between Eurasian and North AmericanH. vulgarisstrains (H. carnea, AEP), and between theH. vulgarisandH. oligactisspecies. The AT-rich microsatellite sequences located in theAIPgene (Aryl Hydrocarbon Receptor Interaction Protein, ms-AIP) also differ between Eurasian and North AmericanH. vulgarisstrains. Finally, the AT-rich microsatellite located in theMyb-Like cyclin D-binding transcription factor1gene (ms-DMTF1) gene helps to distinguish certain transgenicAEPlines. This study shows that the analysis of microsatellite sequences provides a barcoding tool that is sensitive and robust for the identification ofHydrastrains. It is also capable of identifying cryptic species by tracing microevolutionary events within the genusHydra.
Overexposure to apoptosis via disrupted glial specification perturbsDrosophilamacrophage function and reveals roles of the CNS during injury
Authors: Armitage Emma Louise , Roddie Hannah Grace , Evans Iwan Robert
Published: March 2020

Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. Here we seek to understand how apoptotic cells affect macrophage function in the context of a genetically-tractableDrosophilamodel in which macrophages encounter excessive amounts of apoptotic cells. We show that loss of the glial transcription factorrepo, and corresponding removal of the contribution these cells make to apoptotic cell clearance, causes macrophages in the developing embryo to be challenged with large numbers of apoptotic cells. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells and their developmental dispersal and migration is perturbed. We also show that the requirement to deal with excess apoptosis caused by a loss ofrepofunction leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within arepomutant background. In investigating the underlying cause of these impaired inflammatory responses, we demonstrate that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together these results demonstrate important insights into macrophage biology and howrepomutants can be used to study macrophage-apoptotic cell interactions in the fly embryo.

Furthermore, this work shows how these multipurpose cells can be ‘overtasked’ to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo.
A comparative analysis ofCaenorhabditisandDrosophilatranscriptional changes in response to pathogen infection
Authors: Unckless Robert L. , Lansdon Patrick A. , Ackley Brian D.
Published: March 2020

Drosophila melanogasterandCaenorhabditis elegansare well-used invertebrate models for studying the innate immune system. The organisms are susceptible to bacterial pathogens that includePseudomonasspecies,(entomophilia – Drosophila) or (aeruginosa – Caenorhabditis), E. faecalisandP. rettgeri, which are or are related to human pathogens. Further, the consequences of exposure to these pathogens, in terms of organismal survival, are roughly equivalent when compared. That is, worms and flies are more susceptible to infection byPseudomonasthanE. faecalis, whereas organismal survival onE. faecalisandP. rettgeriare roughly the same in both. To better understand how these organisms are coordinating their responses to these bacterial pathogens we examined transcriptomes in infected animals. We grouped our analysis based on protein orthology. Of the 3611 pairs analyzed, we found genes whose responses were conserved across the different species at a higher than expected rate for two of the three pathogens. Interestingly within the animals, genes with 1:1 orthologs between species behaved differently. Such genes were more likely to be expressed inD. melanogaster, and less likely to be expressed inC. elegans. From this analysis we found that the gene nucleobindin (nucb-1/NUCB1inC. elegansandD. melanogaster, respectively) was upregulated in both species in response to Gram negative bacteria. We used RNAi to knock downnucb-1and found the treated animals were more susceptible to infection by the Gram negative pathogenP. rettgerithan controls. These results provide insight into some of the conserved mechanisms of pathogen defense, but also suggest that these divergent organisms have evolved specific means to orchestrate the defense against pathogens.

Article Summary

We analyzed transcriptomic data fromC. elegansandD. melanogasterto compare the expression of orthologous pairs of genes in response to bacterial pathogens. Our results indicated that only a handful of genes that are orthologous between species are differentially expressed in response to pathogens, but that the pattern of expression was different when comparing one-to-one orthologs versus those that are restricted to one of the two organisms. These results suggest that, although broad patterns of susceptibility to bacterial pathogens are conserved, the regulatory framework by which the organisms fight pathogens is less well conserved. Further our results suggest a more complete analysis of the evolutionary changes in organismal responses to pathogens is required.
Novel Functional metabolites that affect biofilm formation are regulated by bioavailable iron with siderophore-dependent pathway
Authors: Guo Rui , Luo Xilin , Liu Jingjing , Lu Haitao
Published: March 2020

Biofilms are broadly formed by diverse microorganisms under stressful environments and are basically surrounded by an EPS matrix, enabling bacterial cells to confer more resistance to biocides, antibiotics and other invasions than their planktonic counterparts. However, biofilm formation causes problems in various fields, including clinical infections, environmental pollution, agricultural production and industrial contamination. Unfortunately, the mechanism of biofilm formation has not been completely elucidated, and currently, we lack an efficient strategy to tackle these tough problems and destroy biofilms. In the present study, we sought to decipher the mechanism of biofilm formation through the regulation of functional metabolites regulated by iron. By exposing bacterial cells to various concentrations of iron, we found that iron can regulate biofilm formation, and phenotypic changes were obviously dependent on iron concentration. A functional metabolome assay was further implemented to investigate the regulatory mechanism of iron on biofilm formation; we verified that siderophores (linear enterobactin, yersiniabactin, di-glucosylated-salmochelin and HPTT-COOH) mostly account for the transportation of iron into bacterial cells. Then, bioavailable iron was recruited by bacterial cells to direct the biosynthesis and expression of five functional metabolites (L-tryptophan, 5’-MTA, spermidine, CMP and L-leucine), which were identified as new effectors that directly regulate biofilm formation. Taken together, this study is the first to identify five new metabolic effectors to efficiently regulate biofilm formation, the biosynthesis and expression of these functional metabolites can be targeted to tackle the challenging problems associated with biofilm formation in different fields.
Autoencoder networks extract latent variables and encode these variables in their connectomes
Authors: Farrell Matthew , Recanatesi Stefano , Reid R. Clay , Mihalas Stefan , Shea-Brown Eric
Published: March 2020

Spectacular advances in imaging and data processing techniques are revealing a wealth of information about brain connectomes. This raises an exciting scientific opportunity: to infer the underlying circuit function from the structure of its connectivity. A potential roadblock, however, is that – even with well constrained neural dynamics – there are in principle many different connectomes that could support a given computation. Here, we define a tractable setting in which the problem of inferring circuit function from circuit connectivity can be analyzed in detail: the function of input compression and reconstruction, in an autoencoder network with a single hidden layer. Here, in general there is substantial ambiguity in the weights that can produce the same circuit function, because largely arbitrary changes to “input” weights can be undone by applying the inverse modifications to the “output” weights. However, we use mathematical arguments and simulations to show that adding simple, biologically motivated regularization of connectivity resolves this ambiguity in an interesting way: weights are constrained such that the latent variable structure underlying the inputs can be extracted from the weights by using nonlinear dimensionality reduction methods.
Chromosomal Resistance to Metronidazole inClostridioides difficilecan be Mediated By Epistasis Between Iron Homeostasis and Oxidoreductases
Authors: Deshpande Aditi , Wu Xiaoqian , Huo Wenwen , Palmer Kelli L. , Hurdle Julian G.
Published: March 2020

Chromosomal resistance to metronidazole has emerged in clinicalClostridioides difficile, but the genetic mechanisms remain unclear. This is further hindered by the inability to generate spontaneous metronidazole-resistant mutants in the lab to aid genetic studies. We therefore constructed a mismatch repair mutator, in non-toxigenic ATCC 700057, to unbiasedly survey the mutational landscape forde novoresistance mechanisms. In separate experimental evolutions, the mutator adopted a deterministic path to resistance, with truncation of ferrous iron transporter FeoB1 as a first-step mechanism of low level resistance. Allelic deletion offeoB1in ATCC 700057 reduced intracellular iron content, appearing to shift cells toward flavodoxin-mediated oxidoreductase reactions, which are less favorable for metronidazole’s cellular action. Higher level resistance evolved from sequential acquisition of mutations to catalytic domains of pyruvate-ferredoxin oxidoreductase (PFOR encoded bynifJ); a synonymous codon change toxdhA1(xanthine dehydrogenase subunit A), likely affecting its translation; and lastly, frameshift and point mutations that inactivated the iron-sulfur cluster regulator (IscR). Gene silencing ofnifJ, xdhA1oriscRwith catalytically dead Cas9 revealed that resistance involving these genes only occurred whenfeoB1was inactivated i.e. resistance was only seen in anfeoB1-deletion mutant and not the isogenic wild-type parent. These findings show that metronidazole resistance inC. difficileis complex, involving multi-genetic mechanisms that could intersect with iron-dependent metabolic pathways.
Suitability of Deep Weakly Supervised Learning to detect Acute Ischemic Stroke and Hemorrhagic Infarction Lesions Using Diffusion-weighted Imaging
Authors: Cao Chen , Liu Zhiyang , Liu Guohua , Jin Song , Xia Shuang
Published: March 2020

Objectives

The automatic detection of acute ischemic stroke (AIS) and hemorrhagic infarction (HI) based on deep learning could avoid missed diagnosis. The fully supervised learning requires the amount of time and the expertise to manually outline lesions, which limits its applicability. The weakly supervised learning has the potential to reduce the labeling workload. The purpose of this study was to evaluate a weakly supervised method in detection of AIS and HI location using DWI.

Methods

We proposed to adopt weakly supervised learning to spatially-locate AIS lesions by residual neural network (ResNet) and visual geometry group (VGG) network. On an AIS dataset, the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity and precision were calculated. Next, ResNet, which presented superior performance on the AIS dataset, was further applied to an HI dataset.

Results

In the AIS dataset, the AUCs of ResNet and VGG on identifying image slices with AIS were 0.97 and 0.94, respectively. On spatially-locating the AIS lesions, ResNet provided higher sensitivity and a lower missed diagnosis rate than VGG, especially for pontine AIS lesions. In the HI dataset, the sensitivity of ResNet was 87.73% for AIS detection, and 86.20% for HI detection, respectively.

Conclusions

Weakly supervised learning can effectively detect the location of AIS and HI lesions in DWI, which is of paramount importance in avoiding misdiagnosis in clinical scenario.

Key points

The deep weakly supervised learning can reduce the labeling workload; ResNet can obtain more exact results, especially for pontine AIS lesions; Weakly supervised learning can effectively detect AIS and HI lesions in DWI
Safety and exceptional immunogenicity of novel 5T4 viral vectored vaccination regimes in early stage prostate cancer: a phase I clinical trial
Authors: Cappuccini Federica , Bryant Richard , Pollock Emily , Carter Lucy , Verrill Clare , Hollidge Julianne , Poulton Ian , Baker Megan , Mitton Celia , Baines Andrea , Meier Armin , Schmidt Guenter , Harrop Richard , Protheroe Andrew , MacPherson Ruth , Kennish Steven , Morgan Susan , Vigano Selena , Romero Pedro , Evans Thomas , Catto James W.F. , Hamdy Freddie , Hill Adrian V.S. , Redchenko Irina
Published: March 2020

Prostate cancer (PCa) has been under investigation as a target for antigen-specific immunotherapies in metastatic disease settings for a decade. However, neither of the two clinically most developed prostate cancer vaccines, Sipuleucel-T and ProstVac, induce strong T cell immunity. In this first-in-man study, VANCE, we evaluated a novel vaccination platform based on two replication-deficient viruses, chimpanzee adenovirus (ChAd) and MVA (Modified Vaccinia Ankara), targeting the oncofetal self-antigen 5T4 in early stage PCa. Forty patients, either newly diagnosed with early stage prostate cancer and scheduled for radical prostatectomy or patients with stable disease on an active surveillance protocol, were recruited to the study to assess the vaccine safety and T cell immunogenicity. Secondary and exploratory endpoints included immune infiltration into the prostate, prostate specific antigen (PSA) change and assessment of phenotype and functionality of antigen-specific T cells. The vaccine had an excellent safety profile. Vaccination-induced 5T4-specific T cell responses were measured in blood by ex vivo IFN-γ ELISpot and were detected in the majority of patients with a mean level in responders of 198 spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis demonstrated the presence of both CD8+ and CD4+ polyfunctional 5T4-specific T cells in the circulation. 5T4-reactive tumour infiltrating lymphocytes (TILs) were isolated from post-treatment prostate tissue. Some of the patients had a transient PSA rise 2-8 weeks following vaccination, possibly indicating an inflammatory response in the target organ. The potent T cell responses elicited support the evaluation of these vectored vaccine in efficacy trials.
Organ-restricted vascular delivery of nanoparticles for lung cancer therapy
Authors: Bölükbas Deniz A. , Datz Stefan , Meyer-Schwickerath Charlotte , Morrone Carmela , Doryab Ali , Gößl Dorothee , Vreka Malamati , Yang Lin , Argyo Christian , van Rijt Sabine H. , Lindner Michael , Eickelberg Oliver , Stoeger Tobias , Schmid Otmar , Lindstedt Sandra , Stathopoulos Georgios T. , Bein Thomas , Wagner Darcy E. , Meiners Silke
Published: March 2020

Nanomedicines hold immense promise for a number of devastating diseases due to the ability to custom-design both the carrier and cargo. However, their clinical implementation has been hampered by physicochemical and biological barriers and off-target deposition which impair cell specific targeting, especially in internal organs. This study reports a new delivery approach using organ-restricted vascular delivery to allow for direct administration and recirculation of stimuli-responsive nanoparticles to promote cellular uptake into an organ of interest. Using this technique, nanoparticles reach the interior of dense tumors and are selectively taken up by lung cancer cells. Importantly, this surgical approach is essential as the same nanoparticles do not reach lung tumor cells upon systemic or intratracheal administration. Organ-restricted vascular delivery thus opens up new avenues for optimized nanotherapies for cancer and other diseases.
Nascent transcript folding plays a major role in determining RNA polymerase elongation rates
Authors: Turowski Tomasz W. , Petfalski Elisabeth , Goddard Benjamin D. , French Sarah L. , Helwak Aleksandra , Tollervey David
Published: March 2020

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed thisin vivo, analyzing RNAPI inS.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5’ bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C-content.In vitroexperiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI fromS.pombewas similarly sensitive to transcript folding, as wereS.cerevisiaeRNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding.

Abstract Figure

HIGHLIGHTS

Structures in the nascent RNA correlate with rapid elongation by RNAPIin vivo

Stable RNA structures limit RNAPI backtrackingin vitro

GC content in the transcription bubble tunes transcription elongation rate

Nascent transcript folding modulates dynamics of all three RNAPsin vivo
Phenotype Discovery from Population Brain Imaging
Authors: Gong Weikang , Beckmann Christian F. , Smith Stephen M.
Published: March 2020

Neuroimaging allows for the non-invasive study of the brain in rich detail. Data-driven discovery of patterns of population variability in the brain has the potential to be extremely valuable for early disease diagnosis and understanding the brain. The resulting patterns can be used as imaging-derived phenotypes (IDPs), and may complement existing expert-curated IDPs. However, population datasets, comprising many different structural and functional imaging modalities from thousands of subjects, provide a computational challenge not previously addressed. Here, for the first time, a multimodal independent component analysis approach is presented that is scalable for data fusion of voxel-level neuroimaging data in the full UK Biobank (UKB) dataset, that will soon reach 100,000 imaged subjects. This new computational approach can estimate modes of population variability that enhance the ability to predict thousands of phenotypic and behavioural variables using data from UKB and the Human Connectome Project. A high-dimensional decomposition achieved improved predictive power compared with widely-used analysis strategies, single-modality decompositions and existing IDPs. In UKB data (14,503 subjects with 47 different data modalities), many interpretable associations with non-imaging phenotypes were identified, including multimodal spatial maps related to fluid intelligence, handedness and disease, in some cases where IDP-based approaches failed.
Transient interactions and influence among bacteria in field-grown Arabidopsis thaliana tissues
Authors: Beilsmith Kathleen , Perisin Matthew , Bergelson Joy
Published: March 2020

Interactions between bacteria are thought to play an important role in the assembly of plant microbial communities (1), yet the extent of temporal variation in these interactions is unclear. We inferred interactions from sequence-based counts of bacteria in a series of Arabidopsis thaliana tissue samples spanning major developmental transitions in the plant life cycle (2). Bacterial interactions were transient, even among variants found together at consecutive developmental stages. The overwhelming majority of these interactions were positive, indicating that competition for the plant niche might be a less important driver of bacterial abundances than cooperation or common responses to host and environmental factors. Over time, interaction networks diverged from an initial scale-free structure and became increasingly modular. In all networks, we found evidence of a hierarchical structure in which hub bacteria bridged network modules. However, the identities of bacteria in these influential roles also varied during plant growth.
Potassium Glutamate and Glycine Betaine Induce Self-Assembly of Sliding Clamps into Higher Order Oligomers
Authors: Purohit Anirban , Douma Lauren G. , Bloom Linda B. , Levitus Marcia
Published: March 2020

Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of theEscherichia coliβ-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of β is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. Replacing NaCl by KGlu, the primary cytoplasmic salt inE. coli, results in the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with theS. cerevisiaePCNA sliding clamp, suggesting that KGlu effects are not specific to β. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly whenE. coliis exposed to high concentrations of extracellular solute. Possible biological implications are discussed.
High throughput functional variant screens via in-vivo production of single-stranded DNA
Authors: Schubert Max G. , Goodman Daniel B. , Wannier Timothy M. , Kaur Divjot , Farzadfard Fahim , Lu Timothy K. , Shipman Seth L. , Church George M.
Published: March 2020

Tremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits inEscherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.
Comprehensive in virio structure probing analysis of the influenza A virus identifies a functional RNA structure involved in replication and segment interactions
Authors: Takizawa Naoki , Higashi Koichi , Kawaguchi Risa Karakida , Gotoh Yasuhiro , Suzuki Yutaka , Hayashi Tetsuya , Kurokawa Ken
Published: March 2020

The influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures on vRNA are thought to be involved in the viral proliferation process, such as intersegment interactions that are necessary for segment bundling. However, the functional RNA structure on vRNA is not well known because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins in a sequence-independent manner. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)-seq. By integrating DMS-seq and SHAPE-seq analyses with robust statistical analysis, we inferred quite a few bases paired regions including a pseudoknot structure on segment 5. Notably, when cells were infected with the recombinant virus which had mutations in the pseudoknot structure, the impairment of replication and packaging was observed on the other specific segment. Moreover, we analyzed the comprehensive intersegment RNA interactions in virion by ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq). Our LIGR-seq analysis revealed that the intersegment interactions of the specific segment became less frequent and rearranged in the recombinant virus in concordance with the strength of genome packaging impairment. Our data provide evidence that the functional RNA structure motif on the influenza A virus genome can affect the efficiency of replication and segment bundling through the segment interactions.
Differentiation drives widespread rewiring of the neural stem cell chaperone network
Authors: Vonk Willianne I. M. , Rainbolt T. Kelly , Dolan Patrick T. , Webb Ashley E. , Brunet Anne , Frydman Judith
Published: March 2020

Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Life-long maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPCs chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.
Sperm migration in the genital tract -in silicoexperiments identify key factors for reproductive success
Authors: Diemer Jorin , Hahn Jens , Goldenbogen Björn , Müller Karin , Klipp Edda
Published: March 2020

Sperm migration in the female genital tract controls sperm selection and, therefore, reproductive success as male gametes are conditioned for fertilization while their number is dramatically reduced. Mechanisms underlying sperm migration are mostly unknown, sincein vivoinvestigations are mostly unfeasible for ethical or practical reasons. By presenting a spatio-temporal model of the mammalian female genital tract combined with agent-based description of sperm motion and interaction as well as parameterizing it with bovine data, we offer an alternative possibility for studying sperm migrationin silico. The model incorporates genital tract geometry as well as biophysical principles of sperm motion observedin vitrosuch as positive rheotaxis and thigmotaxis. This model for sperm migration from vagina to oviducts was successfully tested againstin vivodata from literature. We found that physical sperm characteristics such as velocity and directional stability as well as sperm-fluid interactions and wall alignment are critical for success, i.e. sperms reaching the oviducts. Therefore, we propose that these identified sperm parameters should be considered in detail for conditioning sperm in artificial selection procedures since the natural processes are normally bypassed in reproductivein vitrotechnologies. The tremendous impact of mucus flow to support sperm accumulation in the oviduct highlights the importance of a species-specific optimum time window for artificial insemination regarding ovulation. Predictions from our extendablein silicoexperimental system will improve assisted reproduction in humans, endangered species, and livestock.
A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP
Authors: Felipe-Medina Natalia , Caburet Sandrine , Sánchez-Sáez Fernando , Condezo Yazmine B. , de Rooij Dirk , Gómez-H Laura , García-Valiente Rodrigo , Todeschini Anne-Laure , Duque Paloma , Sánchez-Martín Manuel , Shalev Stavit A. , Llano Elena , Veitia Reiner , Pendás Alberto M.
Published: March 2020

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with 3 cases of POI, we identified the candidate missense variant S167L inHSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse, compared to a newHSF2BPknock-out mouse showed that it behaves as a hypomorphic allele. HSF2BP-S167L females show reduced fertility with small litter sizes. To obtain mechanistic insights, we identified C19ORF57/MIDAP as a strong interactor and stabilizer of HSF2BP by forming a higher-order macromolecular structure involving BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L mutation showed a strongly decreased expression of both MIDAP and HSF2BP at the recombination nodules. Although HSF2BP-S167L does not affect heterodimerization between HSF2BP and MIDAP, it promotes a lower expression of both proteins and a less proficient activity in replacing RPA by the recombinases RAD51/DMC1, thus leading to a lower frequency of cross-overs. Our results provide insights into the molecular mechanism of two novel actors of meiosis underlying non-syndromic ovarian insufficiency.

Felipe-Medina et al. describe a missense variant in the meiotic geneHSF2BPin a consanguineous family with Premature Ovarian Insufficiency, and characterize it as an hypormorphic allele, thatin vivoimpairs its dimerization with a novel meiotic actor, MIDAP/ C19ORF57, and affect recombination at double-strand DNA breaks.
An Artificial Cofactor catalyzing the Baylis-Hillman Reaction using Designed Streptavidin as Protein Host
Authors: Lechner Horst , Emann Vincent R. , Breuning M. , Höcker Birte
Published: March 2020

An artificial cofactor based on an organocatalyst embedded in a protein was used to conduct the Baylis-Hillman reaction in a buffered system. As protein host we chose streptavidin, since it can be easily crystallized and thereby supports the design process. The protein host around the cofactor was rationally designed based on high-resolution crystal structures obtained after each variation of the amino acid sequence. Additionally, DFT-calculated intermediates and transition states were used to rationalize activity. Finally, repeated cycles of structure determination and redesign led to a system with 24 to 35-fold increased activity over the bare cofactor and to the most active proteinogenic catalyst for the Baylis-Hillman reaction known today.
Global Profiling of 2-hydroxyisobutyrylome in Common Wheat
Authors: Zhang Ning , Zhang Lingran , Li Linjie , Geng Junyou , Zhao Lei , Ren Yan , Dong Zhongdong , Chen Feng
Published: March 2020

As a novel post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) has been found to play a role in active gene transcription in mammalian cells and yeast, but the function of Khib proteins in plants remains unknown. In this study, we used western blot to demonstrate that Khib is an evolutionarily-conserved PTM in wheat and its donators, with the highest Khib abundance occurring in hexaploidy wheat. Additionally, global profiling using affinity purification and mass spectroscopy of 2-hydroxyisobutyrylome revealed that there were 3348 lysine modification sites from 1074 proteins in common wheat (Triticum aestivumL.). Moreover, bioinformatic data indicated that Khib proteins participate in a wide variety of biological and metabolic pathways. Immunoprecipitation and western blot confirmed that Khib proteins had anin vivoorigin. A comparison of Khib and other major PTMs revealed that Khib proteins were simultaneously modified by multiple PTMs. Using mutagenesis experiments and Co-IP, we demonstrated that Khib on K206 is a key regulatory modification of phosphoglycerate kinase enzymatic activity and found that de-Khib on K206 affects protein interactions. Furthermore, Khib production of low-molecular-weight proteins was a response to the deacetylase inhibitors nicotinamide and trichostatin A. This study provides evidence that enhances our current understanding of Khib in wheat plants, including the cooperation between this PTM and metabolic regulation.
Photoperiod stress alters the cellular redox status and is associated with an increased peroxidase and decreased catalase activity
Authors: Abuelsoud Walid , Cortleven Anne , Schmülling Thomas
Published: March 2020

Periodic changes of light and dark regulate numerous processes in plants. Recently, a novel type of stress caused by an extended light period has been discovered inArabidopsisand was named photoperiod stress. Photoperiod stress causes the induction of numerous stress response genes during the night following the extended light period of which many are indicators of oxidative stress. The next day, stress-sensitive genotypes display reduced photosynthetic efficiency and programmed cell death in leaves. Here, we have analysed further the consequences of photoperiod stress and report that it causes changes of the cellular redox status. A prolonged light period caused a strong reduction of the AsA redox during the following night indicating that it induces an oxidizing cellular environment. Further, photoperiod stress was associated with an increased activity of peroxidases and a decreased activity of catalases. Increased peroxidase activity was localized to the apoplast and might be causal for the oxidative stress induced by photoperiod stress.
The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
Authors: Marquina Daniel , Buczek Mateusz , Ronquist Fredrik , Łukasik Piotr
Published: March 2020

1.Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70 % ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA. Highly concentrated ethanol (95 – 100 %), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking.

2.We tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After preservation and treatments (various levels of disturbance), we counted the number of appendages (legs, wings, antennae, heads) that specimens had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard.

3.We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90 % made the insects more brittle, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95 % ethanol ranged from 90 % to as low as 50 %.

4.While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens.
FliI6-FliJ molecular motor assists with unfolding in the type III secretion export apparatus
Authors: Kucera Jiri , Terentjev Eugene M.
Published: March 2020

The role of rotational molecular motors of the ATP synthase class is integral to the metabolism of cells. Yet the function of FliI6-FliJ complex - a homolog of theF1ATPase motor - within the flagellar export apparatus remains unclear. We use a simple two-state model adapted from studies of linear molecular motors to identify key features of this motor. The two states are the ‘locked’ ground state where the FliJ coiled coil filament experiences fluctuations in an asymmetric torsional potential, and a ‘free’ excited state in which FliJ undergoes rotational diffusion. Michaelis-Menten kinetics was used to treat transitions between these two states, and obtain the average angular velocity of the FliJ filament within the FliI6stator:ωmax≈ 9.0 rps. The motor was then studied under external counter torque conditions in order to ascertain its maximal power output:Pmax≈ 42 kBT/s, and the stall torque:Gstall≈ 3 kBT/rad. Two modes of action within the flagellar export apparatus are proposed, in which the motor performs useful work either by continuously ‘grinding’ through the resistive environment, or by exerting equal and opposite stall force on it. In both cases, the resistance is provided by flagellin subunits entering the flagellar export channel prior to their unfolding. We therefore propose that the function of the FliI6-FliJ complex is to lower the energy barrier and therefore assist in unfolding of the flagellar proteins before feeding them into the transport channel.
Role ofCNC1gene in TDP-43 aggregation-induced oxidative stress-mediated cell death inS. cerevisiaemodel of ALS
Authors: Bharathi Vidhya , Girdhar Amandeep , Patel Basant K
Published: March 2020

TDP-43 is a multi-functional ribonucleoprotein that is also found deposited as hyper-phosphorylated and ubiquitinated TDP-43 inclusions in the brain and spinal cord of the patients of the motor neuron diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Till date, how the cell death ensues is not fully deciphered although several molecular mechanisms of the TDP-43 toxicity such as impairments of endocytosis and chromatin remodelling, mis-regulations of autophagy and proteasome function, mis-localization to the mitochondria and generation of oxidative stress etc., have been proposed. A predominantly nuclear protein, Cyclin C, can regulate the oxidative stress response by affecting the transcription of stress response genes and also by translocation to the cytoplasm for the activation of the mitochondrial fragmentation-dependent cell death pathway. Using the well-established yeast model of TDP-43 aggregation and toxicity, we examined here whether upon TDP-43 aggregation, the cell survival depends on the presence of theCNC1gene that encodes Cyclin C protein or other genes that encode proteins that function in conjunction with Cyclin C, such as theDNM1, FIS1andMED13genes. We found that the TDP-43 toxicity is significantly reduced in the yeast deleted for theCNC1orDNM1genes. Importantly, the rescue of TDP-43 toxicity in these yeast deletion backgrounds required the presence of functional mitochondria. Also, the deletion ofYBH3gene, which encodes for a protein involved in the mitochondria-dependent apoptosis, also reduced the TDP-43 toxicity. Furthermore, Cyclin C-YFP was observed to localize from the nucleus to the cytoplasm in response to the TDP-43 co-expression. Also, this cytoplasmic localization of Cyclin C was prevented by the addition of an anti-oxidant molecule, N-acetyl-cysteine. Taken together, our data suggest that Cyclin C, Dnm1 and Ybh3 proteins are important in mediating the TDP-43-induced oxidative stress-mediated cell death in theS. cerevisiaemodel.
Simultaneous monitoring of the same animals with PIT-tags and sensor nodes causes no system interference
Authors: Ripperger Simon P , Duda Niklas , Kölpin Alexander , Carter Gerald G
Published: March 2020

Recent technological advances have multiplied the variety of biologgers used in wildlife research, particularly with small-bodied animals. Passive integrated transponders (PIT) have been used for decades to log visits of tagged animals at reader-equipped artificial feeders or roost boxes. More recently, novel miniaturized sensor nodes can collect data on social encounters among tagged individuals in any location. Combining these systems allows researchers to gather high-resolution tracking data on certain individuals from their long-term PIT-tagged animal populations. However, there can be a risk of interference among tracking systems. Here we tested whether placing an additional biologging sensor on top of a PIT-tag might attenuate the magnetic field reaching the PIT-tag and in turn hamper reading success of the radio-frequency identification (RFID) reader. We also evaluated data transmission by a digital sensor node in the presence of a magnetic field created by the RFID-antenna. The combination of this RFID-system and wireless biologging sensors works without error, suggesting that the simultaneous use of PIT-tags and other digital biologgers, e.g. miniaturized GPS-loggers, should also work together properly when communication channels do not overlap. The combination of long-term monitoring with PIT tags and short-term tracking with biologging sensor nodes creates exciting new opportunities to gather rich social data from individuals not present at RFID reader stations.
Automated search of stimulation targets with closed-loop transcranial magnetic stimulation
Authors: Tervo Aino E. , Metsomaa Johanna , Nieminen Jaakko O. , Sarvas Jukka , Ilmoniemi Risto J.
Published: March 2020

Transcranial magnetic stimulation (TMS) protocols often include a manual search of an optimal location and orientation of the coil or peak stimulating electric field to elicit motor responses in a target muscle. This target search is laborious, and the result is user-dependent. Here, we present a closed-loop search method that utilizes automatic electronic adjustment of the stimulation based on the previous responses. The electronic adjustment is achieved by multi-locus TMS, and the adaptive guiding of the stimulation is based on the principles of Bayesian optimization to minimize the number of stimuli (and time) needed in the search. We compared our target-search method with other methods, such as systematic sampling in a predefined cortical grid. Validation experiments on five healthy volunteers and further offline simulations showed that our adaptively guided search method needs only a relatively small number of stimuli to provide outcomes with good accuracy and precision. The automated method enables fast and user-independent optimization of stimulation parameters in research and clinical applications of TMS.
Semantic Segmentation of HeLa Cells: An Objective Comparison between one Traditional Algorithm and Three Deep-Learning Architectures
Authors: Karabağ Cefa , Jones Martin L. , Peddie Christopher J. , Weston Anne E. , Collinson Lucy M. , Reyes-Aldasoro Constantino Carlos
Published: March 2020

In this work, images of a HeLa cancer cell were semantically segmented with one traditional image-processing algorithm and three deep learning architectures: VGG16, ResNet18 and Inception-ResNet-v2. Three hundred slices, each 2000 × 2000 pixels, of a HeLa Cell were acquired with Serial Block Face Scanning Electron Microscopy. The deep learning architectures were pre-trained with ImageNet and then fine-tuned with transfer learning. The image-processing algorithm followed a pipeline of several traditional steps like edge detection, dilation and morphological operators. The algorithms were compared by measuring pixel-based segmentation accuracy and Jaccard index against a labelled ground truth. The results indicated a superior performance of the traditional algorithm (Accuracy = 99%, Jaccard = 93%) over the deep learning architectures: VGG16 (93%, 90%), ResNet18 (94%, 88%), Inception-ResNet-v2 (94%, 89%).