bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubules originate asymmetrically at the somatic Golgi and are guided via Kinesin2 to maintain polarity within neurons
Authors: Mukherjee Amrita , Brooks Paul , Bernard Fred , Guichet Antoine , Conduit Paul T.
Published: November 2019

Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. We show that γ-tubulin localises asymmetrically to the somatic Golgi withinDrosophilaneurons. Microtubules originate from the Golgi with an initial growth preference towards the axon. Their growing plus ends also turn towards and into the axon, adding to the plus-end-out microtubule pool. Any plus ends that reach a dendrite, however, do not readily enter, maintaining minus-end-out polarity. Both turning towards the axon and exclusion from dendrites depend on Kinesin-2, a plus-end-associated motor that guides growing plus ends along adjacent microtubules. We propose that Kinesin-2 engages with a polarised microtubule network within the soma to guide growing microtubules towards the axon; while at dendrite entry sites engagement with microtubules of opposite polarity generates a backward stalling force that prevents entry into dendrites and thus maintains minus-end-out polarity within proximal dendrites.
RH mapping by sequencing: chromosome-scale assembly of the duck genome
Authors: Rao Man , Vignal Alain , Morisson Mireille , Fillon Valérie , Leroux Sophie , Lhuillier Émeline , Esquerré Diane , Bouchez Olivier , Li Ning , Faraut Thomas
Published: November 2019

Like many other species, the duck genome has been sequenced thanks to the technological breakthrough provided by the emergence of Next Generation Sequencing (NGS). The resulting de novo assemblies are however made of thousands of scattered scaffolds. To achieve chromosome-scale contiguity, long-range intermediate genome maps remain indispensable. Radiation Hybrid (RH) maps have been used to assist the generation of chromosome-scale genome assemblies by taking advantage of the high density SNP chips that provide a large number of markers that can be efficiently genotyped on the panel.

In the absence of such a resource in duck, we sequenced 100 hybrid clones of a duck RH panel enabling direct genotyping of the assembly scaffolds on the panel. The rationale is to use scaffolds as markers and to genotype the scaffolds by sequencing the clones: the presence/absence of a scaffold in a particular sequenced hybrid is attested by the presence/absence of reads mapping specifically to this scaffold. The detection of scaffolds exhibiting a chromosomal breakage resulting from the irradiation process revealed itself to be a critical issue of this genotyping by sequencing process. This process resulted in the construction of RH vectors for 2,027 scaffolds, representing a total of about 1 Gb of sequences (95% of the current Duck genome assembly). The subsequent linkage analysis enabled the construction of RH maps and therefore to organize, i.e. order and orient, the scaffolds into pseudomolecules associated to the corresponding duck chromosomes. We describe here the whole mapping process, from sequence-based genotyping to the construction of comparative maps, as well as few examples of intra-chromosomal rearrangements that have been identified by the comparison with the chicken, turkey and zebra finch genomes and subsequently confirmed by FISH.

We describe a method to order and orient sequence scaffolds into super-scaffolds spanning entire chromosomes. The method, which requires a pre-existing RH panel and sequence scaffolds from an NGS assembly, relies on a shallow sequencing of the RH clones. This approach was applied to the duck genome and produced chromosome-scale scaffolds for 29 out of the 41 duck chromosomes.
TUTase mediated site-directed access to clickable chromatin employing CRISPR-dCas9
Authors: George Jerrin Thomas , Azhar Mohd. , Aich Meghali , Sinha Dipanjali , Ambi Uddhav B. , Maiti Souvik , Chakraborty Debojyoti , Srivatsan Seergazhi G.
Published: November 2019

Locus-specific interrogation of the genome using programmable CRISPR-based technologies is tremendously useful in dissecting the molecular basis of target gene function and modulating its downstream output. Although these tools are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using this technique is not well developed due to inadequate labeling technologies. Here, we report the development of a modular technology, sgRNA-Click (sgR-CLK), which harnesses the power of bioorthogonal click chemistry for remodeling CRISPR to display synthetic molecules on target genes. A terminal uridylyl transferase (TUTase) was repurposed to construct an sgRNA containing multiple minimally invasive bioorthogonal clickable handles, which served as a Trojan horse on CRISPR-dCas9 system to guide synthetic tags site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Our results demonstrate that sgR-CLK could provide a simplified solution for site-directed display of small molecules to study as well as modulate the function of gene targets.
A principal component approach to improve association testing with polygenic risk scores
Authors: Coombes Brandon J. , Biernacka Joanna M.
Published: November 2019

Polygenic risk scores (PRSs) have become an increasingly popular approach for demonstrating polygenic influences on complex traits and for establishing common polygenic signals between different traits. PRSs are typically constructed using pruning and thresholding (P+T), but the best choice of parameters is uncertain; thus multiple settings are used and the best is chosen. This optimization can lead to inflated type I error. To correct this, permutation procedures can be used but they can be computationally intensive. Alternatively, a single parameter setting can be chosen a priori for the PRS, but choosing suboptimal settings result in loss of power. We propose computing PRSs under a range of parameter settings, performing principal component analysis (PCA) on the resulting set of PRSs, and using the first PRS-PC in association tests. The first PC reweights the variants included in the PRS with new weights to achieve maximum variation over all PRS settings used. Using simulations, we compare the performance of the proposed PRS-PCA approach with a permutation test and a priori selection of p-value threshold. We then apply the approach to the Mayo Clinic Bipolar Disorder Biobank study to test for PRS association with psychosis using a variety of PRSs constructed from summary statistics from the largest studies of psychiatric disorders and related traits. The PRS-PCA approach is simple to implement, outperforms the other strategies in most scenarios, and provides an unbiased estimate of prediction performance. We therefore recommend it to be used PRS association studies where multiple phenotypes and/or PRSs are being investigated.
Tuning cell motility via cell tension with a mechanochemical cell migration model
Authors: Tao K. , Wang J. , Kuang X. , Wang W. , Liu F. , Zhang L.
Published: November 2019

Cell migration is orchestrated by a complicated mechanochemical system. However, few cell migration models take account of the coupling between a biochemical network and mechanical factors. Here, we construct a mechanochemical cell migration model to study the cell tension effect on cell migration. Our model incorporates the interactions between Rac-GTP, Rac-GDP, F-actin, myosin, and cell tension, and it is based on phase field approach hence very convenient in describing the cell shape change. This model captures common features of cell polarization, cell shape change, and cell migration modes. It shows cell tension inhibits migration ability monotonically when cells are applied with persistent external stimuli. On the other hand, if random internal noise is significant, the regulation of cell tension exerts a non-monotonic effect on cell migration. As the elevation of cell tension impedes the formation of multiple protrusions hence enhances the streamline position of the cell body. Therefore the migration ability could be maximized at intermediate cell tension under random internal noise. These model predictions are consistent with our singlecell experiments and other experimental results.

Statement of significance

Cell migration plays a vital role in many biological processes such as tumor metastasis. It is a complicated process regulated by dynamic coupling between the biochemical network and mechanical forces. However, few cell migration models take account of both factors. Here, we construct a mechanochemical cell migration model to study how cell migration is regulated by cell tension. Our model predicts that cell tension not only inhibits cell movement under persistent external stimuli but also prompts cell migration under random internal noise when cell tension is low. Therefore an optimized cell tension could maximize the migration ability under random internal noise. We further confirmed these model predictions are consistent with our single-cell experiments and other published experimental results.
Translational regulation of Pmt1 and Pmt2 by Bfr1 affects unfolded protein O-mannosylation
Authors: Castells-Ballester Joan , Rinis Natalie , Kotan Ilgin , Gal Lihi , Bausewein Daniela , Kats Ilia , Zatorska Ewa , Kramer Günter , Bukau Bernd , Schuldiner Maya , Strahl Sabine
Published: November 2019

O-mannosylation is implicated in protein quality control inSaccharomyces cerevisiaedue to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding GFP as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance, but has no effect onPMT1andPMT2transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.
Proximate causes of infertility and embryo mortality in captive zebra finches
Authors: Pei Yifan , Forstmeier Wolfgang , Wang Daiping , Martin Katrin , Rutkowska Joanna , Kempenaers Bart
Published: November 2019

Abstract

Some species show high rates of reproductive failure, which is puzzling because natural selection works against such failure in every generation. Hatching failure is common in both captive and wild zebra finches (Taeniopygia guttata), yet little is known about its proximate causes. Here we analyze data on reproductive performance (fate of >23,000 eggs) based on up to 14 years of breeding of four captive zebra finch populations. We find that virtually all aspects of reproductive performance are negatively affected by inbreeding (mean r = -0.117), by an early-starting, age-related decline (mean r = -0.132), and by poor early-life nutrition (mean r = - 0.058). However, these effects together explain only about 3% of the variance in infertility, offspring mortality, fecundity and fitness. In contrast, individual repeatability of different fitness components varied between 15% and 50%. As expected, we found relatively low heritability in fitness components (median: 7% of phenotypic, and 29% of individually repeatable variation). Yet, some of the heritable variation in fitness appears to be maintained by antagonistic pleiotropy (negative genetic correlations) between male fitness traits and female and offspring fitness traits. The large amount of unexplained variation suggests a potentially important role of local dominance and epistasis, including the possibility of segregating genetic incompatibilities.
Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming
Authors: Sampaio RV , Sangalli JR , De Bem THC , Ambrizi DR , del Collado M , Bridi A , Ávila ACFCM , Macabelli CH , Oliveira LJ , da Silveira JC , Chiaratti MR , Perecin F , Bressan FF , Smith LC , Ross PJ , Meirelles FV
Published: November 2019

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone metyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 causes diverse modifications to the chromatin during early development, suggesting an intense epigenetic crosstalk during nuclear reprogramming.
Exposure during embryonic development to Roundup® Power 2.0 affects lateralization, level of activity and growth, but not defensive behaviour of marsh frog tadpoles
Authors: Bolis Alessandro , Gazzola Andrea , Pellitteri-Rosa Daniele , Colombo Anita , Bonfanti Patrizia , Bellati Adriana
Published: November 2019

As glyphosate-based herbicides, sold under the commercial name Roundup®, represent the most used herbicides in the world, contamination of the freshwater environment by glyphosate has become a widespread issue. In Italy, glyphosate was detected in half of the surface waters monitoring sites and its concentrations were higher than environmental quality standards in 24.5% of them. It can last from days to months in water, leading to exposure for aquatic organisms and specifically to amphibians’ larvae that develop in shallow water bodies with proven effects to development and behaviour. In this study, we tested the effects of a 96h exposure during embryonic development of marsh frog’s tadpoles to three ecologically relevant Roundup® Power 2.0 concentrations. As expected, given the low concentrations tested, no mortality was observed. Morphological measurements highlighted a reduction in the total length in tadpoles exposed to 7.6 mg a.e./L, while an increase was observed at lower concentrations of 0.7 and 3.1 mg a.e./L compared to control group. Tadpoles raised in 7.6 mg a.e./L also showed a smaller tail membrane than those raised in the control solution. Regarding behaviour, we tested tadpoles in two different sessions (Gosner stages 25 and 28/29) for lateralization, antipredator response and basal activity. Lower intensity of lateralization was detected in tadpoles raised at the highest Roundup® concentration in the first session of observation, while no significant difference among treatments was observed in the second one. In both sessions, effects of glyphosate embryonic exposure on antipredator response, measured as the proportional change in activity after the injection of tadpole-fed predator (Anax imperator) cue, were not detected. Tadpoles exposed during embryonic development to Roundup® exhibited lower basal activity than the control group, with the strongest reduction for the 7.6 mg a.e./L treatment. Our results reinforce the concern of glyphosate contamination impact on amphibians.
Estimating Genetic Inheritance in Case-control Studies
Authors: Li Na , Zhu Jiayan , Li Zhengbang
Published: November 2019

Case-control genetic association study is an efficient tool to search for the deleterious genetic variants predispose to human complex diseases, where the additive mode of inheritance is commonly assumed. However, how the genetic variants influence the occurrence of a certain disease is impossible to know beforehand. We show numerically that the existing procedures using the Hardy-Weinberg equilibrium test to choose the genetic model might be inconsistent. We propose a new method to choose the genetic model by adopting co-dominant code for risk allele and logistic regression model. Extensive computer simulation results demonstrate superiorities of the new method. Applications to six single nucleotide polymorphisms(SNPs) for breast cancer and eight SNPs for Type 2 Diabetes further show good performances of proposed method. In order to specify a genetic model for an allele, our method has some merits and is consistent as sample size is large. We propose to apply our method in related fields.
A Multiple-trait Bayesian Variable Selection Regression Method for Integrating Phenotypic Causal Networks in Genome-Wide Association Studies
Authors: Wang Zigui , Chapman Deborah , Morota Gota , Cheng Hao
Published: November 2019

Bayesian regression methods that incorporate different mixture priors for marker effects are used in multi-trait genomic prediction. These methods can also be extended to genome-wide association studies (GWAS). In multiple-trait GWAS, incorporating the underlying causal structures among traits is essential for comprehensively understanding the relationship between genotypes and traits of interest. Therefore, we develop a GWAS methodology, SEM-BayesCΠ, which, by applying the structural equation model (SEM), can be used to incorporate causal structures into a multi-trait Bayesian regression method using mixture priors. The performance of SEM-BayesCΠ was demonstrated by comparing its GWAS results with those from multi-trait BayesCΠ. Through the inductive causation (IC) algorithm, three potential causal structures were inferred of 0.9 highest posterior density (HPD) interval. SEM-BayesCΠ provides a more comprehensive understanding of the genotype-phenotype mapping than multi-trait BayesCΠ by performing GWAS based on indirect, direct and overall marker effects. The software tool JWAS offers open-source routines to perform these analyses.
The Reissner Fiber is Highly Dynamic in vivo and Controls Morphogenesis of the Spine
Authors: Troutwine Benjamin , Gontarz Paul , Minowa Ryoko , Monstad-Rios Adrian , Konjikusic Mia J. , Sepich Diane S. , Kwon Ronald Y. , Solnica-Krezel Lilianna , Gray Ryan S.
Published: November 2019

Spine morphogenesis requires the integration of multiple musculoskeletal tissues with the nervous system. Cerebrospinal fluid (CSF) physiology is important for development and homeostasis of the central nervous system and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an enigmatic glycoprotein thread called the Reissner fiber, which is secreted from the subcomissural organ (SCO) in the brain and extends caudally through the central canal to where it terminates at the base of the spinal cord. In zebrafish,scospondinnull mutants are unable to assemble the Reissner fiber and fail to extend a straight body axis during embryonic development [3]. Here, we describe zebrafish hypomorphic missense alleles, which assemble the Reissner fiber and straighten the body axis during early embryonic development, yet progressively lose the fiber, concomitant with the emergence of body curvature, alterations in neuronal gene expression, and scoliosis in adults. Using an endogenously taggedscospondin-GFPzebrafish knock-in line, we directly visualized Reissner fiber dynamics during the normal development and during the progression of scoliosis, and demonstrate that the Reissner fiber is critical for the morphogenesis of the spine. Our study establishes a framework for future investigations of mechanistic roles of the Reissner fiber including its dynamic properties, molecular interactions, and how these processes are involved in the regulation of spine morphogenesis and scoliosis.

Highlights

Hypomorphic mutations in zebrafishscospondinresult in progressive scoliosis

The disassembly of the Reissner fiber inscospondinhypomorphic mutants results in the strong upregulation of neuronal receptors and synaptic transport components

An endogenous fluorescent knock-in allele ofscospondinreveals dynamic properties of the Reissner fiber during zebrafish development

Loss of the Reissner fiber during larval development is a common feature of zebrafish scoliosis models
To composite or replicate: how sampling method and protocol differences alter stream bioassessment metrics
Authors: Tronstad Lusha , Wilmot Oliver , Thornbrugh Darren , Hotaling Scott
Published: November 2019

Aquatic invertebrates are excellent indicators of ecosystem quality; however, choosing a sampling method can be difficult. Each method and associated protocol has advantages and disadvantages, and finding the approach that minimizes biases yet fulfills management objectives is crucial. To test the effects of both sampling methods and sample handling – i.e., to composite samples or leave them as replicates – we collected aquatic invertebrates from the Niobrara River at Agate Fossil Beds National Monument, Nebraska using three methods and two sample handling protocols. We compared aquatic invertebrate assemblages collected with a Hester-Dendy multi-plate sampler, Hess sampler and a D-frame dipnet. We calculated six common bioassessment metrics from composite (combined) and replicate (separate) samples. Hess samples contained the highest taxonomic richness (capturing 77% of all taxa observed) and dipnet samples the least (47%). Hester-Dendy samples had the greatest proportion of Ephemeroptera, and Ephemeroptera, Plecoptera and Trichoptera (EPT). Dipnet samples had the lowest evenness values. In terms of sample handling, composite samples had inflated richness, diversity and evenness compared to replicate samples, but bioassessment metrics calculated from proportions or averages (i.e. Hilsenhoff’s Biotic Index and the proportion of EPT taxa) did not differ between them. The proportion of invertebrate groups from composite samples were not statistically different among sampling methods, but several groups differed between replicate samples collected by different methods. Ultimately, we recommend collecting replicate samples with a Hess sampler when the goal of the study is to detect ecosystem change, among locations or differences in variables of interest.
A Division of Labor in the Recruitment and Topological Organization of a Bacterial Morphogenic Complex
Authors: Caccamo Paul D. , Jacq Maxime , VanNieuwenhze Michael S. , Brun Yves V.
Published: November 2019

Bacteria come in an array of shapes and sizes, but the mechanisms underlying diverse morphologies are poorly understood. The peptidoglycan (PG) cell wall is the primary determinant of cell shape. At the molecular level, morphological variation often results from the regulation of enzymes involved in cell elongation and division. These enzymes are spatially controlled by cytoskeletal scaffolding proteins, that both recruit and organize the PG synthesis complex. How then do cells define alternative morphogenic processes that are distinct from cell elongation and division? To address this, we have turned to the specific morphotype of Alphaproteobacterial stalks. Stalk synthesis is a specialized form of zonal growth, which requires PG synthesis in a spatially constrained zone to extend a thin cylindrical projection of the cell envelope. The morphogen SpmX defines the site of stalk PG synthesis, but SpmX is a PG hydrolase. How then does a non-cytoskeletal protein, SpmX, define and constrain PG synthesis to form stalks? Here we report that SpmX and the bactofilin BacA act in concert to regulate stalk synthesis in Asticcacaulis biprosthecum. We show that SpmX recruits BacA to the site of stalk synthesis. BacA then serves as a stalk-specific topological organizer for PG synthesis activity, including its recruiter SpmX, at the base of the stalk. In the absence of BacA, cells produce “pseudostalks” that are the result of unconstrained PG synthesis. Therefore, the protein responsible for recruitment of a morphogenic PG remodeling complex, SpmX, is distinct from the protein that topologically organizes the complex, BacA.
Evaluating the efficacy of multiple myeloma cell lines as models for patient tumors via transcriptomic correlation analysis
Authors: Sarin Vishesh , Yu Katharine , Ferguson Ian D. , Gugliemini Olivia , Nix Matthew A. , Hann Byron C. , Sirota Marina , Wiita Arun P.
Published: November 2019

Multiple myeloma (MM) cell lines are routinely used to model the disease. However, a long-standing question is how well these cell lines truly represent tumor cells in patients. Here, we employ a recently-described method of transcriptional correlation profiling to compare similarity of 66 MM cell lines to 779 newly-diagnosed MM patient tumors. We found that individual MM lines differ significantly with respect to patient tumor representation, with median R ranging from 0.35-0.54. ANBL-6 was the “best” line, markedly exceeding all others (p < 2.2e-16). Notably, some widely-used cell lines (RPMI-8226, U-266) scored poorly in our patient similarity ranking (48 and 52 of 66, respectively). Lines cultured with interleukin-6 showed significantly improved correlations with patient tumor (p = 9.5e-4). When common MM genomic features were matched between cell lines and patients, only t(4;14) and t(14;16) led to increased transcriptional correlation. To demonstrate utility of our top-ranked line for preclinical studies, we showed that intravenously-implanted ANBL-6 proliferates in hematopoietic organs in immunocompromised mice. Overall, our large-scale quantitative correlation analysis, utilizing emerging datasets, provides a resource informing the MM community of cell lines that may be most reliable for modeling patient disease while also elucidating biological differences between cell lines and tumors.
Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex
Authors: Schaefer Kristina N. , Pronobis Mira , Williams Clara E. , Zhang Shiping , Bauer Lauren , Goldfarb Dennis , Yan Feng , Major M. Ben , Peifer Mark
Published: November 2019

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis, and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector β-catenin. The multiprotein destruction complex binds and phosphorylates β-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase, for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing β-catenin. Recent work clarified underlying mechanisms, but important questions remain. We explore β-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin’s RGS domain plays an important role. We find that elevating Dishevelled levels inDrosophilaembryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. SIM imaging in mammalian cells suggests that this may result by promoting Dishevelled: Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.
Chromosome-level assembly of Drosophila bifasciata reveals important karyotypic transition of the X chromosome
Authors: Bracewell Ryan , Tran Anita , Chatla Kamalakar , Bachtrog Doris
Published: November 2019

The Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromere, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.
Breast tumor stiffness instructs bone metastasis via mechanical memory
Authors: Watson Adam W , Grant Adam , Parker Sara S , Harman Michael W , Roman Mackenzie R , Forte Brittany L , Gowan Cody C , Castro-Portuguez Raul , Franck Christian , Cusanovich Darren , Padi Megha , Romanoski Casey , Mouneimne Ghassan
Published: November 2019

The mechanical microenvironment of primary breast tumors plays a substantial role in promoting tumor progression. While the transitory response of cancer cells to pathological stiffness in their native microenvironment has been well described, it is still unclear how mechanical stimuli in the primary tumor influence distant, late-stage metastatic phenotypes across time and space in absentia. Here, we show that primary tumor stiffness promotes stable, non-genetically heritable phenotypes in breast cancer cells. This mechanical memory instructs cancer cells to adopt and maintain increased cytoskeletal dynamics, traction force, and 3D invasion in vitro, in addition to promoting osteolytic bone metastasis in vivo. Furthermore, we established a mechanical conditioning (MeCo) score comprised of mechanically-regulated genes as a global gene expression measurement of tumor stiffness response. Clinically, we show that a high MeCo score is strongly associated with bone metastasis in patients. Using a discovery approach, we mechanistically traced mechanical memory in part to ERK-mediated mechanotransductive activation of RUNX2, an osteogenic gene bookmarker and bone metastasis driver. The combination of these RUNX2 traits permits the stable transactivation of osteolytic target genes that remain upregulated after cancer cells disseminate from their activating microenvironment in order to modify a distant microenvironment. Using genetic, epigenetic, and functional approaches, we were able to simulate, repress, select and extend RUNX2-mediated mechanical memory and alter cancer cell behavior accordingly. In concert with previous studies detailing the influence of biochemical properties of the primary tumor stroma on distinct metastatic phenotypes, our findings detailing the influence of biomechanical properties support a generalized model of cancer progression in which the integrated properties of the primary tumor microenvironment govern the secondary tumor microenvironment, i.e., soil instructs soil.
Time is vision: functional preservation and enhanced capacity for recovery in subacute occipital stroke
Authors: Saionz Elizabeth L. , Tadin Duje , Melnick Michael D. , Huxlin Krystel R.
Published: November 2019

Stroke damage to the primary visual cortex (V1) causes a loss of vision known as hemianopia or cortically-induced blindness (CB). While early, spontaneous, perimetric improvements can occur, by 6 months post-stroke, the deficit is considered chronic and permanent. Despite evidence from sensorimotor stroke showing that early injury responses heighten neuroplastic potential, to date, rehabilitation research has focused only on chronic CB patients. Consequently, little is known about the functional properties of subacute, post-stroke visual systems, and whether they can be harnessed to enhance visual recovery. Here, for the first time, we show that conscious visual discrimination abilities are partially preserved inside subacute, perimetrically-defined blind fields, disappearing by 6 months post-stroke. Complementing this discovery, we show that global motion discrimination training initiated subacutely leads to comparable magnitude of recovery as that initiated in chronic CB. However, it does so 6 times faster, generalizes to deeper, untrained regions of the blind field, and to other [untrained] aspects of motion perception, preventing their degradation upon reaching the chronic period. Untrained subacutes exhibited only spontaneous improvements in perimetry - spontaneous recovery of motion discriminations was never observed. Thus, in CB, the early post-stroke period appears characterized by gradual - rather than sudden - loss of visual processing. Subacute training stops this degradation, and is dramatically more efficient at eliciting recovery than identical training in the chronic period. Finally, spontaneous improvements in subacutes appear restricted to luminance detection, whereas recovering discrimination abilities requires deliberate training. Simply stated, after an occipital stroke, “time is VISION”.

One Sentence Summary

The first 3 months after an occipital stroke are characterized by a gradual - not sudden - loss of visual perceptual abilities and increased rehabilitative potential if visual discrimination training is administered in the blind field.
Endogenous responses in brain pH andPO2in a rodent model of birth asphyxia
Authors: Pospelov Alexey S. , Puskarjov Martin , Kaila Kai , Voipio Juha
Published: November 2019

Aim

To study brain-sparing physiological responses in a rodent model of birth asphyxia which reproduces the asphyxia-defining systemic hypoxia and hypercapnia.

Methods

Steady or intermittent asphyxia was induced for 15-45 min in anesthetized 6 and 11 days old rats and neonatal guinea pigs using gases containing 5% or 9% O2plus 20% CO2(in N2). Hypoxia and hypercapnia were induced with low O2and high CO2, respectively. Oxygen partial pressure (PO2) and pH were measured with microsensors within the brain and subcutaneous (“body”) tissue. Blood lactate was measured after asphyxia.

Results

Brain and bodyPO2fell to apparent zero with little recovery during 5% O2asphyxia and 5% or 9% O2hypoxia, and increased more than twofold during 20% CO2hypercapnia. Unlike bodyPO2, brainPO2recovered rapidly to control after a transient fall (rat), or was slightly higher than control (guinea pig) during 9% O2asphyxia. Asphyxia (5% O2) induced a respiratory acidosis paralleled by a progressive metabolic (lact)acidosis that was much smaller within than outside the brain. Hypoxia (5% O2) produced brain-confined alkalosis. Hypercapnia outlasting asphyxia suppressed pH recovery and prolonged the post-asphyxiaPO2overshoot. All pH changes were accompanied by consistent shifts in the blood-brain barrier potential.

Conclusion

Regardless of brain maturation stage, hypercapnia can restore brainPO2and protect the brain against metabolic acidosis despite compromised oxygen availability during asphyxia. This effect extends to recovery phase if normocapnia is restored slowly, and it is absent during hypoxia, demonstrating that exposure to hypoxia does not mimic asphyxia.
RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells
Authors: Kovač Adrian , Miskey Csaba , Menzel Michael , Grueso Esther , Gogol-Döring Andreas , Ivics Zoltán
Published: November 2019

Two different approaches of genomic modification are currently used for genome engineering and gene therapy: integrating vectors, which can efficiently integrate large transgenes but are unspecific with respect to their integration sites, and nuclease-based approaches, which are highly specific but not efficient at integrating large genetic cargoes. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, occurred in a relatively narrow, ∼200 bp window around the targeted sites and displayed an asymmetric pattern with a bias towards sites that are downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for efficient and specific gene insertion for precision genetic engineering.
Unspecific expression in limited excitatory cell populations in interneuron-targeting Cre-driver lines can have large functional effects
Authors: Müller-Komorowska Daniel , Opitz Thoralf , Elzoheiry Shehabeldin , Schweizer Michaela , Beck Heinz
Published: November 2019

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Transgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. Particularly, the investigation of interneurons has benefitted from the ability to target genetic constructs to defined cell types. However, the cell type specificity of some mouse lines has been called into questions. Here we show for the first time the functional consequences of unspecific expression in a somatostatin-Cre (SST-Cre) mouse line. We find large optogenetically evoked excitatory currents originating from unspecifically targeted CA3 pyramidal cells. We also used public Allen Brain Institute data to estimate expression specificity in other Cre lines. Another SST-Cre mouse lines shows comparable unspecificity, whereas a Parvalbumin-Cre mouse line shows much less unspecific expression. Finally, we make suggestions to ensure that the results from in-vivo use of Cre mouse lines are interpretable.
Rhomboid protease RHBDL4 promotes retrotranslocation of aggregation-prone proteins for degradation
Authors: Bock Josephine , Kühnle Nathalie , Knopf Julia D. , Landscheidt Nina , Lee Jin-Gu , Ye Yihong , Lemberg Marius K.
Published: November 2019

Protein degradation is fundamentally important to ensure cell homeostasis. In the endoplasmic reticulum (ER), the ER-associated degradation (ERAD) pathway targets incorrectly folded and unassembled proteins into the cytoplasm for turnover by the proteasome. In contrast, lysosomal degradation serves as a failsafe mechanism for removing proteins that resist ERAD by forming aggregates. Previously, we showed that the ER- resident rhomboid protease RHBDL4, together with p97, mediates membrane protein degradation. However, whether RHBDL4 acts in concert with additional ERAD components is unclear, and its full substrate spectrum remains to be defined. Here, we show that besides membrane proteins, RHBDL4 cleaves aggregation-prone luminal ERAD substrates. Because RHBDL4 with mutations in the rhomboid domain leads to stabilization of substrates at the cytoplasmic side, we hypothesize that analogue to the homologue ERAD factor derlin, RHBDL4 is directly involved in substrate retrotranslocation. RHBDL4’s interaction with the erlin ERAD complex and reciprocal interaction of rhomboid substrates with erlins suggest that RHBDL4 and erlins form a complex that clips substrates and thereby rescues aggregation-prone peptides in the ER lumen from terminal aggregation.
Semaphorin 3A induces cytoskeletal paralysis in tumor-specific CD8+ T cells
Authors: Barnkob Mike B , Michaels Yale S , André Violaine , Macklin Philip S , Gileadi Uzi , Valvo Salvatore , Rei Margarida , Kulicke Corinna , Chen Ji-Li , Jain Vitul , Woodcock Victoria , Colin-York Huw , Hadjinicolaou Andreas V , Kong Youxin , Mayya Viveka , Bull Joshua A , Rijal Pramila , Pugh Christopher W , Townsend Alain R , Olsen Lars R , Fritzsche Marco , Fulga Tudor A , Dustin Michael L , Jones E Yvonne , Cerundolo Vincenzo
Published: November 2019

Semaphorin-3A (Sema3A) regulates tumor angiogenesis, but its role in modulating anti-tumor immunity is unclear. We demonstrate that Sema3A secreted within the tumor microenvironment (TME) suppresses tumor-specific CD8+ T cell function via Neuropilin-1 (NRP1), a receptor that is upregulated upon activation with T cells’ cognate antigen. Sema3A inhibits T cell migration, assembly of the immunological synapse, and tumor killing. It achieves these functional effects through hyper-activating the acto-myosin system in T cells leading to cellular paralysis. Finally, using a clear cell renal cell carcinoma patient cohort, we demonstrate that human tumor-specific CD8+ T cells express NRP1 and are trapped in Sema3A rich regions of tumors. Our study establishes Sema3A as a potent inhibitor of anti-tumor immunity.
A cell fate switch in theC. elegansseam cell lineage occurs through modulation of the Wnt asymmetry pathway in response to temperature increase
Authors: Hintze Mark , Koneru Sneha L. , Gilbert Sophie P.R. , Katsanos Dimitris , Barkoulas Michalis
Published: November 2019

Populations often display consistent developmental phenotypes across individuals despite the inevitable biological stochasticity. Nevertheless, developmental robustness has limits and systems can fail upon change in the environment or the genetic background. We use here the seam cells, a population of epidermal stem cells inCaenorhabditis elegans, to study the influence of temperature change and genetic variation on cell fate. Seam cell development has mostly been studied so far in the lab reference strain (N2), grown at 20° temperature. We demonstrate that an increase in culture temperature to 25°, introduces variability in the wild-type seam cell lineage with a proportion of animals showing an increase in seam cell number. We map this increase to lineage-specific symmetrisation events of normally asymmetric cell divisions at the final larval stage, leading to the retention of seam cell fate in both daughter cells. Using genetics and single molecule imaging, we demonstrate that this symmetrisation occurs via changes in the Wnt asymmetry pathway, leading to aberrant Wnt target activation in anterior cell daughters. We find that intrinsic differences in the Wnt asymmetry pathway already exist between seam cells at 20° and this may sensitise cells towards a cell fate switch at increased temperature. Finally, we demonstrate that wild isolates ofC. elegansdisplay variation in seam cell sensitivity to increased culture temperature, although seam cell numbers are comparable when raised at 20°. Our results highlight how temperature can modulate cell fate decisions in an invertebrate model of stem cell patterning.
Microtubule growth rates are sensitive to global and local changes in microtubule plus-end density
Authors: Geisterfer Zachary M. , Zhu Daniel , Mitchison Timothy , Oakey John , Gatlin Jesse C.
Published: November 2019

The microtubule (MT) cytoskeleton plays critically important roles in numerous cellular functions in eukaryotes, and it does so across a functionally diverse and morphologically disparate range of cell types [1]. In these roles, MT assemblies must adopt distinct morphologies and physical dimensions to perform specific functions [2-5]. As such, these macromolecular assemblies—as well as the dynamics of the individual MT polymers from which they are made—must scale and change in accordance with cell size and geometry. As first shown by Inoue using polarization microscopy, microtubules assemble to a steady state in mass, leaving enough of their subunits soluble to allow rapid growth and turnover. This suggests some negative feedback that limits the extent of assembly, for example decrease in growth rate, or increase in catastrophe rate, as the soluble subunit pool decreases. Such feedbacks might be global or local. Although these ideas have informed the field for decades, they have not been observed experimentally. Here we describe an experimental system designed to examine these long-standing ideas and determine a role for MT plus-end density in regulating MT growth rates.
A machine learning strategy that leverages large datasets to boost statistical power in small-scale experiments
Authors: Fondrie William E. , Noble William S.
Published: November 2019

Machine learning methods have proven invaluable for increasing the sensitivity of peptide detection in proteomics experiments. Most modern tools, such as Percolator and PeptideProphet, use semi-supervised algorithms to learn models directly from the datasets that they analyze. Although these methods are effective for many proteomics experiments, we suspected that they may be suboptimal for experiments of smaller scale. In this work, we found that the power and consistency of Percolator results was reduced as the size of the experiment was decreased. As an alternative, we propose a different operating mode for Percolator: learn a model with Per-colator from a large dataset and use the learned model to evaluate the small-scale experiment. We call this a “static modeling” approach, in contrast to Percolator’s usual “dynamic model” that is trained anew for each dataset. We applied this static modeling approach to two settings: small, gel-based experiments and single-cell proteomics. In both cases, static models increased the yield of detected peptides and eliminated the model-induced variability of the standard dynamic approach. These results suggest that static models are a powerful tool for bringing the full benefits of Percolator and other semi-supervised algorithms to small-scale experiments.

Abstract Figure
Profilin 1 Controls the Assembly, Organization, and Dynamics of Leading Edge Actin Structures Through Internetwork Competition and Collaboration
Authors: Skruber Kristen , Warp Peyton V. , Shklyarov Rachael , Thomas James D. , Swanson Maurice S. , Henty-Ridilla Jessica L. , Read Tracy-Ann , Vitriol Eric A.
Published: November 2019

How actin monomers are distributed to different networks remains poorly understood. One emerging concept is that the monomer pool is limited and heterogenous, causing biased assembly and internetwork competition. However, most knowledge regarding monomer distribution comes from studies where competing networks are discrete. In metazoans, many actin-based structures are complex, containing competing networks that overlap and are functionally interdependent. Addressing how monomers control the assembly and organization of these complex structures is critical to understanding how actin functions in cells. Here, we identify the monomer-binding protein profilin 1 (PFN1) as a major determinant of actin assembly, organization, and network homeostasis in mammalian cells. At the leading edge, PFN1 controls the localization and activity of the assembly factors Arp2/3 and Mena/VASP, with discrete stages of internetwork competition and collaboration occurring at different PFN1 concentrations. This causes substantial changes to leading edge actin architecture and the types of structures that form there.