bluuebunny/crossref_metadata_2025 huggingface dataset:
GPU Accelerated Adaptive Banded Event Alignment for Rapid Comparative Nanopore Signal Analysis
Authors: Gamaarachchi Hasindu , Lam Chun Wai , Jayatilaka Gihan , Samarakoon Hiruna , Simpson Jared T. , Smith Martin A. , Parameswaran Sri
Published: September 2019

Nanopore sequencing has the potential to revolutionise genomics by realising portable, real-time sequencing applications, including point-of-care diagnostics and in-the-field genotyping. Achieving these applications requires efficient bioinformatic algorithms for the analysis of raw nanopore signal data. For instance, comparing raw nanopore signals to a biological reference sequence is a computationally complex task despite leveraging a dynamic programming algorithm for Adaptive Banded Event Alignment (ABEA)—a commonly used approach to polish sequencing data and identify non-standard nucleotides, such as measuring DNA methylation. Here, we parallelise and optimise an implementation of the ABEA algorithm (termed f5c) to efficiently run on heterogeneous CPU-GPU architectures. By optimising memory, compute and load balancing between CPU and GPU, we demonstrate how f5c can perform ~3-5× faster than the original implementation of ABEA in the Nanopolish software package. We also show that f5c enables DNA methylation detection on-the-fly using an embedded System on Chip (SoC) equipped with GPUs. Our work not only demonstrates that complex genomics analyses can be performed on lightweight computing systems, but also benefits High-Performance Computing (HPC). The associated source code for f5c along with GPU optimised ABEA is available at https://github.com/hasindu2008/f5c.
HY5 is not integral to light mediated stomatal development in Arabidopsis
Authors: Zoulias Nicholas , Brown Jordan , Rowe James , Casson Stuart A.
Published: September 2019

Light is a crucial signal that regulates many aspects of plant physiology and growth including the development of stomata, the pores in the epidermal surface of the leaf. Light signals positively regulate stomatal development leading to changes in stomatal density and stomatal index (SI; the proportion of cells in the epidermis that are stomata). Both phytochrome and cryptochrome photoreceptors are required to regulate stomatal development in response to light. The transcription factor ELONGATED HYPOCOTYL 5 (HY5) is a key regulator of light signalling, acting downstream of photoreceptors. We hypothesised that HY5 could regulate stomatal development in response to light signals due to the putative presence of HY5 binding sites in the promoter of the STOMAGEN (STOM) gene, which encodes a peptide regulator of stomatal development. Our analysis shows that HY5 does have the potential to regulate the STOM promoter in vitro and that HY5 is expressed in both the epidermis and mesophyll. However, analysis of hy5 and hy5 hyh double mutants (HYH; HY5-HOMOLOG), found that they had normal stomatal development under different light conditions and the expression of stomatal developmental genes was not perturbed following light shift experiments. Analysis of stable lines overexpressing HY5 also showed no change in stomatal development or the expression of stomatal developmental genes. We therefore conclude that whilst HY5 has the potential to regulate the expression of STOM, it does not have a major role in regulating stomatal development in response to light signals.
Compilation of 29-yearpostmortemexaminations identifies major shifts in equine parasite prevalence from 2000 onwards
Authors: Sallé G. , Guillot J. , Tapprest J. , Foucher N. , Sevin C. , Laugier C.
Published: September 2019

Horses are infected by a wide range of parasite species that form complex communities. Parasite control imposes significant constraints on parasite communities whose monitoring remains however difficult to track through time.Postmortemexamination is a reliable method to quantify parasite communities. Here, we compiled 1,673 necropsy reports accumulated over 29 years, in the reference necropsy centre from Normandy (France). The burden of non-strongylid species was quantified and the presence of strongylid species was noted. Details of horse deworming history and the cause of death were registered. Building on these data, we investigated the temporal trend in non-strongylids epidemiology and we determined the contribution of parasites to the death of horses throughout the study period. Data analyses revealed the seasonal variations of non-strongylid parasite abundance and reduced worm burden in race horses. Beyond these observations, we found a shift in the species responsible for fatal parasitic infection from the year 2000 onward, whereby fatal cyathostominosis andParascarisspp. infection have replaced death cases caused byS. vulgarisand tapeworms. Concomitant break in the temporal trend of parasite species prevalence was also found within a 10-year window (1998-2007) that has seen the rise ofParascarisspp. and the decline of bothGasterophilusspp. and tapeworms. A few cases of parasite persistence following deworming were identified that all occurred after 2000. Altogether, these findings provide insights into major shifts in non-strongylid parasite prevalence and abundance over the last 29 years. They also underscore the critical importance ofParascarisspp. in young equids.
ArdC protein overpasses the recipient hsdRMS restriction system broadening conjugation host range
Authors: González-Montes Lorena , del Campo Irene , de la Cruz Fernando , Moncalian Gabriel
Published: September 2019

Plasmids, when transferred by conjugation, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida, but not from E. coli to E. coli. Surprisingly, expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. Thus, ArdC has antirestriction activity against HsdRMS system, and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and in the presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity is not needed for antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggers an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies.
FACT mediates cohesin function on chromatin
Authors: Garcia-Luis Jonay , Lazar-Stefanita Luciana , Gutierrez-Escribano Pilar , Thierry Agnes , García Alicia , González Sara , Sánchez Mar , Jarmuz Adam , Montoya Alex , Dore Marian , Kramer Holger , Karimi Mohammad Mehdi , Antequera Francisco , Koszul Romain , Aragon Luis
Published: September 2019

Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion 1,2 and the organisation of higher-order structures during interphase 3 and mitosis 4,5. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes 6-8. Here we show that cohesin role in chromosome organisation requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin-dependent compartmentalization of chromosomes into loop domains.
Temperature response of wheat affects final height and the timing of stem elongation under field conditions
Authors: Kronenberg Lukas , Yates Steven , Boer Martin P. , Kirchgessner Norbert , Walter Achim , Hund Andreas
Published: September 2019

In wheat, temperature affects the timing and intensity of stem elongation (SE). Genetic variation for this process is therefore important for adaptation. This study investigates the genetic response to temperature fluctuations during SE and its relationship to phenology and height. Canopy height of 315 wheat genotypes (GABI wheat panel) was scanned twice weekly in the field phenotyping platform (FIP) of ETH Zurich using a LIDAR. Temperature response was modelled using linear regressions between SE and mean temperature in each measurement interval. This led to a temperature–responsive (slope) and a temperature-irresponsive (intercept) component.

The temperature response was highly heritable (H2 = 0.81) and positively related to a later start and end of SE as well as final height. Genome-wide association mapping revealed three temperature-responsive and four temperature-irresponsive quantitative trait loci (QTL). Furthermore, putative candidate genes for temperature-response QTL were frequently related to the flowering pathway in A. thaliana, whereas temperature-irresponsive QTLs corresponded with growth and reduced height genes. In combination with Rht and Ppd alleles, these loci, together with the loci for the timing of SE accounted for 71% of the variability in height.

This demonstrates how high-throughput field phenotyping combined with environmental covariates can contribute to a smarter selection of climate-resilient crops.

Highlight

We measured ambient temperature response of stem elongation in wheat grown under field conditions. The results indicate that temperature response is highly heritable and linked to the flowering pathway.
Direct Arp2/3-vinculin binding is essential for cell spreading, but only on compliant substrates and in 3D
Authors: Isogai Tadamoto , Dean Kevin M. , Roudot Philippe , Shao Qiuyan , Cillay Justin D. , Welf Erik. S. , Driscoll Meghan K. , Royer Shaina P. , Mittal Nikhil , Chang Bo-Jui , Han Sangyoon J. , Fiolka Reto , Danuser Gaudenz
Published: September 2019

Cells modify their shape in response to the extracellular environment through dynamic remodeling of the actin cytoskeleton by actin-binding proteins (ABPs) 1–4. The relation between actin dynamics and spreading is well-understood for cells on flat glass coverslips; much less is known about cell morphogenesis in compliant three-dimensional environments, and, in particular, how ABPs contribute to this process 5. Here, we knocked-out a diverse set of ABPs, and evaluated the effect of each on cell spreading on planar glass surfaces (2D) and in reconstituted collagen gels (3D). Our morphometric analyses identify the Arp2/3 complex and its associated regulatory genes among the ABPs that contribute most strongly to cell spreading in 3D, but marginally in 2D. Cells lacking Arp3 have reduced spreading specifically in 3D, and display stiffness-dependent cell-matrix adhesion defects. Through manipulation of vinculin activity, we determine that the Arp3 knock-out phenotype largely arises from the lack of direct interaction between vinculin and Arp2/3 complex. This interaction is dispensable for cell spreading in 2D. These data highlight that actin architectural features necessary for adhesion formation and cell spreading in 3D are efficiently compensated on flat and stiff surfaces.
Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling
Authors: Forsthoefel David J. , Cejda Nicholas I. , Khan Umair W. , Newmark Phillip A.
Published: September 2019

Organ regeneration requires precise coordination of new cell differentiation and remodeling of uninjured tissue to faithfully re-establish organ morphology and function. An atlas of gene expression and cell types in the uninjured state is therefore an essential pre-requisite for understanding how damage is repaired. Here, we use laser-capture microdissection (LCM) and RNA-Seq to define the transcriptome of the intestine ofSchmidtea mediterranea,a planarian flatworm with exceptional regenerative capacity. Bioinformatic analysis of 1,844 intestine-enriched transcripts suggests extensive conservation of digestive physiology with other animals, including humans. Comparison of the intestinal transcriptome to purified absorptive intestinal cell (phagocyte) and published single-cell expression profiles confirms the identities of known intestinal cell types, and also identifies hundreds of additional transcripts with previously undetected intestinal enrichment. Furthermore, by assessing the expression patterns of 143 transcriptsin situ, we discover unappreciated mediolateral regionalization of gene expression and cell-type diversity, especially among goblet cells. Demonstrating the utility of the intestinal transcriptome, we identify 22 intestine-enriched transcription factors, and find that several have distinct functional roles in the regeneration and maintenance of goblet cells. Furthermore, depletion of goblet cells inhibits planarian feeding and reduces viability. Altogether, our results show that LCM is a viable approach for assessing tissue-specific gene expression in planarians, and provide a new resource for further investigation of digestive tract regeneration, the physiological roles of intestinal cell types, and axial polarity.
Splotch: Robust estimation of aligned spatial temporal gene expression data
Authors: Äijö Tarmo , Maniatis Silas , Vickovic Sanja , Kang Kristy , Cuevas Miguel , Braine Catherine , Phatnani Hemali , Lundeberg Joakim , Bonneau Richard
Published: September 2019

Spatial genomics technologies enable new approaches to study how cells interact and function in intact multicellular environments but present a host of technical and computational challenges. Here we describe Splotch, a novel computational framework for the analysis of spatially resolved transcriptomics data. Splotch aligns transcriptomics data from multiple tissue sections and timepoints to generate improved posterior estimates of gene expression. We demonstrate alignment of a large corpus of single-cell RNA-seq data into an automatically generated spatial-temporal coordinate and study optimal design for spatial transcriptomics experiments.
Genetic perturbations of disease risk genes in mice capture transcriptomic signatures of late-onset Alzheimer’s disease
Authors: Pandey Ravi S. , Graham Leah , Uyar Asli , Preuss Christoph , Howell Gareth R. , Carter Gregory W.
Published: September 2019

Background

New genetic and genomic resources have identified multiple genetic risk factors for late-onset Alzheimer’s disease (LOAD) and characterized this common dementia at the molecular level. Experimental studies in model organisms can validate these associations and elucidate the links between specific genetic factors and transcriptomic signatures. Animal models based on LOAD-associated genes can potentially connect common genetic variation with LOAD transcriptomes, thereby providing novel insights into basic biological mechanisms underlying the disease.

Methods

We performed RNA-Seq on whole brain samples from a panel of six-month-old female mice, each carrying one of the following mutations: homozygous deletions of Apoe and Clu; hemizygous deletions of Bin1 and Cd2ap; and a transgenic APOEε4. Similar data from a transgenic APP/PS1 model was included for comparison to early-onset variant effects. Weighted gene co-expression network analysis (WGCNA) was used to identify modules of correlated genes and each module was tested for differential expression by strain. We then compared mouse modules with human postmortem brain modules from the Accelerating Medicine’s Partnership for AD (AMP-AD) to determine the LOAD-related processes affected by each genetic risk factor.

Results

Mouse modules were significantly enriched in multiple AD-related processes, including immune response, inflammation, lipid processing, endocytosis, and synaptic cell function. WGCNA modules were significantly associated with Apoe−/−, APOEε4, Clu−/−, and APP/PS1 mouse models. Apoe−/−, GFAP-driven APOEε4, and APP/PS1 driven modules overlapped with AMP-AD inflammation and microglial modules; Clu−/− driven modules overlapped with synaptic modules; and APP/PS1 modules separately overlapped with lipid-processing and metabolism modules.

Conclusions

This study of genetic mouse models provides a basis to dissect the role of AD risk genes in relevant AD pathologies. We determined that different genetic perturbations affect different molecular mechanisms comprising AD, and mapped specific effects to each risk gene. Our approach provides a platform for further exploration into the causes and progression of AD by assessing animal models at different ages and/or with different combinations of LOAD risk variants.
Using Bifurcation Theory for Exploring Pain
Authors: Verma Parul , Kienle Achim , Flockerzi Dietrich , Ramkrishna Doraiswami
Published: September 2019

Pain is a common sensation which inescapably arises due to injuries, as well as, various diseases and disorders. However, for the same intensity of disturbance arising due to the forgoing causes, the threshold for pain sensation and perception varies among individuals. Here, we present a computational approach using bifurcation theory to understand how the pain sensation threshold varies and how it can be controlled, the threshold being quantified by the electrical activity of a pain-sensing neuron. To this end, we explored the bifurcations arising from a mathematical model representing the dynamics of this neuron. Our findings indicate that the bifurcation points are sensitive to specific model parameters. This demonstrates that the pain sensation threshold can change as shown in experimental studies found in literature. Further investigation using our bifurcation approach coupled with experimental studies can facilitate rigorous understanding of pain response mechanism and provide strategies to control the pain sensation threshold.
Induction of Activity Synchronization among Primed Hippocampal Neurons out of Random Dynamics is Key for Trace Memory Formation and Retrieval
Authors: Zhou Yuxin , Qiu Liyan , Wang Haiying , Chen Xuanmao
Published: September 2019

Memory is thought to be encoded by sparsely distributed neuronal ensembles in memory-related regions. However, it is unclear how memory-eligible neurons react incrementally during learning to encode trace fear memory, and how they respond to cues to retrieve the memory. We implemented fiber-optic confocal fluorescence endoscopy to directly visualize calcium dynamics of hippocampal CA1 neurons in freely behaving mice, which were subjected to trace fear conditioning. Here we report that the overall activity levels of CA1 principal neurons showed a right-skewed lognormal-like distribution. A small portion of highly active neurons (termed Primed Neurons) exhibited high sensitivity to sensory stimuli and marked activity plasticity. The Primed Neurons maintained random activity status for at least 5 hours in multiple contexts, including those prior to training and prior to recall. Repetitive training induced Primed Neurons to shift from random activity to a well-tuned synchronization. Importantly, the emergence of activity synchronization coincided with the appearance of mouse freezing behaviors. In recall, a partial synchronization among the same population of Primed Neurons was induced from originally random activity, which also coincided with mouse freezing behaviors. Additionally, training-induced synchronization facilitated robust calcium entry into individual Primed Neurons. In contrast, most CA1 neurons stayed silent and did not respond significantly to tone and foot-shock throughout the training and recall testing cycles. In conclusion, highly active Primed Neurons are preferably recruited to encode trace fear memory, and induction of activity synchronization among Primed Neurons out of random dynamics is critical for trace memory formation and memory retrieval.

Significance Statement

The overall activity levels of hippocampal principal CA1 neurons show a right-skewed lognormal-like distribution.

A small portion of CA1 neurons (termed Primed Neurons) exhibit high activity and marked activity plasticity during learning and recall.

Primed Neurons in the hippocampus are preferably recruited to encode trace fear memory, whereas the majority of CA1 neurons show little activity and little activity change during fear conditioning and recall testing.

Induction of activity synchronization among Primed Neurons out of random dynamics is critical for trace memory formation and memory retrieval.

Training-induced synchronization drastically increases calcium entry into Primed Neurons, which may promote memory consolidation.
QTL mapping of intestinal neutrophil variation and inflammation between threespine stickleback populations reveals links to neurodegenerative disease
Authors: Beck Emily A. , Currey Mark C. , Small Clayton M. , Cresko William A.
Published: September 2019

Host selection is often required to foster beneficial microbial symbionts and suppress deleterious pathogens. In animals, the host immune system is at the center of this relationship. Failed host immune system-microbial interactions can result in a persistent inflammatory response in which the immune system indiscriminately attacks resident microbes, and at times the host cells themselves, leading to diseases such as Ulcerative Colitis, Crohn’s Disease, and Psoriasis. Host genetic variation has been linked to both microbiome diversity and to severity of such inflammatory disease states in humans. However, the microbiome and inflammatory states manifest as quantitative traits, which encompass many genes interacting with one another and the environment. The mechanistic relationships among all of these interacting components are still not clear. Developing natural genetic models of host-microbe interactions is therefore fundamental to understanding the complex genetics of these and other diseases. Threespine stickleback (Gasterosteus aculeatus) fish are a tractable model for attacking this problem because of abundant population-level genetic and phenotypic variation in the gut inflammatory response. Previous work in our laboratory identified genetically divergent stickleback populations exhibiting differences in intestinal neutrophil activity. We took advantage of this diversity to genetically map variation in an emblematic element of gut inflammation – intestinal neutrophil recruitment – using an F2-intercross mapping framework. We identified three regions of the genome associated with increased intestinal inflammation containing several promising candidate genes. Within these regions we found candidates in the Coagulation/Complement System, NFkB and MAPK pathways along with several genes associated with neurodegenerative diseases commonly accompanying intestinal inflammation as a secondary symptom. These findings highlight the utility of using naturally genetically diverse ‘evolutionary mutant models’ such as threespine stickleback to better understand interactions among host genetic diversity and microbiome variation in health and disease states.
PTPN2 deletion in T cells promotes anti-tumour immunity and CAR T cell efficacy in solid tumours
Authors: Wiede Florian , Lu Kun-Hui , Du Xin , Liang Shuwei , Hochheiser Katharina , Dodd Garron T. , Goh Pei , Kearney Conor , Meyran Deborah , Beavis Paul A. , Henderson Melissa A. , Park Simone L. , Waithman Jason , Zhang Sheng , Zhang Zhong-Yin , Oliaro Jane , Gebhardt Thomas , Darcy Phillip K. , Tiganis Tony
Published: September 2019

Although adoptive T cell therapy has shown remarkable clinical efficacy in hematological malignancies, its success in combating solid tumours has been limited. Here we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling thereby enhancing both CAR T cell activation and homing to CXCL9/10 expressing tumours to eradicate HER-2+ mammary tumoursin vivo. Our findings define PTPN2 as a target for bolstering T-cell mediated anti-tumour immunity and CAR T cell therapy against solid tumours.
Dopamine response gene pathways in dorsal striatum MSNs from a gene expression viewpoint: cAMP-mediated gene networks
Authors: Babenko Vladimir , Galyamina Anna , Rogozin Igor , Smagin Dmitry , Kudryavtseva Natalia
Published: September 2019

A mouse model of chronic social conflicts was used to analyze dorsal striatum neurons implicated in cAMP-mediated phosphorylation activation pathways specific for Medium Spiny Neurons (MSNs). Based on expression correlation analysis, we succeeded in dissecting Drd1- and Drd2-dopaminoceptive neurons (D1 and D2, correspondingly) gene pathways. We also found that D1 neurons feature previously reported two states, passive and active ones, represented in our analysis by distinct, negatively correlated gene clusters.

The correlation based gene pathways strongly corroborate the phosphorylation cascades highlighted in the previous studies, implying that the expression-based viewpoint corresponds to phosphorylation/dephosphorylation interplay in each type of neurons. Notably, D2 neurons showed the largest Ppp1r1b (encoding DARPP-32) expression modulation impact, implying that Ppp1r1b expression dynamics is mostly associated with neuroendocrine response mediated by Penk/Pdyn genes expression in D2 neurons.

We observed that under defeat stress in chronic social conflicts mice exhibited reduced motor activity as well as overall depression of dopamine-mediated MSNs activity, while aggressive mice exhibited motor hyperactivity and an increase in both D1-active phase and D2 MSNs genes expression.

Based on alternative transcript isoforms expression analysis, it was assumed that many genes (Drd1, Adora1, Pde10, Ppp1r1b, Gnal), specifically those in D1 neurons, apparently remain transcriptionally repressed via the reversible mechanism of promoter CpG island silencing, resulting in alternative promoter usage following profound reduction in their expression rate.

Significance statement

Medium Spiny Neurons (MSNs) comprise the main body of dorsal striatum neurons and represent dopaminoceptive GABAergic neurons. The cAMP- mediated cascade of excitation and inhibition responses involved in dopaminergic neurotransmission is crucial for neuroscience research due to its involvement in the motor and behavioral functions. In particular, all types of addictions are related to MSNs. Shedding the light on the mechanics of the above-mentioned cascade is of primary importance for this research domain. In this paper MSNs steady states will be elucidated based on pooled tissue RNA-Seq data not explicitly outlined before and connected with dynamic dopamine neurotransmission cycles.
Kinetics of the immune response toEimeria maximain relatively resistant and susceptible chicken lines
Authors: Bremner Abi , Kim Sungwon , Morris Katrina , Nolan Matthew J. , Borowska Dominika , Wu Zhiguang , Tomley Fiona , Blake Damer P. , Hawken Rachel , Kaiser Pete , Vervelde Lonneke
Published: September 2019

Eimeria maximais a common cause of coccidiosis in chickens, a disease which has a huge economic impact on poultry production. Knowledge of immunity toE. maximaand the specific mechanisms that contribute to differing levels of resistance observed between chicken breeds and between congenic lines derived from a single breed of chickens is required. This study aimed to define differences in the kinetics of the immune response of two inbred lines of White Leghorn chickens that exhibit differential resistance (line C.B12) or susceptibility (line 15I) to infection byE. maxima. Line C.B12 and 15I chickens were infected withE. maximaand transcriptome analysis of infected jejunal tissue was carried out at 2, 4, 6 and 8 days post-infection (dpi). RNA-Seq analysis revealed differences in the rapidity and magnitude of cytokine transcription responses post-infection between the two lines. In particular, IFN-γ and IL-10 transcripts in the jejunum accumulated earlier in line C.B12 (at 4 dpi) compared to line 15I (at 6 dpi). Line C.B12 chickens exhibited increases ofIFNGandIL10mRNA in the jejunum at 4 dpi, whereas in line 15I transcription was delayed but increased to a greater extent. RT-qPCR and ELISAs confirmed the results of the transcriptomic study. Higher serum IL-10 correlated strongly with higherE. maximareplication in line 15I compared to line C.B12 chickens. Overall, the findings suggest early induction of the IFN-γ and IL-10 responses, as well as immune-related genes at 4 dpi identified by RNA-Seq, may be key to resistance toE. maxima.
A cell-free platform based on nisin biosynthesis for discovering novel lanthipeptides and guiding their overproductionin vivo
Authors: Liu Ran , Zhang Yuchen , Zhai Guoqing , Fu Shuai , Xia Yao , Hu Ben , Cai Xuan , Zhang Yan , Li Yan , Deng Zixin , Liu Tiangang
Published: September 2019

Lanthipeptides have extensive therapeutic and industrial applications; however, since many are bactericidal, traditionalin vivoplatforms are limited in their capacity to discover and mass produce novel lanthipeptides as bacterial organisms are often critical components in these systems. We developed a cell-free protein synthesis (CFPS) platform that enables rapid genome mining, screening and guiding overproduction of lanthipeptidesin vivo. For proof-of-concept studies, the type I lanthipeptide, nisin, was selected. Four novel lanthipeptides with anti-bacterial activity were identified among all nisin analogs in the NCBI database in a single day. Further, we coupled the CFPS platform with a screening assay for gram-negative bacterial growth and identified a potent nisin mutant, M5. The titer of nisin and nisin analogs significantly improved with CFPS platform guidance. Owing to the similarities in biosynthesis, our CFPS platform is broadly applicable to other lanthipeptides, provides a universal method for lanthipeptides discovery and overproduction.
PD-L1 expression in 522 selected sarcomas with subset analysis of recurrent or metastatic matched samples and association with tumour-infiltrating lymphocytes
Authors: Vargas Ana Cristina , Maclean Fiona M , Sioson Loretta , Tran Dinh , Bonar Fiona , Mahar Annabelle , Cheah Alison L. , Russell Peter , Grimison Peter , Richardson Louise , Gill Anthony J
Published: September 2019

We assessed the frequency of programmed death-ligand 1 (PD-L1) expression by immunohistochemistry (IHC) in a cohort of 522 sarcomas from 457 patients, incuding a subset of 46 patients with 63 matched samples from local recurrence or metastases with primary tumours and/or metachronous metastases. We also investigated the correlation of PD-L1 with the presence and degree of tumour-infiltrating lymphocytes (TILs) in a subset of cases. IHC was performed using the PD-L1 SP263 companion kit (VENTANA) on tissue microarrays from an archival cohort. Evaluation of PD-L1 and TILs was performed on full sections for a subset of 23 cases. Fisher’s exact and Mann Whitney test were used to establish significance (P <0.05). PD-L1 positive expression (≥1%) was identified in 31% of undifferentiated pleomorphic sarcomas, 29% of angiosarcomas, 26% of rhabdomyosarcomas, 18% of myxofibrosarcomas, 11% of leiomyosarcomas and 10% of dedifferentiated liposarcomas. Negative expression was present in all atypical lipomatous tumous/well-differentiated lipoasarcomas, myxoid liposarcomas, synovial sarcomas, pleomorphic liposarcomas, and Ewing sarcomas. PD-L1 IHC was concordant in 81% (38 of 47) of matched/paired samples. PD-L1 IHC was discordant in 19% (9 of 47 matched/paired samples), displaying differences in the proportion of cells expressing PD-L1 amongst paired samples with the percentage of PD-L1-positive cells increasing in the metastatic/recurrent site compared to the primary in 6 of 9 cases (67%). Significant correlation between PD-L1 expression and the degree of TILs was exclusively identified in the general cohort of leiomyosarcomas, but not in other sarcoma subtypes or in metastatic/recurrent samples. We conclude that the prevalence of PD-L1 expression in selected sarcomas is variable and likely to be clone dependent. Importantly, we demonstrated that PD-L1 can objectively increase in a small proportion of metastases/recurrent sarcomas, offering the potential of treatment benefit to immune checkpoint inhibitors in this metastatic setting.
Lgr5 Controls Extracellular Matrix Production By Stem Cells In The Developing Intestine
Authors: Vallone Valeria Fernandez , Leprovots Morgane , Gerbier Romain , Ribatallada-Soriano Didac , Lefort Anne , Libert Frédérick , Vassart Gilbert , Garcia Marie-Isabelle
Published: September 2019

The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos revealed that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, altered stem cell fate towards the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses revealed that fetal ISCs generate their own extracellular matrix components, a property lost in adult ISCs, which adopt a definitive epithelialized phenotype and an inflammatory response signature. Absence of Lgr5 in fetal ISCs resulted in reduced extracellular matrix production and accelerated ISC maturation, indicating that Lgr5 regulates the ISC niche. Finally, evidences are provided that Rspondin 2 negatively regulates the pool of ISCs in organoids via Lgr5, revealing a sophisticated regulatory process for Wnt signaling in ISC.
Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins
Authors: Qin Liang , Ben Bdira Fredj , Sterckx Yann G. J. , Volkov Alexander N. , Vreede Jocelyne , Giachin Gabriele , van Schaik Peter , Ubbink Marcellus , Dame Remus T.
Published: September 2019

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive.

Here, we focus on the H-NS family protein MvaT from P. aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strengths on electrostatic interactions between the appositively charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.
Biochemical and structural analysis reveals the Neurofibromin (NF1) protein forms a high-affinity dimer
Authors: Sherekar Mukul , Han Sae-Won , Ghirlando Rodolfo , Messing Simon , Drew Matthew , Juneja Puneet , O’Neill Hugh , Stanley Christopher , Bhowmik Debsindhu , Ramanathan Arvind , Subramaniam Sriram , Nissley Dwight V. , Gillette William , McCormick Frank , Esposito Dominic
Published: September 2019

Neurofibromin is the protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to aberrant signaling through the RAS-MAPK pathway due to disruption of the Neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of the majority of Neurofibromin—to date, biochemical and structural data exist only for the GAP domain and the region containing a Sec-PH motif. To better understand the role of this large protein, we carried out a series of biochemical and biophysical studies which demonstrate that full length Neurofibromin forms a high-affinity dimer. Neurofibromin dimerization also occurs in cells, and likely has biological and clinical implications. Analysis of purified full-length and truncated variants of Neurofibromin by negative stain electron microscopy reveals the overall architecture of the dimer and predicts the potential interactions which contribute to the dimer interface. Structures resembling high-affinity full-length dimers could be reconstituted by mixing N- and C-terminal protein domains in vitro. Taken together these data suggest how Neurofibromin dimers might form and be stabilized within the cell.
The kinesin-14, Ncd, drives a right-handed, helical motion of antiparallel microtubules around each other
Authors: Mitra Aniruddha , Gandhimathi Rojapriyadharshini , Ruhnow Felix , Renger Roman , Diez Stefan
Published: September 2019

Within the mitotic spindle, several kinesin motors crosslink and slide microtubules. While some of them (e.g. kinesin-5, kinesin-8 and kinesin-14) have been shown to exhibit sideways components in their step cycles, the impact of the resulting off-axis power strokes on motility and force generation in the spindle has not been investigated so far. Here, we develop and utilize a novel three-dimensional in vitro motility assay to explore the kinesin-14, Ncd, driven sliding of crosslinked, fluorescently-labeled microtubules. We find that free microtubules, sliding in an antiparallel orientation on microtubules suspended between nanofabricated ridges, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. In contrast, microtubules crosslinked in parallel orientation are static with neither longitudinal nor helical motion. Further, our technique allows us to measure the in situ spatial extension of the motors between the crosslinked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.
Comparative analysis of inducible promoters in cyanobacteria
Authors: Behle Anna , Saake Pia , Axmann Ilka M.
Published: September 2019

Research in the field of synthetic biology highly depends on efficient, well-characterized promoters. While great progress has been made with other model organisms such asEscherichia coli, photosynthetic cyanobacteria still lag behind. Commonly used promoters that have been tested in cyanobacteria show weaker dynamic range or no regulation at all. Alternatives such as native metal-inducible promoters pose the problem of inducer toxicity.

Here, we evaluate four different inducible promoters, both previously published and new, using the modular plasmid pSHDY, in the model cyanobacteriumSynechocystis sp. PCC 6803 - namely the vanillate-inducible promoter PvanCC, the rhamnose-inducible Prha, and the aTc-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages, as well as dynamic range and strength of each promoter in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in order to facilitate the construction of more complex regulatory synthetic networks, as well as more complicated biotechnological pathways for cyanobacteria.
Serotonin receptor 4 in mature excitatory hippocampal neurons modulates mood and anxiety
Authors: Karayol Remzi , Medrihan Lucian , Warner-Schmidt Jennifer L. , Rao Meghana N. , Holzner Eva B. , Greengard Paul , Heintz Nathaniel , Schmidt Eric F.
Published: September 2019

Serotonin receptor 4 (5-HT4R) plays an important role in regulating mood, anxiety, and cognition, and drugs that activate this receptor have fast-acting antidepressant (AD)-like effects in preclinical models. However, 5-HT4R is widely expressed throughout the central nervous system (CNS) and periphery, making it a poor therapeutic target and difficult to pinpoint the cell types and circuits underlying its effects. Therefore, we generated a Cre-dependent 5-HT4R knockout mouse line to dissect the function of 5-HT4R in specific brain regions and cell types. We show that the loss of functional 5-HT4R specifically in the mature excitatory neurons of hippocampus led to robust AD-like behavioral responses and an elevation in baseline anxiety. 5-HT4R was necessary to maintain the proper excitability of mature dentate gyrus (DG) granule cells and cell type specific molecular profiling revealed a dysregulation of genes necessary for normal neural function and plasticity in cells lacking 5-HT4R. These adaptations were accompanied by an increase in the number of immature neurons in ventral, but not dorsal, dentate gyrus, indicating a broad impact of 5-HT4R loss on the local cellular environment. This study is the first to use conditional genetic targeting to demonstrate a direct role for hippocampal 5-HT4R signaling in modulating mood and anxiety. Our findings also underscore the need for cell type-based approaches to elucidate the complex action of neuromodulatory systems on distinct neural circuits.
Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2-like RNA Helicase
Authors: Absmeier Eva , Santos Karine F. , Wahl Markus C.
Published: September 2019

RNA-dependent NTPases can act as RNA/RNA-protein remodeling enzymes and typically exhibit low NTPase activity in the absence of RNA/RNA-protein substrates. How futile intrinsic NTP hydrolysis is prevented is frequently not known. The ATPase/RNA helicase Brr2 belongs to the Ski2-like family of nucleic acid-dependent NTPases and is an integral component of the spliceosome. Comprehensive nucleotide binding and hydrolysis studies are not available for a member of the Ski2-like family. We present crystal structures of Chaetomium thermophilum Brr2 in the apo, ADP-bound and ATPyS-bound states, revealing nucleotide-induced conformational changes and a hitherto unknown ATPyS binding mode. Our results in conjunction with Brr2 structures in other molecular contexts reveal multiple molecular mechanisms that contribute to the inhibition of intrinsic ATPase activity, including an N-terminal region that restrains the RecA-like domains in an open conformation and exclusion of an attacking water molecule, and suggest how RNA substrate binding can lead to ATPase stimulation.

HIGHLIGHTS

Crystal structures of Brr2 in complex with different adenine nucleotides.

The Brr2 N-terminal region counteracts conformational changes induced by ATP binding.

Brr2 excludes an attacking water molecule in the absence of substrate RNA.

Different helicase families resort to different NTPase mechanisms.
Bidirectional Wnt signaling between endoderm and mesoderm confer tracheal identity in mouse and human
Authors: Kishimoto Keishi , Furukawa Kana T. , Madrigal Agustin Luz , Yamaoka Akira , Matsuoka Chisa , Habu Masanobu , Alev Cantas , Zorn Aaron M. , Morimoto Mitsuru
Published: September 2019

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. Loss of β-catenin in fetal mouse mesoderm caused loss of Tbx4+tracheal mesoderm and tracheal cartilage agenesis. The Tbx4 expression relied on endodermal Wnt activity and its downstream Wnt ligand but independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Repopulatingin vivomodel, activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generated tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation was required along with Wnt to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.
Profiling cellular diversity in sponges informs animal cell type and nervous system evolution
Authors: Musser Jacob M. , Schippers Klaske J. , Nickel Michael , Mizzon Giulia , Kohn Andrea B. , Pape Constantin , Hammel Jörg U. , Wolf Florian , Liang Cong , Hernández-Plaza Ana , Achim Kaia , Schieber Nicole L. , Francis Warren R. , Vargas R. Sergio , Kling Svenja , Renkert Maike , Feuda Roberto , Gaspar Imre , Burkhardt Pawel , Bork Peer , Beck Martin , Kreshuk Anna , Wörheide Gert , Huerta-Cepas Jaime , Schwab Yannick , Moroz Leonid L. , Arendt Detlev
Published: September 2019

The evolutionary origin of metazoan cell types such as neurons, muscles, digestive, and immune cells, remains unsolved. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identify 18 distinct cell types comprising four major families. This includes nitric-oxide sensitive contractile cells, digestive cells active in macropinocytosis, and a family of amoeboid-neuroid cells involved in innate immunity. We uncover ‘presynaptic’ genes in an amoeboid-neuroid cell type, and ‘postsynaptic’ genes in digestive choanocytes, suggesting asymmetric and targeted communication. Corroborating this, long neurite-like extensions from neuroid cells directly contact and enwrap choanocyte microvillar collars. Our data indicate a link between neuroid and immune functions in sponges, and suggest that a primordial neuro-immune system cleared intruders and controlled ciliary beating for feeding.
Mesoscale, Cantilever-Based Indentation Device for Mechanical Characterization of Soft Matter and Biological Tissue
Authors: Rubiano Andres , Simmons Chelsey S.
Published: September 2019

Tissue engineering has been driving a growing interest in mesoscale tissue mechanics (10−4 – 10−2 m), requiring tools to compare modulus between irregularly shaped primary tissue explants and synthetic scaffolds. We have designed and built a simple cantilever-based mesoscale indentation device to record force-displacement data during spring-loading, stress-relaxation, and creep experiments. Its simple design enables quantification of a wide range of soft matter moduli, from ~500 Pa collagen hydrogels to ~2 MPa silicones, by its compatibility with cantilevers of different stiffnesses and indentation probes of different sizes. A piezo-electric stage is used to drive a cylindrical or spherical indentation tip into the sample, while custom programming in LabVIEW through a data acquisition card enables stage control and acquisition of cantilever deflection using a capacitive sensor. Cantilever stiffness, deflection, and piezoelectric stage positions, acquired at a rate of 10Hz, are used to calculate force and indentation depth throughout indentation cycles. Using xyz manual coarse stages, tissue properties can be mapped across the sample surface. We have also built in commands to tune initial tip location using the piezo-stage to more easily find the sample surface, which is critical for accurate application of contact models. Here, we provide detailed information on how to design, build, and code a system for mesoscale indentation.