bluuebunny/crossref_metadata_2025 huggingface dataset:
Repurposing of the tamoxifen metabolites to treat methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecalis infections
Authors: Miró-Canturri Andrea , Vila-Domínguez Andrea , Ayerbe-Algaba Rafael , Pachón J , Jiménez-Mejías Manuel E. , Smani Younes
Published: May 2021

Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM) and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyse the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus spp.

MRSE (n=17) and Enterococcus spp. (E. faecalis n=8, and E. faecium n=10) strains were used. MIC of the mixture of DTAM, HTAM and ENDX, and vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays.

MIC50 and MIC90 of tamoxifen metabolites were 1 mg/L for MRSE strains and 2 mg/L for Enterococcus spp. strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 2x and 4xMIC for MRSE (SE385 and SE742) and E. faecalis (EVR1 and EVR2) strains. This antimicrobial activity of tamoxifen metabolites paralleled an increased membrane permeability of SE385 and EVR2 strains.

Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drugs treatment against these bacterial pathogens.
A Deep Recurrent Reinforced Learning model to compare the efficacy of targeted local vs. national measures on the spread of COVID-19 in the UK
Authors: Dong Tim , benedetto umberto , Sinha Shubhra , Dimagli Arnaldo , Caputo Massimo , Angelini Gianni D
Published: May 2021

Objectives We have developed a deep learning model that provides predictions of the COVID-19 related number of cases and mortality in the upcoming 5 weeks and simulates the effect of policy changes targeting COVID-19 spread.
Methods We developed a Deep Recurrent Reinforced Learning (DRRL) based model. The data used to train the DRRL model was based on various available datasets that have the potential to influence the trend in the number of COVID-19 cases and mortality. Analyses were performed based on the simulation of policy changes targeting COVID-19 spread, and the geographical representation of these effects.
Results Model predictions of the number of cases and mortality of COVID-19 in the upcoming 5 weeks closely matched the actual values. Local lockdown with social distancing (LD_SD) was found to be ineffective compared to national lockdown. The ranking of effectiveness of supplementary measures for LD_SD were found to be consistent across national hotspots and local areas. Measure effectiveness were ranked from most effective to least effective: 1) full lockdown; 2) LD_SD with international travel -50%; 3) LD_SD with 100% quarantine; 4) LD_SD with closing school -50%; 5) LD_SD with closing pubs -50%. There were negligible differences observed between LD_SD, LD_SD with -50% food & Accommodation and LD_SD with -50% Retail.
Conclusions The second national lockdown should be followed by measures which are more effective than LD_SD alone. Our model suggests the importance of restrictions on international travel and travel quarantines, thus suggesting that follow-up policies should consist of the combination of LD_SD and a reduction in the number of open airports within close proximity of the hotspot regions. Stricter measures should be placed in terms travel quarantine to increase the impact of this measure. It is also recommended that restrictions should be placed on the number of schools and pubs open.
TheUrtica DioicaAgglutinin Prevents Rabies Virus Infection in a Muscle Explant Model
Authors: Wang Xinyu , Terrie Lisanne , Wu Guanghui , Van Damme Els J.M. , Fooks Anthony R , Banyard Ashley C , Thorrez Lieven , Jochmans Dirk , Neyts Johan
Published: May 2021

Infection with the rabies virus (RABV) results, once symptoms develop, in a 100% lethal neurological disease. Post-exposure prophylaxis (PEP), consists of a combination of vaccination and anti-rabies immunoglobulins (RIGs); it is 100% effective if administered early after infection. Because of the limited availability, alternatives for RIGs are needed. To that end we evaluated a panel of 33 different lectins for their effect on RABV infection in cell culture. Several lectins, with either mannose or GlcNAc specificity elicited anti-RABV activity of which the GlcNAc specificUrtica dioicaAgglutinin (UDA) was selected for further studies. UDA was found to prevent entry of the virus into the host cell. To further assess the potential of UDA, a physiologically relevant RABV infection muscle explant model was developed. Strips of dissected swine skeletal muscle that were kept in culture medium could be productively infected with the RABV. When the infection of the muscle strips was carried out in the presence of UDA, RABV replication was completely prevented. We thus developed a physiologically relevant RABV muscle infection model. UDA (i) may serve as a reference for further studies and (ii) holds promise as a cheap and simple to produce alternative for RIGs in PEP.
Fission yeast polycystin Pkd2p promotes the cell expansion and antagonizes the Hippo pathway SIN
Authors: Sinha Debatrayee , Ivan Denisa , Gibbs Ellie , Chetluru Madhurya , Goss John , Chen Qian
Published: May 2021

Polycystins are conserved mechanosensitive channels whose mutations lead to the common human renal disorder ADPKD. Previously we discovered that the plasma membrane-localized fission yeast homologue Pkd2p is an essential protein required for cytokinesis, but the mechanism remains unclear. Here, we isolated a novel temperature-sensitive mutant pkd2-B42. Among its strong growth defects, the most unique was that many mutant cells often lost significant portion of their volume in just 5 minutes followed by a gradual recovery, a process that we termed Deflation. Unlike cell lysis, deflation did not result in the plasma membrane rupture and it occurred independently from the cell cycle progression. The tip extension of pkd2-B42 cells was 80% slower than the wild type and their turgor pressure was 50% lower. Both pkd2-B42 and the other mutant pkd2-81KD partially rescued the mutants of the yeast Hippo signaling pathway Septation Initiation Network, by preventing cell lysis, enhancing septum formation, and doubling the number of Sid2/Mob1 molecules at the spindle pole bodies. We conclude that Pkd2p promotes cell size expansion during interphase by regulating turgor pressure and antagonizes SIN during cytokinesis.

Summary statement

Mutations of polycystins lead to human genetic disorder ADPKD. We discovered that the fission yeast homologue Pkd2p promotes the cell expansion during interphase growth and antagonizes the Hippo pathway SIN during cytokinesis.
An Integrative Apoptotic Reaction Model for extrinsic and intrinsic stimuli
Authors: Corbat Agustin A. , Silberberg Mauro , Grecco Hernán E.
Published: May 2021

Apoptosis, a form of programmed cell death central to all multicellular organisms, plays a key role during organism development and is often misregulated in cancer. Devising a single model applicable to distinct stimuli and conditions has been limited by lack of robust observables. Indeed, previous numerical models have been tailored to fit experimental datasets in restricted scenarios, failing to predict response to different stimuli. We quantified the activity of three caspases simultaneously upon intrinsic or extrinsic stimulation to assemble a comprehensive dataset. We measured and modeled the time between maximum activity of intrinsic, extrinsic and effector caspases, a robust observable of network dynamics, to create the first integrated Apoptotic Reaction Model (ARM). Observing how effector caspases reach maximum activity first irrespective of stimuli used, led us to identify and incorporate a missing feedback into a successful model for extrinsic stimulation. By simulating different recently performed experiments, we corroborated that ARM adequately describes them. This integrated model provides further insight into the indispensable feedback from effector caspase to initiator caspases.
Personality profiling may help select better cleaner fish for sea-lice control in salmon farming
Authors: Whittaker Benjamin Alexander , Consuegra Sofia , de Leaniz Carlos Garcia
Published: May 2021

Lumpfish (Cyclopterus lumpus) are increasingly being used as cleaner fish to control parasitic sea-lice in salmon farming, but cleaning rates are very variable and not all individuals eat sea-lice, which increases the risk of emaciation and has ethical and practical implications. Selecting good cleaners is a priority to make the industry more sustainable, but there is little information on what behaviours make cleaner fish effective under a commercial setting. We examined variation in lumpfish personalities according to the five-factor personality model that takes into account differences in activity, anxiety (shelter use, thigmotaxis), aggression, sociality, and boldness (neophobia). We then quantified how variation in lumpfish personalities influenced interactions with naïve Atlantic salmon (Salmo salar), without the confounding effects of variation in sea-lice loads. Variation in activity, sociality, aggression and neophobia, but not in anxiety, was repeatable, which is consistent with a heritable basis. Neophilic, non-aggressive lumpfish spent more time inspecting salmon than neophobic and aggressive individuals, but salmon fled in the presence of the most active and social individuals, suggesting there may be an optimal cleaner fish personality amenable to artificial selection. The personality screening protocols developed in this study could inform a more efficient use of cleaner fish in salmon farming and reduce the number of individuals required to control sea-lice.
Genetic isolation by distance underlies color pattern divergence in red-eyed treefrogs (Agalychnis callidryas)
Authors: Clark Meaghan I. , Bradburd Gideon S. , Akopyan Maria , Vega Andres , Rosenblum Erica Bree , Robertson Jeanne M.
Published: May 2021

Investigating the spatial distribution of genetic and phenotypic variation can provide insights into the evolutionary processes that shape diversity in natural systems. We characterized patterns of genetic and phenotypic diversity to learn about drivers of color-pattern diversification in red-eyed treefrogs (Agalychnis callidryas) in Costa Rica. Along the Pacific coast, red-eyed treefrogs have conspicuous leg color patterning that transitions from orange in the north to purple in the south. We measured phenotypic variation of frogs, with increased sampling at sites where the orange-to-purple transition occurs. At the transition zone, we discovered the co-occurrence of multiple color-pattern morphs. To explore possible causes of this variation, we generated a SNP dataset to analyze population genetic structure, measure genetic diversity, and infer the processes that mediate genotype-phenotype dynamics. We investigated how patterns of genetic relatedness correspond with individual measures of color pattern along the coast, including testing for the role of hybridization in geographic regions where orange and purple phenotypic groups co-occur. We found no evidence that color-pattern polymorphism in the transition zone arose through recent hybridization. Instead, a strong pattern of genetic isolation by distance (IBD) indicates that color-pattern variation was either retained through other processes such as ancestral color polymorphisms or ancient secondary contact, or else it was generated by novel mutations. We found that phenotype changes along the Pacific coast more than would be expected based on genetic divergence and geographic distance alone. Combined, our results suggest the possibility of selective pressures acting on color pattern at a small geographic scale.
Assessing and removing the effect of unwanted technical variations in microbiome data
Authors: Fachrul Muhamad , Méric Guillaume , Inouye Michael , Pamp Sünje Johanna , Salim Agus
Published: May 2021

Varying technologies and experimental approaches used in microbiome studies often lead to irreproducible results due to unwanted technical variations. Such variations, often unaccounted for and of unknown source, may interfere with true biological signals, resulting in misleading biological conclusions. In this work, we aim to characterize the major sources of technical variations in microbiome data and demonstrate how a state-of-the art approach can minimize their impact on downstream analyses. We analyzed 184 pig faecal metagenomes encompassing 21 specific combinations of deliberately introduced factors of technical and biological variations. We identify several known experimental factors, specifically storage conditions and freeze-thaw cycles, as a likely major source of unwanted variation in metagenomes. We also observed that these unwanted technical variations do not affect taxa uniformly, with freezing samples affecting taxa of class Bacteroidia the most, for example. Additionally, we benchmarked the performance of a novel batch correcting tool used in this study, RUV-III-NB (https://github.com/limfuxing/ruvIIInb/), to other popular batch correction methods, including ComBat, ComBat-seq, RUVg, and RUVs. While RUV-III-NB performed consistently robustly across our sensitivity and specificity metrics, most other methods did not remove unwanted variations optimally, with RUVg even overcorrecting and removing some of the true biological signals from the samples. Our analyses suggests that a careful consideration of possible technical confounders is critical in the experimental design of microbiome studies to ensure accurate biological reading of microbial taxa of interest, and that the inclusion of technical replicates is necessary to efficiently remove unwanted variations computationally.
A barley gene cluster for the biosynthesis of diterpenoid phytoalexins
Authors: Liu Yaming , Balcke Gerd U. , Porzel Andrea , Mahdi Lisa , Scherr-Henning Anja , Bathe Ulschan , Zuccaro Alga , Tissier Alain
Published: May 2021

Phytoalexins are specialized metabolites that are induced upon pathogen infection and contribute to the defense arsenal of plants. Maize and rice produce multiple diterpenoid phytoalexins and there is evidence from genomic sequences that other monocots may also produce diterpenoid phytoalexins. Here we report on the identification and characterization of a gene cluster in barley (Hordeum vulgare cv. Golden Promise) that is involved in the production of a set of labdane-related diterpenoids upon infection of roots by the fungal pathogen Bipolaris sorokiniana. The cluster is localized on chromosome 2, covers over 600 kb and comprises genes coding for a (+)-copalyl diphosphate synthase (HvCPS2), a kaurene synthase like (HvKSL4) and several cytochrome P450 oxygenases (CYPs). Expression of HvCPS2 and HvKSL4 in yeast and Nicotiana benthamiana resulted in the production of a single major product, whose structure was determined to be of the cleistanthane type and was named hordediene. Co-expression of HvCPS2, HvKSL4 and one of the CYPs from the cluster (CYP89E31) afforded two additional products, hordetriene and 11-hydroxy-hordetriene. Both of these compounds could be detected in extracts of barley roots infected by B. sorokiniana, validating the function of these genes in planta. Furthermore, diterpenoids with multiple oxidations and with molecular masses of 316, 318 and 332 were induced in infected barley roots and secreted in the medium, indicating that additional oxidases, possibly from the same genomic cluster are involved in the production of these phytoalexins. Our results provide the basis for further investigation of the role of this gene cluster in the defense of barley against pathogens and more generally in the interaction with the microbiome.
Automated Pipeline for Infants Continuous EEG (APICE): a flexible pipeline for developmental studies
Authors: Fló Ana , Gennari Giulia , Benjamin Lucas , Dehaene-Lambertz Ghislaine
Published: May 2021

Infant electroencephalography (EEG) presents several challenges compared with adult data. Recordings are typically short. Motion artifacts heavily contaminate the data. The EEG neural signal and the artifacts change throughout development. Traditional data preprocessing pipelines have been developed mainly for event-related potentials analyses, and they required manual steps, or use fixed thresholds for rejecting epochs. However, larger datasets make the use of manual steps infeasible, and new analytical approaches may have different preprocessing requirements. Here we propose an Automated Pipeline for Infants Continuous EEG (APICE). APICE is fully automated, flexible, and modular. Artifacts are detected using multiple algorithms and adaptive thresholds, making it suitable to different age groups and testing procedures without redefining parameters. Artifacts detection and correction of transient artifacts is performed on continuous data, allowing for better data recovery and providing flexibility (i.e., the same preprocessing is usable for different analyses). Here we describe APICE and validate it using two infant datasets of different ages tested in different experimental paradigms. We also tested the combination of APICE with common data cleaning methods such as Independent Component Analysis and Denoising Source Separation. APICE uses EEGLAB and compatible custom functions. It is freely available athttps://github.com/neurokidslab/eeg_preprocessing, together with example scripts.
Identification of a novel lineage bat SARS-related coronaviruses that use bat ACE2 receptor
Authors: Guo Hua , Hu Ben , Si Hao-rui , Zhu Yan , Zhang Wei , Li Bei , Li Ang , Geng Rong , Lin Hao-Feng , Yang Xing-Lou , Zhou Peng , Shi Zheng-Li
Published: May 2021

Severe respiratory disease coronavirus-2 (SARS-CoV-2) causes the most devastating disease, COVID-19, of the recent century. One of the unsolved scientific questions around SARS-CoV-2 is the animal origin of this virus. Bats and pangolins are recognized as the most probable reservoir hosts that harbor the highly similar SARS-CoV-2 related viruses (SARSr-CoV-2). Here, we report the identification of a novel lineage of SARSr-CoVs, including RaTG15 and seven other viruses, from bats at the same location where we found RaTG13 in 2015. Although RaTG15 and the related viruses share 97.2% amino acid sequence identities to SARS-CoV-2 in the conserved ORF1b region, but only show less than 77.6% to all known SARSr-CoVs in genome level, thus forms a distinct lineage in theSarbecovirusphylogenetic tree. We then found that RaTG15 receptor binding domain (RBD) can bind to and useRhinolophus affinisbat ACE2 (RaACE2) but not human ACE2 as entry receptor, although which contains a short deletion and has different key residues responsible for ACE2 binding. In addition, we show that none of the known viruses in bat SARSr-CoV-2 lineage or the novel lineage discovered so far use human ACE2 efficiently compared to SARSr-CoV-2 from pangolin or some of the SARSr-CoV-1 lineage viruses. Collectively, we suggest more systematic and longitudinal work in bats to prevent future spillover events caused by SARSr-CoVs or to better understand the origin of SARS-CoV-2.
Analysis of Normal Levels of Urine and Plasma Free Glycosaminoglycans in Adults
Authors: Bratulic Sinisa , Limeta Angelo , Maccari Francesca , Galeotti Fabio , Volpi Nicola , Levin Max , Nielsen Jens , Gatto Francesco
Published: May 2021

Plasma and urine glycosaminoglycans (GAGs) are long linear sulfated polysaccharides recognized as potential non-invasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition, the so-called GAGome, a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their urine and plasma free GAGomes using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS) method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine, and hemoglobin or erythrocyte counts in plasma. We found higher free GAGome concentration – but not composition - in males. Partitioned by gender, we established reference intervals for all detectable free GAGome features in urine and plasma. We carried out a transference analysis in healthy individuals from two distinct geographical sites, including the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal plasma and urine free GAGomes reference intervals and represents a critical resource for physiology and biomarker research.
Different individual-level responses of great black-backed gulls (Larus marinus) to shifting local prey availability
Authors: Maynard Laurie D. , Gulka Julia , Jenkins Edward , Davoren Gail K.
Published: May 2021

To grow, survive and reproduce under anthropogenic-induced changes, individuals must respond quickly and favourably to the surrounding environment. A species that feeds on a wide variety of prey types (i.e. generalist diet) may be comprised of generalist individuals, specialist individuals that feed on different prey types, or a combination of the two. If individuals within a population respond differently to an environmental change, population-level responses may not be detectable. By tracking foraging movements of eight great black-backed gulls (Larus marinus), a generalist species, we compared group-level and individual-level responses to an increase in prey biomass (capelin; Mallotus villosus) during the breeding season in coastal Newfoundland, Canada. As hypothesized, shifts in prey availability resulted in significantly different individual responses in foraging behaviour and space use, which was not detectable when data from individuals were combined. Some individuals maintained similar foraging areas, foraging trip characteristics (e.g., trip length, duration) and habitat use with increased capelin availability, while others shifted foraging areas and habitats resulting in either increased or decreased trip characteristics. We show that individual specialization can be non-contextual in some gulls, whereby these individuals continuously use the same feeding strategy despite significant change in prey availability conditions. Findings also indicate high response diversity among individuals to shifting prey conditions that a population- or group-level study would not have detected, emphasizing the importance of examining individual-level strategies for future diet and foraging studies on generalist species.
Linking animal migration and ecosystem processes: data-driven simulation of propagule dispersal by migratory herbivores
Authors: Somveille Marius , Ellis-Soto Diego
Published: May 2021

Animal migration is a key process underlying active subsidies and species dispersal over long distances, which affects the connectivity and functioning of ecosystems. Despite much research describing patterns of where animals migrate, we still lack a framework for quantifying and predicting how animal migration affects ecosystem processes.

In this study, we aim to integrate animal movement behavior and ecosystem functioning by developing a predictive modeling framework that can inform ecosystem management and conservation. Our framework models individual-level migration trajectories between populations’ seasonal ranges as well as the resulting dispersal and fate of propagules carried by the migratory animals, and it can be calibrated using empirical data at every step of the modeling process.

As a case study, we applied our framework to model the spread of guava seeds, Psidium guajava, by a population of migratory Galapagos tortoises, Chelonoidis porteri, across Santa Cruz Island. Galapagos tortoises are large herbivores that transport seeds and nutrients across the island, while Guava is one of the most problematic invasive species in the Galapagos archipelago.

Our model is able to predict the pattern of spread of guava seeds alongside tortoises’ downslope migration range, and it identified areas most likely to see germination success and establishment. Our results show that Galapagos tortoises’ seed dispersal may particularly contribute to guava range expansion on Santa Cruz Island, due to both long gut retention time and tortoise’s long-distance migration across vegetation zones. In particular, we predict that tortoises are dispersing a significant amount of guava seeds into the Galapagos National Park, which has important consequences for the native flora.

The flexibility and modularity of our framework allows for the integration of multiple data sources. It also allows for a wide range of applications to investigate how migratory animals affect ecosystem processes, including propagule dispersal but also other processes such as nutrient transport across ecosystems. Our framework is also a valuable tool for predicting how animal-mediated propagule dispersal can be affected by environmental change. These different applications can have important conservation implications for the management of ecosystems that include migratory animals.
Interrogation of cancer gene dependencies reveals novel paralog interactions of autosome and sex chromosome encoded genes
Authors: Köferle Anna , Schlattl Andreas , Hörmann Alexandra , Spreitzer Fiona , Popa Alexandra , Thatikonda Venu , Puchner Teresa , Oberndorfer Sarah , Wieshofer Corinna , Corcokovic Maja , Reiser Christoph , Wöhrle Simon , Popow Johannes , Pearson Mark , Mair Barbara , Neumüller Ralph A.
Published: May 2021

Genetic networks are characterized by extensive buffering. During tumour evolution, disruption of these functional redundancies can createde novovulnerabilities that are specific to cancer cells. In this regard, paralog genes are of particular interest, as the loss of one paralog gene can render tumour cells dependent on a remaining paralog. To systematically identify cancer-relevant paralog dependencies, we searched for candidate dependencies using CRISPR screens and publicly available loss-of-function datasets. Our analysis revealed >2,000 potential candidate dependencies, several of which were subsequently experimentally validated. We provide evidence thatDNAJC15-DNAJC19, FAM50A-FAM50BandRPP25-RPP25Lare novel cancer relevant paralog dependencies. Importantly, our analysis also revealed unexpected redundancies between sex chromosome genes. We show that chrX- and chrY- encoded paralogs, as exemplified byZFX-ZFY, DDX3X-DDX3YandEIF1AX-EIF1AY, are functionally linked so that tumour cell lines from male patients with Y-chromosome loss become exquisitely dependent on the chrX-encoded gene. We therefore propose genetic redundancies between chrX- and chrY- encoded paralogs as a general therapeutic strategy for human tumours that have lost the Y-chromosome.
Afferent connections of cytoarchitectural area 6M and surrounding cortex in the marmoset: putative homologues of the supplementary and pre-supplementary motor areas
Authors: Bakola Sophia , Burman Kathleen J. , Bednarek Sylwia , Chan Jonathan M. , Jermakow Natalia , Worthy Katrina H. , Majka Piotr , Rosa Marcello G.P.
Published: May 2021

Cortical projections to the caudomedial frontal cortex were studied using retrograde tracers in marmosets. We tested the hypothesis that cytoarchitectural area 6M includes homologues of the supplementary and pre-supplementary motor areas (SMA and preSMA) of other primates. We found that, irrespective of the injection sites’ location within 6M, over half of the labeled neurons were located in motor and premotor areas. Other connections originated in prefrontal area 8b, ventral anterior and posterior cingulate areas, somatosensory areas (3a and 1-2), and areas on the rostral aspect of the dorsal posterior parietal cortex. Although the origin of afferents was similar, injections in rostral 6M received higher percentages of prefrontal afferents, and fewer somatosensory afferents, compared to caudal injections, compatible with differentiation into SMA and preSMA. Injections rostral to 6M (area 8b) revealed a very different set of connections, with increased emphasis in prefrontal and posterior cingulate afferents, and fewer parietal afferents. The connections of 6M were also quantitatively different from those of M1, dorsal premotor areas, and cingulate motor area 24d. These results show that the cortical motor control circuit is conserved in simian primates, indicating that marmosets can be valuable models for studying movement planning and control.
Plasmodiophora brassicae infection threshold – how many resting spores are required for infection of canola (Brassica napus)
Authors: Zahr Kher , Yang Yalong , Sarkes Alian , Dijanovic Snezana , Fu Heting , Harding Michael W. , Feindel David , Feng Jie
Published: May 2021

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other brassica crops. Improved understanding of host and pathogen biology is frequently useful in guiding management strategies. In order to better understand infection thresholds, seven-day old seedlings of canola cultivar Westar were inoculated with serially diluted concentrations of P. brassicae resting spores. Controlled soil and plant inoculation assays were performed and the plants maintained in a greenhouse for 42 days and clubroot disease severity evaluated visually. Clubroot symptoms were observed in soils containing as low as one spore/mL soil and on plants inoculated with as few as ≤ 100 resting spores. These thresholds were lower than any previously reported. The results indicated the importance of highly sensitive detection methods for P. brassicae diagnosis and quantification methods for clubroot risk prediction in soils. Furthermore, these results highlighted the low probability of obtaining P. brassicae single spore isolates.
Morpho-functional characterization of the endo-lysosomal system by high-throughput correlative light-electron microscopy
Authors: van der Beek Jan , de Heus Cecilia , Liv Nalan , Klumperman Judith
Published: May 2021

Rab5, EEA1 and APPL1 are frequently used in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as marker for late endosomes and lysosomes. However, since these proteins localize poorly in immuno-electron microscopy, systematic studies on their ultrastructural distributions are lacking. Here we address this gap by presenting a quantitative, high-throughput, on-section correlative light-electron microscopy (CLEM) approach using the sensitivity of fluorescence microscopy to infer label to hundreds of organelles classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 co-localizes with Rab5 on especially early endosomes, but unexpectedly also labels Rab5-negative late endosomes and even lysosomes. APPL1 is restricted to small Rab5-positive, vesicular profiles without any visible content or ultrastructural marks. Rab7 primarily labels late endosomes and lysosomes. Our studies reveal the first ultrastructural distribution of key endosomal proteins at their endogenous levels and introduce CLEM as sensitive alternative for quantitative immuno-EM.
Molecular basis for PICS-mediated piRNA biogenesis and cell division
Authors: Wang Xiaoyang , Zeng Chenming , Liao Shanhui , Zhu Zhongliang , Zhang Jiahai , Tu Xiaoming , Feng Xuezhu , Guang Shouhong , Xu Chao
Published: May 2021

By incorporating two mutually exclusive factors, PID-1 and TOST-1, C. elegans PICS complex plays important roles in piRNA biogenesis, chromosome segregation and cell division, respectively. We firstly mapped the interaction network between PICS subunits. By solving the several complex structures, including those of TOFU-6/PICS-1, ERH-2/PICS-1, and ERH-2/TOST-1, we uncover the mechanisms underlying the interactions between PICS subunits. Our biochemical experiment demonstrates that PICS exists as an octamer consisting of two copies of each subunits. Combining structural analyses with mutagenesis experiments, we identified residues of PICS subunits that are critical for maintaining intact PICS complex in vitro. Furthermore, using genetics, cell biology and imaging experiments, we found that those mutants impairing the in vitro interaction network within PICS, also lead to abnormal dysfunction PICS in vivo, including mislocalization of PICS, and reduced levels of piRNAs or abnormal chromosome segregation and cell division. Therefore, our work provides structural insights into understanding the PICS-mediated piRNA biogenesis and cell division.
Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36
Authors: van den Heuvel Jasmin , Ashiono Caroline , Gillet Ludovic , Dörner Kerstin , Wyler Emanuel , Zemp Ivo , Kutay Ulrike
Published: May 2021

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.
The Role of ATP in the RNA Translocation Mechanism of SARS-CoV-2 NSP13 Helicase
Authors: Weber Ryan , McCullagh Martin
Published: May 2021

The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but non-structural protein 13 (nsp13) stands out as a highly conserved and yet under studied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound presenting a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 μs of Gaussian accelerated molecular dynamics simulation (at least 6 μs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key aspects of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inch-worm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV and V and suggest a powerstroke–like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.
Generation of human chambered cardiac organoids from pluripotent stem cells for improved modelling of cardiovascular diseases
Authors: Ho Beatrice Xuan , Pang Jeremy , Phua Qian Hua , Liew Lee Chuen , Poh Boon Min , Chen Ying , Loh Yuin-Han , An Omer , Yang Henry He , Seshachalam Veerabrahma Pratap , Koh Judice LY , Chan Woon-Khiong , Ng Shi-Yan , Soh Boon-Seng
Published: May 2021

Recent progress on murine and human cardiac organoids have provided understanding to the developmental processes of the heart. However, there is still an unfulfilled need for improved modelling of cardiovascular diseases using human cardiac organoids. Herein, we report successful generation of intrinsically formed human chambered cardiac organoids (CCO) and highlight its utility in modelling disease. Single cell transcriptomic profiling of CCOs showed appropriate cardiovascular cell type composition exhibiting improved maturation. Functionally, CCOs recapitulated clinical cardiac hypertrophy by exhibiting thickened chamber walls, reduced ejection fractions, increased myofibrillar disarray and tachycardia. Therefore, CCOs improve current capabilities of disease modelling as an in vitro model bridging the gap to in vivo models, with the ability to assess functional parameters that previously can only be achieved in animal systems.
A framework to identify antigen-expanded T Cell Receptor (TCR) clusters within complex repertoires
Authors: Ceglia Valentina , Kelley Erin J , Boyle Annalee S , Levy Yves , Zurawski Gerard , Altin John A
Published: May 2021

Common approaches for monitoring T cell responses are limited in their multiplexity and sensitivity. In contrast, deep sequencing of the T Cell Receptor (TCR) repertoire offers a global view whose theoretical sensitivity is limited only by the depth of available sampling. However, assignment of antigen specificities within TCR repertoires has become a bottleneck. Here, we combine antigen-driven expansion, deep TCR sequencing and a novel analysis framework to show that homologous ‘Clusters of Expanded TCRs (CETs)’ can be confidently identified without cell isolation, and assigned to antigen against a background of non-specific clones. We show that clonotypes within each CET respond to the same epitope, and that protein antigens stimulate multiple CETs reactive to constituent peptides. Finally, we demonstrate the personalized assignment of antigen-specificity to rare clones within fully-diverse unexpanded repertoires. The method presented here may be used to monitor T cell responses to vaccination and immunotherapy with high fidelity.
ESCA pipeline: Easy-to-use SARS-CoV-2 genome Assembler
Authors: Rueca Martina , Giombini Emanuela , Messina Francesco , Bartolini Barbara , Di Caro Antonino , Capobianchi Maria R. , Gruber Cesare E. M.
Published: May 2021

Early sequencing and quick analysis of SARS-CoV-2 genome are contributing to understand the dynamics of COVID19 epidemics and to countermeasures design at global level. Amplicon-based NGS methods are widely used to sequence the SARS-CoV-2 genome and to identify novel variants that are emerging in rapid succession, harboring multiple deletions and amino acid changing mutations. To facilitate the analysis of NGS sequencing data obtained from amplicon-based sequencing methods, here we propose an easy-to-use SARS-CoV-2 genome Assembler: the ESCA pipeline. Results showed that ESCA can perform high quality genome assembly from IonTor-rent and Illumina raw data, and help the user in easily correct low-coverage regions. Moreover, ESCA includes the possibility to compare assembled genomes of multi sample runs through an easy table format.

Script and manuals are available on GitHub: https://github.com/cesaregruber/ESCA
Cap-dependent translation initiation monitored in living cells
Authors: Gandin Valentina , English Brian P. , Freeman Melanie , Leroux Louis-Philippe , Preibisch Stephan , Walpita Deepika , Jaramillo Maritza , Singer Robert H.
Published: May 2021

Despite extensive biochemical, genetic, and structural studies, a complete understanding of mRNA translation initiation is still lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-molecule resolution are necessary to fully elucidate regulation of this paramount process. We fused tags suitable for live imaging to eIF4E, eIF4G1 and 4E-BP1 without affecting their function. We combined Fluorescence Correlation Spectroscopy (FCS) and Single-Particle Tracking (SPT) to interrogate the binding dynamics of initiation factors to the 5’cap. Both FCS and SPT were able to detect eIF4E:eIF4G1 binding to the mRNA in the cytoplasm of proliferating cells and neuronal processes. Upon inhibition of phosphorylation by mTOR, 4E-BP1:eIF4E complexes rapidly dissociated from the 5’cap followed by eIF4G1 dissociation. Imaging of the binding dynamics of individual translation factors in living cells revealed the temporal regulation of translation at unprecedented resolution.
Neural Representations of Task-relevant and Task-irrelevant Features of Attended Objects
Authors: Chen Jiageng , Scotti Paul S , Dowd Emma W , Golomb Julie D
Published: May 2021

Visual attention plays an essential role in selecting task-relevant and ignoring task-irrelevant information, for both object features and their locations. In the real world, multiple objects with multiple features are often simultaneously present in a scene. When spatial attention selects an object, how are the task-relevant and task-irrelevant features represented in the brain? Previous literature has shown conflicting results on whether and how irrelevant features are represented in visual cortex. In an fMRI task, we used a modified inverted encoding model (IEM, e.g., Sprague & Serences, 2015) to test whether we can reconstruct the task-relevant and task-irrelevant features of spatially attended objects in a multi- feature (color + orientation), multi-item display. Subjects were briefly shown an array of three colored, oriented gratings. Subjects were instructed as to which feature (color or orientation) was relevant before each block, and on each trial were asked to report the task-relevant feature of the object that appeared at a spatially pre-cued location, using a continuous color or orientation wheel. By applying the IEM, we achieved reliable feature reconstructions for the task-relevant features of the attended object from visual ROIs (V1 and V4v) and Intraparietal sulcus. Preliminary searchlight analyses showed that task-irrelevant features of attended objects could be reconstructed from activity in some intraparietal areas, but the reconstructions were much weaker and less reliable compared with task-relevant features. These results suggest that both relevant and irrelevant features may be represented in visual and parietal cortex but in different forms. Our method provides potential tools to noninvasively measure unattended feature representations and probe the extent to which spatial attention acts as a "glue" to bind task-relevant and task-irrelevant features.
Coordinated regulation of CB1 cannabinoid receptors and anandamide metabolism stabilizes network activity during homeostatic downscaling
Authors: Ye Michael , Monroe Sarah K. , Gay Sean M. , Armstrong Michael L. , Youngstrom Diane E. , Urbina Fabio L. , Gupton Stephanie L. , Reisdorph Nichole , Diering Graham H.
Published: May 2021

Neurons express overlapping homeostatic mechanisms to regulate synaptic function and network properties in response to perturbations of neuronal activity. Endocannabinoids (eCBs) are bioactive lipids synthesized in the post-synaptic compartments to regulate synaptic transmission, plasticity, and neuronal excitability primarily through retrograde activation of pre- synaptic cannabinoid receptor type 1 (CB1). The eCB system is well-situated to regulate neuronal network properties and coordinate pre- and post-synaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. To address this issue, we used western blot and targeted lipidomics to measure adaptations in eCB system to bicuculline (BCC)-induced chronic hyperexcitation in mature (>DIV21) cultured rat cortical neurons, and used multielectrode array recording and live-cell imaging of glutamate dynamics to test the effects of pharmacological manipulations of eCB on network activities. We show that BCC-induced chronic hyperexcitation triggers homeostatic downscaling and a coordinated adaptation to enhance tonic eCB signaling. Hyperexcitation triggers first the downregulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide, then an accumulation of anandamide and related metabolites, and finally a delayed upregulation of surface and total CB1. Additionally, we show that BCC-induced downregulation of surface AMPA-type glutamate receptors (AMPARs) and upregulation of CB1 occur through independent mechanisms. Finally, we show that endocannabinoids support baseline network activities before and after downscaling and is engaged to suppress network activities during adaptation to hyperexcitation. We discuss the implications of our findings in the context of downscaling and homeostatic regulation of oscillatory network activities.

Significance statement

Neurons are remarkably resilient to perturbations in network activities thanks to the expression of overlapping homeostatic adaptations. In response to network hyperactivity or silencing, neurons respond through regulating excitatory and inhibitory post-synaptic neurotransmitter receptors density, probability of pre-synaptic neurotransmitter release, and/or membrane excitability. The endocannabinoid system is a prominent signaling pathway at many synapses that is known to be involved in multiple forms of short- and long-term synaptic plasticity. Here we find that components of the endocannabinoid system are upregulated in response to chronic hyperexcitation of cultured cortical neurons, and that endocannabinoid signaling is required to maintain network activity but also suppresses network events during hyperexcitation. This work supports a novel tonic homeostatic function for the endocannabinoid system in neurons.
Constraints and Opportunities for Detecting Land Surface Phenology in Drylands
Authors: Taylor Shawn D. , Browning Dawn M. , Baca Ruben A. , Gao Feng
Published: May 2021

Land surface phenology (LSP) enables global scale tracking of ecosystem processes, but its utility is limited in drylands due to low vegetation cover and resulting low annual amplitudes of vegetation indices (VIs). Due to the importance of drylands for biodiversity, food security, and the carbon cycle it is necessary to understand limitations in measuring dryland dynamics. Here, using simulated data and multi-temporal unmanned aerial vehicle (UAV) imagery of a desert shrubland, we explore the feasibility of detecting LSP with respect to fractional vegetation cover, plant functional types, VI uncertainty, and two different detection algorithms. Using simulated data we found that plants with distinct VI signals, such as deciduous shrubs, can require up to 60% fractional cover to consistently detect LSP. Evergreen plants, with lower seasonal VI amplitude, require considerably higher cover and can have undetectable phenology even with 100% vegetation cover. Our evaluation of two algorithms showed that neither performed the best in all cases. Even with adequate cover, biases in phenological metrics can still exceed 20 days, and can never be 100% accurate due to VI uncertainty from shadows, sensor view angle, and atmospheric interference. We showed how high-resolution UAV imagery enables LSP studies in drylands, and highlighted important scale effects driven by within canopy VI variation. With high-resolution imagery the open canopies of drylands are beneficial as they allow for straightforward identification of individual plants, enabling the tracking of phenology at the individual level. Drylands thus have the potential to become an exemplary environment for future LSP research.