bluuebunny/crossref_metadata_2025 huggingface dataset:
Label-free Cell Tracking Enables Collective Motion Phenotyping in Epithelial Monolayers
Authors: Gu Shuyao , Lee Rachel M. , Benson Zackery , Ling Chenyi , Vitolo Michele I. , Martin Stuart S. , Chalfoun Joe , Losert Wolfgang
Published: December 2021

Collective cell migration is an umbrella term for a rich variety of cell behaviors, whose distinct character is essential for biological function, notably for cancer metastasis. One essential feature of collective behavior is the motion of cells relative to their immediate neighbors. We introduce an AI-based pipeline to segment and track cell nuclei from phase contrast images. Nuclei segmentation is based on a U-Net convolutional neural network trained on images with nucleus staining. Tracking, based on the Crocker-Grier algorithm, quantifies nuclei movement and allows for robust downstream analysis of collective motion. Since the AI algorithm required no new training data, our approach promises to be applicable to and yield new insights for vast libraries of existing collective motion images. In a systematic analysis of a cell line panel with oncogenic mutations, we find that the collective rearrangement metric, D2min, which reflects non-affine motion, shows promise as an indicator of metastatic potential.

Graphical Abstract

Highlights

Versatile AI-based algorithm can robustly identify individual cells and track their motion from phase contrast images.

Analysis of motion of cells relative to nearby neighbors distinguishes weakly tumorigenic (KRas) and metastatic (KRas/PTEN-/-) cell lines.
Identifying C. elegans Lifespan Mutants by Screening for Early-Onset Protein Aggregation
Authors: Midkiff Daniel F. , San-Miguel Adriana
Published: December 2021

Genetic screens have been widely used to identify genetic pathways that control specific biological functions. In C. elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Since aged individuals are unable to generate clonal populations, screens that target post-reproductive phenotypes, such as longevity, are challenging. In this work, we developed an approach that combines microfluidic technologies and image processing to perform a high-throughput, automated screen for mutants with shortened lifespan using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively-active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently labeled PAB-1 as a readout. We identified five mutants with increased aggregation levels, of which two exhibited a reduced lifespan. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently-labeled aggregates in populations that do not require conditional sterilization or manual separation of parental and progeny populations. We further analyzed the morphology of protein aggregates and reveal that patterns of aggregation in naturally-aging animals differ from mutants with increased aggregation, suggesting aggregate growth is time-dependent. This screening approach can be customized to other non-developmental phenotypes that appear during adulthood, as well as to other aging markers to identify additional longevity-regulating genetic pathways.
Prefrontal top-down projections control context-dependent strategy selection
Authors: Gschwend Olivier , Yang Tao , van de Lisdonk Daniëlle , Zhang Xian , Sharma Radhashree , Li Bo
Published: December 2021

The rules governing behavior often vary with behavioral contexts. As a consequence, an action rewarded in one context may be discouraged in another. Animals and humans are capable of switching between behavioral strategies under different contexts and acting adaptively according to the variable rules, a flexibility that is thought to be mediated by the prefrontal cortex (PFC)1–4. However, how the PFC orchestrates context-dependent switch of strategies remains unclear. Here we show that pathway-specific projection neurons in the medial PFC (mPFC) differentially contribute to context-instructed strategy selection. In a decision-making task in which mice have been trained to flexibly switch between a previously established rule and a newly learned rule in a context-dependent manner, the activity of mPFC neurons projecting to the dorsomedial striatum encodes the contexts, and further represents decision strategies conforming to the old and new rules. Moreover, the activity of these neuron is required for context-instructed strategy selection. In contrast, the activity of mPFC neurons projecting to the ventral midline thalamus does not discriminate between the contexts, and represents the old rule even if mice have adopted the new one; furthermore, these neurons act to prevent the strategy switch under the new rule. Our results suggest that the mPFC→striatum pathway promotes flexible strategy selection guided by contexts, whereas the mPFC→thalamus pathway favors fixed strategy selection by preserving old rules. Balanced activity between the two pathways may be critical for adaptive behaviors.
Sampling motion trajectories during hippocampal theta sequences
Authors: Ujfalussy Balázs B , Orbán Gergő
Published: December 2021

Efficient planning in complex environments requires that uncertainty associated with current inferences and possible consequences of forthcoming actions is represented. Representation of uncertainty has been established in sensory systems during simple perceptual decision making tasks but it remains unclear if complex cognitive computations such as planning and navigation are also supported by probabilistic neural representations. Here we capitalized on gradually changing uncertainty along planned motion trajectories during hippocampal theta sequences to capture signatures of uncertainty representation in population responses. In contrast with prominent theories, we found no evidence of encoding parameters of probability distributions in the momentary population activity recorded in an open-field navigation task in rats. Instead, uncertainty was encoded sequentially by sampling motion trajectories randomly in subsequent theta cycles from the distribution of potential trajectories. Our analysis is the first to demonstrate that the hippocampus is well equipped to contribute to optimal planning by representing uncertainty.
The perceived position of a moving object is reset by temporal, not spatial limits
Authors: Liu Sirui , Tse Peter U. , Cavanagh Patrick
Published: December 2021

When the internal texture of a Gabor patch drifts orthogonally to its physical path, its perceived motion deviates dramatically from its physical path. The local position shifts accumulate to such an extent that a 45° oblique physical path appears to be vertical. However, at some point, a limit is reached and the path resets back to its veridical location, whereupon a new accumulation starts, making the new perceived path segment appear parallel to the pre-reset segment, but offset horizontally from it. Here, we tested whether spontaneous resets of this motion-induced position shift depend on the time or the distance over which position errors accrue, or both. We introduced a temporal gap in the middle of the path that forced the illusory path to reset back to its veridical physical position. This gap-triggered reset allowed us to measure the magnitude of the illusory offset up to that point. We found that perceived offset was less than expected for the angle of illusory drift, indicating that spontaneous resets had occurred prior to the gap-induced reset. The position offset decreased when the pre-gap duration increased but approximately doubled when the path length doubled. This pattern of perceived offsets is best accounted for by spontaneous resets that occur randomly over time at a constant rate, independently of the distance traveled. Our results suggest a temporal, not spatial, limit for the accumulation of position errors that underlies this illusion.
Prolyl-hydroxylase inhibitor-induced regeneration of alveolar bone and soft tissue in a mouse model of ligature-induced periodontitis
Authors: Zebrowitz Elan , Kajikawa Tetsuhiro , Bedelbaeva Kamila , Aslanukov Azamat , Bollinger Sam , Zhang Yong , Sarfatti David , Cheng Jing , Messersmith Phillip B. , Hajishengallis George , Heber-Katz Ellen
Published: December 2021

Bone injuries and fractures reliably heal through a process of regeneration with restoration to original structure and function when the gap between adjacent sides of a fracture site is small. However, when there is significant volumetric loss of bone, bone regeneration usually does not occur.

In the present studies, we explore a particular case of volumetric bone loss in a mouse model of human periodontal disease (PD) in which alveolar bone surrounding teeth is permanently lost and not replaced. This model employs the placement a ligature around the upper second molar which leads to inflammation and bone breakdown and faithfully replicates the bacterially-induced inflammatory etiology of human PD to induce bone degeneration. After 10 days, the ligature is removed and the mice are treated with a timed-release formulation of a small molecule inhibitor of prolylhydroxylases (PHDi; 1,4-DPCA) previously shown to induce epimorphic regeneration of soft tissue in non-regenerating mice. This PHDi induces high expression of HIF-1α and the regenerative response is completely blocked by siHIF1a. Here, we observe that timed-release 1,4-DPCA rapidly and completely restores bone and soft tissue with normal anatomic fidelity and with increased stem cell markers due to stem cell migration into the site and/or de-differentiation of local tissue, PDL cell proliferation, and increased vascularization. In-vitro studies using gingival tissue show that 1,4-DPCA indeed induces de-differentiation and the expression of stem cell markers but does not exclude the role of migrating stem cells.
Areas Important for Ecological Connectivity Throughout Canada
Authors: Pither Richard , O’Brien Paul , Brennan Angela , Hirsh-Pearson Kristen , Bowman Jeff
Published: December 2021

Governments around the world have acknowledged the importance of conserving ecological connectivity to help reverse the decline of biodiversity. In this study we employed recent methodological developments in circuit theory to conduct the first pan-Canadian analysis of multi-species connectivity for all terrestrial regions of the country, at a spatial grain sufficient to support local land-management decisions. We developed a movement cost surface with a limited number of thematic categories using the most recently updated land cover data available for the country. We divided the country into 17 tiles and used a wall-to-wall, omnidirectional mode of Circuitscape on each tile in order to assess ecological connectivity throughout entire landscapes as opposed to strictly among protected areas. The resulting raw current density map of Canada revealed heterogenous patterns of current density across the country, strongly influenced by geography, natural barriers, and human development. We included a validation analysis of the output current density map with independent wildlife data from across the country and found that mammal and herpetofauna locations were predicted by areas of high current density. We believe our current density map can be used to identify areas important for connectivity throughout Canada and thereby contribute to efforts to conserve biodiversity.
Genetic diversity patterns of human ethnic groups as inferred from the 1000 genomes
Authors: Xia Zhiyi , Huang Shi
Published: December 2021

Human genetic diversity remains to be better understood. We here analyzed data from the 1000 Genomes Project and defined group specific fixed alleles (GSFAs) as those that are likely fixed in one ethnic group but non-fixed in at least one other group. The fraction of derived alleles in GSFAs indicates relative distance to apes because such alleles are absent in apes. Our results show that different groups differed in GSFA numbers consistent with known genetic diversity patterns, but also differed in the fraction of derived alleles in GSFAs throughout the entire genome, with East Asians having the largest fraction, followed by South Asians, Europeans, Native Americans, and Africans. Fast evolving sites such as intergenic regions were enriched with derived alleles and showed greater differences in GSFA numbers between East Asians and Africans. Furthermore, GSFAs in East Asians are mostly not fixed in other groups especially Africans, which was particularly more pronounced for fast evolving noncoding variants, while GSFAs in Africans are mostly also fixed in East Asians. Finally, variants that are likely non-neutral such as those leading to stop codon gain/loss and splice donor/acceptor gain/loss showed patterns similar to those of fast-evolving noncoding variants. These results can be accounted for by the maximum genetic diversity theory but not by the neutral theory or its inference that Eurasians suffered bottlenecks, and have implications for better management of group specific genetic diseases.
Asymmetric Structure of the Native Rhodobacter sphaeroides Dimeric LH1-RC Complex
Authors: Tani K. , Kanno R. , Kikuchi R. , Kawamura S. , Nagashima K. V. P. , Hall M. , Takahashi A. , Yu L.-J. , Kimura Y. , Madigan M. T. , Mizoguchi A. , Humbel B. M. , Wang-Otomo Z.-Y.
Published: December 2021

The light-harvesting-reaction center (LH1-RC) core complex of purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides is characterized by the presence of both a dimeric form and a monomeric form. Following structure determination of the monomeric LH1-RC including its previously unrecognized component designated protein-U (Nat. Commun. 12, 6300, 2021), here we present cryo-EM structures of the dimeric LH1-RC from native Rba. sphaeroides IL106 at 2.75 Å resolution and from an LH1-RC monomer lacking protein-U (ΔU) at 2.64 Å resolution. The native dimeric core complex reveals many asymmetric features in the arrangement of its two monomeric components including the structural integrity of protein-U, the overall LH1 organization, and the rigidities of the proteins and pigments that form the complex. PufX polypeptides play a critical role in connecting two monomers, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in another monomer, in good agreement with biochemical analyses. One of the proteins-U was only partially identified in the dimeric structure, signaling significantly different degrees of disorder in the two monomers. The ΔU LH1-RC monomer revealed a half-moon-shaped structure containing 11 α- and 10 β-polypeptides (compared with 14 of each in the wild type), indicating a critical role for protein-U in controlling the number of αβ-subunits required for correct assembly and stabilization of the LH1-RC dimer. The structural features are discussed in relation to the unusual topology of intracytoplasmic photosynthetic membranes and an assembly model proposed for the native Rba. sphaeroides dimeric LH1-RC complex in membranes of wild-type cells.
Evolution of stickleback spines through independentcis-regulatory changes atHOXDB
Authors: Wucherpfennig Julia I. , Howes Timothy R. , Au Jessica N. , Au Eric H. , Roberts Kingman Garrett A. , Brady Shannon D. , Herbert Amy L. , Reimchen Thomas E. , Bell Michael A. , Lowe Craig B. , Dalziel Anne C. , Kingsley David M.
Published: December 2021

Understanding the genetic mechanisms leading to new traits is a fundamental goal of evolutionary biology. We show thatHOXDBregulatory changes have been used repeatedly in different stickleback fish species to alter the length and number of bony dorsal spines. InGasterosteus aculeatus, a variantHOXDBallele is genetically linked to shortening an existing spine and adding a spine. InApeltes quadracus, a variant allele is associated with lengthening an existing spine and adding a spine. The alleles alter the same conserved non-codingHOXDBenhancer by diverse molecular mechanisms, including SNPs, deletions, and transposable element insertions. The independentcis-acting regulatory changes are linked to anterior expansion or contraction ofHOXDBexpression. Our findings support the long-standing hypothesis that naturalHoxgene variation underlies key morphological patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.

Abstract Figure
Simulation of the omicron variant of SARS-CoV-2 shows broad antibody escape, weakened ACE2 binding, and modest increase in furin binding
Authors: Jawaid M. Zaki , Baidya A. , Mahboubi-Ardakani R. , Davis Richard L. , Cox Daniel L.
Published: December 2021

The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is significant reduction of antibody binding strength corresponding to escape, the omicron spike pays a cost in terms of weaker receptor binding. The furin cleavage domain is the same or weaker binding than the alpha variant, suggesting less viral load and disease intensity than the extant delta variant.
Ethyl-Iophenoxic acid as a serum marker for oral baiting of carnivorous marsupials
Authors: Pye Ruth , Nichols David , Gilbert Amy T. , Flies Andrew S.
Published: December 2021

Context

Ethyl-Iophenoxic acid (Et-IPA) has been widely used as a bait biomarker to determine oral bait consumption by vertebrate wildlife species. Oral bait vaccines have been delivered to numerous wildlife species to protect them from disease. The Tasmanian devil (Sarcophilis harrisii), the largest extant carnivorous marsupial species, is threatened by the transmissible cancers known as devil facial tumour disease (DFTD). Development of a protective DFTD vaccine is underway, and an oral bait has been proposed to deliver the vaccine in the wild. The bait delivery system requires a biomarker that can be detected for several months post-consumption in Tasmanian devils.

Aim

To determine the suitability of Et-IPA as a bait biomarker in the Tasmanian devil.

Method

Two Tasmanian devils were fed 50 mg Et-IPA (4.5 to 7.1 mg Et-IPA/kg bodyweight). Liquid chromatography with tandem mass spectrometry (LC-MS/-MS) was used to directly measure Et-IPA in baseline serum samples and samples collected on days 1, 14, 26 and 56 post-baiting.

Key result

Both devils retained serum Et-IPA concentrations at two orders of magnitude above negative control sera when this study concluded.

Conclusions

Et-IPA is a useful bait biomarker for Tasmanian devils and can be included in future DFTD bait vaccine field trials to determine bait vaccine uptake.
Modelling to infer the role of animals in gambiense human African trypanosomiasis transmission and elimination in DRC
Authors: Crump Ronald E , Huang Ching-I , Spencer Simon E F , Brown Paul E , Shampa Chansy , Miaka Erick Mwamba , Rock Kat S
Published: December 2021

Gambiense human African trypanosomiasis (gHAT) has been targeted for elimination of transmission (EoT) to humans by 2030. Whilst this ambitious goal is rapidly approaching, there remain fundamental questions about the presence of non-human animal transmission cycles and their potential role in slowing progress towards, or even preventing, EoT. In this study we focus on the country with the most gHAT disease burden, the Democratic Republic of Congo (DRC), and use mathematical modelling to assess whether animals may contribute to transmission in specific regions, and if so, how their presence could impact the likelihood and timing of EoT.

By fitting two model variants – one with, and one without animal transmission – to the human case data from 2000–2016 we estimate model parameters for 158 endemic health zones of DRC. We evaluate the statistical support for each model variant in each health zone and infer the contribution of animals to overall transmission and how this could impact predicted time to EoT.

We conclude that there are 24/158 health zones where there is moderate or high statistical support for some animal transmission. However, – even in these regions – we estimate that animals would be extremely unlikely to maintain transmission on their own. Animal transmission could hamper progress towards EoT in some settings, with projections under continuing interventions indicating that the number of health zones expected to achieve EoT by 2030 reduces from 68 to 61 if animals are included in the model. With supplementary vector control (at a modest 60% tsetse reduction) added to medical screening and treatment interventions, the predicted number of health zones meeting the goal increases to 147/158 for the model including animals. This is due to the impact of vector reduction on transmission to and from all hosts.

Author summary

Elimination of African sleeping sickness by 2030 is an ambitious goal, not least because of the unclear role that animals might play in transmission. We use mathematical models, fitted to case data from DRC to assess and quantify the contribution of animals to the human case burden.

We found that 24/158 geographic regions included in this study had statistical evidence of animal transmission, although it appears extremely unlikely that animals could maintain transmission on their own. Animals could, however, delay elimination; using our model without animal transmission we predicted that 68 regions are expected to achieve elimination by 2030, whereas this reduces to 61 with animals. If vector control to reduce fly populations (which transmit the disease to and from hosts) are controlled in addition to medical interventions, then 147 regions are predicted to reach elimination by 2030 even with animal transmission.
Sporadic, late-onset, and multistage diseases
Authors: Webster Anthony J. , Clarke Robert
Published: December 2021

Somatic mutations can cause cancer and have recently been linked with a range of non-malignant diseases. Multistage models can characterise how mutations lead to cancer, and may also be applicable to these other diseases. Here we found the incidence of over 60% of common diseases in UK Biobank were consistent with a multistage model with an ordered sequence of stages, as approximated by a Weibull distribution, with the log of incidence linearly related to the log of age and the slope often interpreted as the number of stages. A model where the stages can occur in any order was also explored, as was stratification by smoking and diabetes status. Most importantly, we find that many diseases are low risk when young but then become inevitable in old age, but many other diseases do not, being more sporadic with a modest and modifiable risk that slowly increases with age.
A world of viruses nested within parasites: Unraveling viral diversity within parasitic flatworms (Platyhelminthes)
Authors: Dheilly Nolwenn M , Lucas Pierrick , Blanchard Yannick , Rosario Karyna
Published: December 2021

Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host-shifts. Yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein we uncover viruses of flatworms (Phylum Platyhelminthes, group Neodermata) that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of Neodermatans to a parasitic lifestyle. Most Neodermatan viruses seem to co-diversify with their host, with the exception of rhabdoviruses which may switch host more often, based on phylogenetic relationships. Neodermatan rhabodviruses also have an ancestral position to vertebrate-associated viruses, including Lyssaviruses, suggesting that vertebrate rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases.
TRPM4 is required for calcium oscillations downstream from the stretch-activated TRPV4 channel
Authors: Yarishkin Oleg , Phuong Tam T. T. , Vazquez-Chona Felix , Bertrand Jacques , Redmon Sarah N. , Lakk Monika , Rudzitis Christopher N. , Baumann Jackson M. , Hwang Eun Mi , Overby Darryl , Križaj David
Published: December 2021

Transduction of mechanical information is influenced by physical, chemical and thermal cues but the molecular mechanisms through which transducer activation shapes temporal signaling remain underexplored. In the present study, electrophysiology, histochemistry and functional imaging were combined with gene silencing and heterologous expression to gain insight into calcium signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated nonselective cation channel. We show that trabecular meshwork (TM) cells, which employ mechanotransduction to actively regulate intraocular pressure, respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. ([Ca2+]i oscillations coincided with a monovalent cation current that was suppressed by BAPTA, Ruthenium Red and 9-phenanthrol, an inhibitor of TRPM4 (Transient Receptor Potential Melastatin 4) channels. Accordingly, TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings indicated that TRPM4-dependent oscillations required release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. These findings provide insight into the complexity of membrane-cytosolic interactions during TRPV4 signaling and may foster strategies to promote homeostatic regulation and counter pathological remodeling within the conventional outflow pathway of the mammalian eye.
Differing Associations between Optic Nerve Head Strains and Visual Field Loss in Normal- and High-Tension Glaucoma Subjects
Authors: Chuangsuwanich Thanadet , Tun Tin A. , Braeu Fabian A. , Wang Xiaofei , Chin Zhi Yun , Panda Satish Kumar , Buist Martin , Strouthidis Nicholas , Perera Shamira , Nongpiur Monisha , Aung Tin , Girard Michael JA
Published: December 2021

Purpose

To study the associations between optic nerve head (ONH) strains under intraocular pressure (IOP) elevation with retinal sensitivity in glaucoma subjects.

Design

Clinic based cross-sectional study.

Participants

229 subjects with primary open angle glaucoma (subdivided into 115 high tension glaucoma (HTG) subjects and 114 normal tension glaucoma (NTG) subjects).

Methods

For one eye of each subject, we imaged the ONH using spectral-domain optical coherence tomography (OCT) under the following conditions: (1) primary gaze and (2) primary gaze with acute IOP elevation (to approximately 33 mmHg) achieved through ophthalmodynamometry. A 3-dimensional (3D) strain-mapping algorithm was applied to quantify IOP-induced ONH tissue strain (i.e. deformation) in each ONH. Strains in the pre-lamina tissue (PLT)and the retina, the choroid, the sclera and the lamina cribrosa (LC) were associated (using linear regression) with measures of retinal sensitivity from the 24-2 Humphrey visual field test (Carl Zeiss Meditec, Dublin, CA, USA). This was done globally, then locally according to the regionalization scheme of Garway-Heath et al.

Main Outcome Measures

Associations between ONH strains and values of retinal sensitivity from visual field testing.

Results

For HTG subjects, we found that (1) there were significant negative linear associations between ONH strains and retinal sensitivity (p<0.001) (on average, a 1% increase in ONH strains corresponded to a decrease in retinal sensitivity of 1.1 dB), (2) high strain regions co-localized with anatomically-mapped regions of high visual field loss, (3) the strongest negative associations were observed in the superior region and in the PLT. In contrast, for NTG subjects, no significant associations between strains and retinal sensitivity were observed except in the supero-temporal region of the LC.

Conclusion

We found significant negative associations between IOP-induced ONH strains and retinal sensitivity in a relatively large glaucoma cohort. Specifically, HTG subjects who experienced higher ONH strains were more likely to exhibit lower retinal sensitivities. Interestingly, this trend was in general less pronounced in NTG subjects, which could suggest a distinct pathophysiology between the two glaucoma subtypes.
Nest aggregations of wild bees and apoid wasps in urban pavements: a “street life” to be promoted in urban planning
Authors: Noël Grégoire , Van Keymeulen Violette , Barbier Yvan , Smets Sylvie , Van Damme Olivier , Colinet Gilles , Ruelle Julien , Francis Frédéric
Published: December 2021

In the last 10 years, knowledges of wild bees and apoid wasps’ community dynamics have gained interest in urban ecology focusing on the availability of floral resources in cities. Although understudied, the urban environment impacts the conditions of their nesting sites. Recent observations in the Brussels-Capital Region (Belgium) showed that urban pavements can be a novel nesting opportunity for Hymenoptera ground-nesting species such as wild bees and apoid wasps. Here, using citizen science, we investigated the richness of ground-nesting species living under urban pavements, the preferences of the sidewalk joint size related to ground-nesting species size and for sidewalk type or for soils texture under the pavements on the nesting site selection. A total of 22 species belonging to 10 Hymenoptera families of wild bees and digger wasps with their associated kleptoparasites were identified on 89 sites in Brussels. Sandstone setts or concrete slabs with an unbound joint size around 1 cm were found to be best suitable urban pavements for the ground-nesting species. The soil texture under the pavement was highly sandy among our samples. Finally, we also suggest engineering management guidelines to support bee and wasp species nesting under urban pavement in highly urbanized areas. Such observations pave the way for much research in the field of urban ecology to conceive multifunctional pavement promoting biodiversity.
Plexins Promote Hedgehog Signaling Through Their Cytoplasmic GAP Activity
Authors: Pinskey Justine M. , Hoard Tyler M. , Zhao Xiao-Feng , Franks Nicole E. , Frank Zoë C. , McMellen Alexandra N. , Giger Roman J. , Allen Benjamin L.
Published: December 2021

Hedgehog signaling controls tissue patterning during embryonic and postnatal development and continues to play important roles throughout life. Characterizing the full complement of Hedgehog pathway components is essential to understanding its wide- ranging functions. Previous work has identified Neuropilins, established Semaphorin receptors, as positive regulators of Hedgehog signaling. Neuropilins require Plexin co- receptors to mediate Semaphorin signaling, but a role for Plexins in Hedgehog signaling has not yet been explored. Here, we provide evidence that multiple Plexins promote Hedgehog signaling in NIH/3T3 fibroblasts and that Plexin loss-of-function in these cells results in significantly reduced Hedgehog pathway activity. Catalytic activity of the Plexin GTPase activating protein (GAP) domain is required for Hedgehog signal promotion, and constitutive activation of the GAP domain further amplifies Hedgehog signaling. Additionally, we demonstrate that Plexins promote Hedgehog signaling at the level of GLI transcription factors and that this promotion requires intact primary cilia.

Finally, we find that Plexin loss-of-function significantly reduces the response to Hedgehogα pathway activation in the mouse dentate gyrus. Together, these data identify Plexins as novel components of the Hedgehog pathway and provide insight into their mechanism of action.
Bacterial origin of thymidylate and folate metabolism in Asgard Archaea
Authors: Filée Jonathan , Becker Hubert F. , Mellottee Lucille , Li Zhihui , Lambry Jean-Christophe , Liebl Ursula , Myllykallio Hannu
Published: December 2021

Little is known about the evolution and biosynthetic function of DNA precursor and the folate metabolism in the Asgard group of archaea. As Asgard occupy a key position in the archaeal and eukaryotic phylogenetic trees, we have exploited very recently emerged genome and metagenome sequence information to investigate these central metabolic pathways. Our genome-wide analyses revealed that the recently cultured Asgard archaeon Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Psyn) contains a complete folate-dependent network for the biosynthesis of DNA/RNA precursors, amino acids and syntrophic amino acid utilization. Altogether our experimental and computational data suggest that phylogenetic incongruences of functional folate-dependent enzymes from Asgard archaea reflect their persistent horizontal transmission from various bacterial groups, which has rewired the key metabolic reactions in an important and recently identified archaeal phylogenetic group. We also experimentally validated the functionality of the lateral gene transfer of Psyn thymidylate synthase ThyX. This enzyme uses bacterial-like folates efficiently and is inhibited by mycobacterial ThyX inhibitors. Our data raise the possibility that the thymidylate metabolism, required for de novo DNA synthesis, originated in bacteria and has been independently transferred to archaea and eukaryotes. In conclusion, our study has revealed that recent prevalent lateral gene transfer has markedly shaped the evolution of Asgard archaea by allowing them to adapt to specific ecological niches.
Dynamic epistasis analysis reveals how chromatin remodeling regulates transcriptional bursting
Authors: Brouwer Ineke , Kerklingh Emma , van Leeuwen Fred , Lenstra Tineke Laura
Published: December 2021

Transcriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in budding yeast to measure how transcriptional bursting changes upon single and double perturbations of chromatin remodeling factors, the transcription factor Gal4 and preinitiation complex (PIC) components. Using dynamic epistasis analysis, we reveal how remodeling of different nucleosomes regulates individual transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC acts synergistically with Gal4 binding to facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. In the absence of remodelers, nucleosomes at canonical TATA boxes are displaced by TBP binding to allow for transcription activation. Overall, our results reveal how promoter nucleosome remodeling, together with transcription factor and PIC binding regulates the kinetics of transcriptional bursting.
Period Doubling Bifurcations in a Forced Cell-Free Genetic Oscillator
Authors: Aufinger Lukas , Brenner Johann , Simmel Friedrich C.
Published: December 2021

Complex non-linear dynamics such as period doubling and chaos have been previously found in computational models of the oscillatory gene networks of biological circadian clocks, but their experimental study is difficult. Here, we present experimental evidence of period doubling in a forced synthetic genetic oscillator operated in a cell-free gene expression system. To this end, an oscillatory negative feedback gene circuit is established in a microfluidic reactor, which allows continuous operation of the system over extended periods of time. We first thoroughly characterize the unperturbed oscillator and find good agreement with a four-species ODE model of the system. Guided by simulations, microfluidics is then used to periodically perturb the system by modulating the concentration of one of the oscillator components with a given amplitude and frequency. When the ratio of the external ‘zeitgeber’ period and the intrinisic period is close to 1, we experimentally find period doubling and quadrupling in the oscillator dynamics, whereas for longer zeitgeber periods, we find stable entrainment. Our theoretical model suggests favorable conditions for which the oscillator can be utilized as an externally synchronized clock, but also demonstrates that related systems could, in principle, display chaotic dynamics.
A putative cap binding protein and the methyl phosphate capping enzyme Bin3/MePCE function in telomerase biogenesis
Authors: Páez-Moscoso Diego J. , Ho David V. , Pan Lili , Hildebrand Katie , Jensen Kristi L. , Levy Michaella J. , Florens Laurence , Baumann Peter
Published: December 2021

Telomerase reverse transcriptase (TERT) and the noncoding telomerase RNA (TR) subunit constitute the core of telomerase. Additional subunits are required for ribonucleoprotein complex assembly and in some cases remain stably associated with the active holoenzyme. Pof8, a member of the LARP7 protein family is such a constitutive component of telomerase in fission yeast. Using affinity purification of Pof8, we have identified two previously uncharacterized proteins that form a complex with Pof8 and participate in telomerase biogenesis. Both proteins participate in ribonucleoprotein complex assembly and are required for wildtype telomerase activity and telomere length maintenance. One factor we named Thc1 (Telomerase Holoenzyme Component 1) shares structural similarity with the nuclear cap binding complex and the poly-adenosine ribonuclease (PARN), the other is the ortholog of the methyl phosphate capping enzyme (Bin3/MePCE) in metazoans and was named Bmc1 (Bin3/MePCE 1) to reflect its evolutionary roots. Thc1 and Bmc1 function together with Pof8 in recognizing correctly folded telomerase RNA and promoting the recruitment of the Lsm2-8 complex and the catalytic subunit to assemble functional telomerase.
Rostral and caudal BLA engage distinct circuits in the prelimbic and infralimbic PFC
Authors: Manoocheri Kasra , Carter Adam G.
Published: December 2021

Connections from the basolateral amygdala (BLA) to medial prefrontal cortex (PFC) regulate memory and emotion and become disrupted in neuropsychiatric disorders. We hypothesized that the diverse roles attributed to interactions between the BLA and PFC reflect multiple circuits nested within a wider network. To assess these circuits, we first used anatomy to show that the rostral BLA (rBLA) and caudal BLA (cBLA) differentially project to prelimbic (PL) and infralimbic (IL) subregions of the PFC, respectively. We then combined in vivo silicon probe recordings and optogenetics to show that rBLA primarily engages PL, whereas cBLA mainly influences IL. Using ex vivo whole-cell recordings and optogenetics, we then assessed which neuronal subtypes are targeted, showing that rBLA preferentially drives layer 2 (L2) cortico-amygdalar (CA) neurons in PL, whereas cBLA drives layer 5 (L5) pyramidal tract (PT) cells in IL. Lastly, we used soma-tagged optogenetics to explore the local circuits linking superficial and deep layers of PL, showing how rBLA can also impact L5 PT cells. Together, our findings delineate how subregions of the BLA target distinct networks within the PFC to have different influence on prefrontal output.
The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin
Authors: Repton Charlotte , Cullen C. Fiona , Costa Mariana F. A. , Spanos Christos , Rappsilber Juri , Ohkura Hiroyuki
Published: December 2021

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to binding microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function. The mass spectrometry data are available from ProteomeXchange, identifier .

Author Summary

Accurate segregation of chromosomes during cell division is fundamental for genome stability. Chromosome segregation is mediated by the spindle, which is made of dynamic microtubules and associated proteins that regulate microtubule behaviour. How these microtubule-associated proteins are regulated is not well understood. Furthermore, as oocytes have an exceptionally large volume of cytoplasm and lack centrosomes, regulation of microtubule-associated proteins is especially crucial for organisation and function of the meiotic spindle. In this study, we showed that 14-3-3, a protein that binds to phosphorylated proteins, plays an important role to regulate multiple microtubule-associated proteins in fly oocytes. The regulated proteins include subunits of the conserved kinase complex called the chromosomal passenger complex. We further found that interaction of one of the subunits with 14-3-3 is regulated by two phosphorylations, and that these phosphorylations are important for localisation and function of this subunit. As these proteins are widely conserved including humans, this study may provide an insight into chromosome mis-segregation in human oocytes, which is very frequent and a major cause of human infertility, miscarriages and congenital birth conditions.
An RNA-binding switch drives ribosome biogenesis and tumorigenesis downstream of RAS oncogene
Authors: Azman Muhammad S. , Dodel Martin , Capraro Federica , Faraway Rupert , Dermit Maria , Fan Wanling , Ule Jernej , Mardakheh Faraz K.
Published: December 2021

Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy is largely unclear. Using quantitative RNA Interactome Capture analysis, we reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of cancer cells, with a network of nuclear proteins centered around Nucleolin displaying enhanced RNA-binding activity. We show that Nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal-RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This Nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced cancer cell proliferation, and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of Nucleolin, and highlight the crucial role of this process in RAS-mediated tumorigenesis.
Swift Formation of Optimal Single Spheroids towards In-Vitro 3-Dimensional Tumour Models
Authors: Habra Kinana , Pearson Joshua R.D. , McArdle Stéphanie E. B.
Published: December 2021

Monolayer cell culture, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more resemble the in vivo tumour growth thereby allowing results obtained with those on proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. However, the cost associated with their generation often involve expensive, plate, media, and growth supplements, which have limited their use for high throughput experiments. The protocol herein presents a novel and rapid generation for single spheroids of various cancer cell lines, U87 MG; SEBTA-027; SF188, brain cancer cells, DU-145, TRAMP-C1, prostate cancer cells, in 96-round bottom well plates. Cells are washed with anti-adherent solution, and the homogeneous compact spheroid morphology was evidenced as early as 24 hours after 10 minutes centrifugation for the seeded cells. By using confocal microscopy, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. The H&E stain of spheroid slices and the western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. The protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals.
Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity
Authors: Jády Beáta E. , Ketele Amandine , Moulis Dylan , Kiss Tamás
Published: December 2021

Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal “antisense” sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5′-UN-3′, including the 5′ substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.