bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine-scale survey of intertidal macroalgae reveals recent changes in a cold-water biogeographic stronghold
Authors: Monteiro Cátia , Pereira Joana , Seabra Rui , Lima Fernando P.
Published: March 2022

Global warming has been causing severe impacts on marine ecosystems, a notorious one being shifts in the geographical ranges of species. The north-western coast of the Iberian Peninsula is an especially interesting zone to study distributional shifts as it has a strong latitudinal thermal gradient, is influenced by the Canary upwelling system (which partially cancels coastal warming) and holds some of the most diverse macroalgae communities in Europe. Notably, it is within this region that many cold-water species, common in northern Europe, have their southernmost distribution refuge. Recent studies hypothesize that the environmental conditions may be nonetheless changing and already threatening this biodiversity hotspot.

The main goal of this study was to carry out a fine-scale assessment of the distributional limits of several macroalgae in North-western Iberia, as well as identify possible population and range shifts using historical data (2001-2005) as reference. In addition, invasive species were also surveyed. We also assessed if the regions of (i) Galicia, (ii) Northern Portugal, and (iii) Central Portugal displayed distinctive characters regarding macroalgae composition and abundance.

We identified an increase in abundance of some invasive macroalgae as well as a decrease in the abundance of some cold-water species. In the most severe cases, cold-water species were extirpated along hundreds of km. The compounded effect of the decrease in the abundance of cold-water species and the increase in the abundance of invasive species is leading to the homogenization of macroalgae communities in north-western Iberia.
Zebrafish her3 knockout impacts developmental and cancer-related gene signatures
Authors: Kent Matthew R. , Calderon Delia , Silvius Katherine M. , LaVigne Collette A. , Cannon Matthew V. , Kendall Genevieve C.
Published: February 2022

HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle. However, the mechanisms of HES3 cooperation in fusion-positive rhabdomyosarcoma are unclear and are likely related to her3/HES3’s role in neurogenesis. To investigate HES3’s function during development, we generated a zebrafish CRISPR/Cas9 knockout of her3, the zebrafish ortholog of HES3. Loss of her3 is not embryonic lethal and adults exhibit expected Mendelian ratios. Embryonic her3 zebrafish mutants are significantly smaller than wildtype and a subset present with lens defects as adults. Transcriptomic analysis of her3 mutant embryos indicates that genes involved in organ development, such as pctp and grinab, are significantly downregulated. Further, differentially expressed genes in her3 knockout embryos are enriched for HOX and SOX10 motifs. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases. Altogether, this new model is a powerful system to study her3/HES3-mediated neural development and its misappropriation in cancer contexts.

Summary Statement

Here, we generate and characterize a zebrafish her3/HES3 knockout to elucidate the functional role of her3/HES3, a transcriptional repressor, in neural development and tumorigenic processes.
A Tensor Decomposition Uncovers the Effect of Ageing on Muscle and Grip-Load Force Couplings During Grasping
Authors: Ye Chang , Saboksayr Seyed Saman , Shaw William , Coats Rachel O. , Astill Sarah L. , Mateos Gonzalo , Delis Ioannis
Published: March 2022

Do motor patterns of grasp-to-lift movements change as a result of ageing? Previous studies often relied on simple temporal and kinetic variables to unveil differences caused by ageing, yet their neuromuscular origins remain largely unknown. Here we employed a bimanual grasping protocol with younger and older adults and combined measurements of muscle activity with grip and load forces to provide a window into the neuromuscular strategies underlying effective grasping. We introduced a tensor decomposition to identify patterns of muscle activity and grip-load force ratios while also characterising their temporal profiles and relative activation across object weights and participants of different age groups. This approach extracted the motor components underpinning object grasping across participants. We then probed age-induced changes in these components. A classification analysis revealed two motor components that are differentially recruited between the two age groups. Linear regression analyses further showed that advanced age and poorer manual dexterity can be predicted by the coupled activation of forearm and hand muscles which is associated with high levels of grip force. Our findings suggest that ageing may induce stronger muscle couplings in distal aspects of the upper limbs, and a less economic grasping strategy to overcome age-related decline in manual dexterity.
Structural basis for the simultaneous recognition of NEMO and acceptor ubiquitin by the HOIP NZF1 domain
Authors: Rahighi Simin , Iyer Mamta , Oveisi Hamid , Nasser Sammy , Duong Vincent
Published: February 2022

Ubiquitination of NEMO by the linear ubiquitin chain assembly complex (LUBAC) is essential for activating the canonical NF-κB signaling pathway. While the NZF1 domain of the HOIP subunit of LUBAC recognizes the NEMO substrate, it is unclear how it cooperates with the catalytic domains in the ubiquitination process. Here, we report a crystal structure of NEMO in complex with HOIP NZF1 and linear diubiquitin chains, in which the two proteins bind to distinct sites on NEMO. Moreover, the NZF1 domain simultaneously interacts with NEMO and Ile44 surface of a proximal ubiquitin from a linear diubiquitin chain, where the C-term tail of the ubiquitin is in the proximity of the NEMO ubiquitination site (Lys285). We further propose a model for the linear ubiquitination of NEMO by HOIP. In the model, NZF1 binds the monoubiquitinated NEMO and recruits the catalytic domains to the ubiquitination site, thereby ensuring site-specific ubiquitination of NEMO.
Rapid weed adaptation and range expansion in response to agriculture over the last two centuries
Authors: Kreiner Julia M. , Latorre Sergio M. , Burbano Hernán A. , Stinchcombe John R. , Otto Sarah P. , Weigel Detlef , Wright Stephen I.
Published: March 2022

North America has seen a massive increase in cropland use since 1800, accompanied more recently by the intensification of agricultural practices. Through genome analysis of present-day and historical samples spanning environments over the last two centuries, we studied the impact of these changes in farming on the extent and tempo of evolution across the native range of common waterhemp (Amaranthus tuberculatus), a now pervasive agricultural weed. Modern agriculture has imposed strengths of selection rarely observed in the wild, with striking shifts in allele frequency trajectories since agricultural intensification in the 1960s. An evolutionary response to this extreme selection was facilitated by a concurrent human-mediated range shift. By reshaping genome-wide diversity across the landscape, agriculture has driven the success of this weed in the 21st-century.

One Sentence Summary

Modern agriculture has shaped the evolution of a native plant into a weed by driving range shifts and strengths of selection rarely observed in the wild.
Multi-omic analysis along the gut-brain axis points to a functional architecture of autism
Authors: Morton James T. , Jin Dong-Min , Mills Robert H. , Shao Yan , Rahman Gibraan , McDonald Daniel , Berding Kirsten , Needham Brittany D. , Zurita María Fernanda , David Maude , Averina Olga V. , Kovtun Alexey S. , Noto Antonio , Mussap Michele , Wang Mingbang , Frank Daniel N. , Li Ellen , Zhou Wenhao , Fanos Vassilios , Danilenko Valery N. , Wall Dennis P. , Cárdenas Paúl , Baldeón Manuel E. , Xavier Ramnik J. , Mazmanian Sarkis K. , Knight Rob , Gilbert Jack A. , Donovan Sharon M. , Lawley Trevor D. , Carpenter Bob , Bonneau Richard , Taroncher-Oldenburg Gaspar
Published: February 2022

Autism is a highly heritable neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in autism, with dozens of cross-sectional microbiome and other omic studies revealing autism-specific profiles along the GBA albeit with little agreement in composition or magnitude. To explore the functional architecture of autism, we developed an age and sex-matched Bayesian differential ranking algorithm that identified autism-specific profiles across 10 cross-sectional microbiome datasets and 15 other omic datasets, including dietary patterns, metabolomics, cytokine profiles, and human brain expression profiles. The analysis uncovered a highly significant, functional architecture along the GBA that encapsulated the overall heterogeneity of autism phenotypes. This architecture was determined by autism-specific amino acid, carbohydrate and lipid metabolism profiles predominantly encoded by microbial species in the generaPrevotella, Enterococcus, Bifidobacterium, andDesulfovibrio, and was mirrored in brain-associated gene expression profiles and restrictive dietary patterns in individuals with autism. Pro-inflammatory cytokine profiling and virome association analysis further supported the existence of an autism-specific architecture associated with particular microbial genera. Re-analysis of a longitudinal intervention study in autism recapitulated the cross-sectional profiles, and showed a strong association between temporal changes in microbiome composition and autism symptoms. Further elucidation of the functional architecture of autism, including of the role the microbiome plays in it, will require deep, multi-omic longitudinal intervention studies on well-defined stratified cohorts to support causal and mechanistic inference.
Decoupling diet from microbiome dynamics results in model mis-specification that implicitly annuls potential associations between the microbiome and disease phenotypes—ruling out any role of the microbiome in autism (Yap et al. 2021) likely a premature conclusion
Authors: Morton James T. , Donovan Sharon M. , Taroncher-Oldenburg Gaspar
Published: March 2022

Recently, and in a tour de force effort, Yap et al. performed a comprehensive association analysis of factors such as demographics, psychometrics, diet, stool metagenomics, stool consistency, and genome-wide SNP genotypes with autism in 247 Australian children. Surprisingly, the authors suggest their data show a strong correlation between diet and autism spectrum disorder (ASD) but only negligible, if any, ASD-specific microbiome signals. While the first conclusion comes as no surprise, we were rather puzzled by the second conclusion given the growing evidence of strong associations between the microbiome and ASD phenotype and the wide consensus on a close connection between diet and microbiome composition and function. The causal model proposed by Yap et al. seemed to imply that diet and the microbiome were two independent variables. A careful review of the approach used by the authors confirmed our suspicions that the statistical models were mis-specified, i.e. they had a questionable biological assumption—the independence of diet and microbiome—embedded in them. We have run side-by-side simulations of the causal linear model proposed by Yap et al. and of an analogous model in which diet and the microbiome are treated as co-dependent variables. We show how the Yap et al. model can preemptively exclude any potential host-microbe interactions if the diet-microbiome independence assumption is violated. We believe large-scale efforts such as the one described by Yap et al. are essential to advance our understanding of the potential role of the microbiome in ASD and other diseases. But these are highly complex systems to analyze and thus ensuring that the statistical methods used are accurate is essential to avoid drawing any potentially misleading conclusions due to subtle causal assumptions propagated by the statistical models themselves.
Early events in reovirus infection influence induction of innate immune response
Authors: Abad Andrew T. , Danthi Pranav
Published: February 2022

Mammalian orthoreovirus (reovirus) is a double-stranded (ds)RNA virus which encapsidates its 10 genome segments within a double-layered viral particle. Reovirus infection triggers an antiviral response in host cells which serves to limit viral replication. This antiviral response is initiated by recognition of the incoming viral genome by host sensors present in the cytoplasm. However, how host sensors gain access to the reovirus genome is unclear as this dsRNA is protected by the viral particle proteins throughout infection. To initiate infection, reovirus particles are endocytosed and the outer viral particle layer is disassembled through the action of host proteases. This disassembly event is required for viral escape into the cytoplasm to begin replication. We show that even after disassembly is complete, endosomal proteases are required to induce an immune response to reovirus. Additionally, counter to dogma, our data demonstrate at least some viral dsRNA genome is exposed and detectable during entry. We hypothesize that some proportion of reovirus particles remain trapped within endosomes, allowing for the breakdown of these particles and release of their genome. We show that rapidly uncoating mutants escape the endosome more rapidly and induce a diminished immune response. Further, we show that particles entering through dynamin-independent pathways evade detection by host sensors. Overall, our data provide new insight into how genomes from entering reovirus particles are detected by host cells.

IMPORTANCE

Viruses must infect host cells to replicate, often killing the host cell in the process. However, hosts can activate defenses to limit viral replication and protect the organism. To trigger these host defenses to viral infections, host cells must first recognize they are infected. Mammalian orthoreovirus (reovirus) is a model system used to study host-virus interactions. This study identifies aspects of host and virus biology which determine the capacity of host cells to detect infection. Notably, entry of reovirus into host cells plays a critical role in determining the magnitude of immune response triggered during infection. Mutants of reovirus which can enter cells more rapidly are better at avoiding detection by the host. Additionally, reovirus can enter cells through multiple routes. Entry through some of these routes also helps reovirus evade detection.
NEURONAL AND ASTROCYTIC TETRAPLOIDY IS INCREASED IN DRUG-RESISTANT EPILEPSY
Authors: Sanz-García Ancor , Sánchez-Jiménez Patricia , Granero-Cremades Inmaculada , De Toledo María , Pulido Paloma , Navas Marta , Frade Jose Maria , Pereboom Desirée , Torres-Díaz Cristina Virginia , Carmen Ovejero-Benito María
Published: March 2022

Background

Epilepsy is one of the most prevalent neurological diseases, a third of patients remain drug-resistant. The exact etiology of drug-resistant epilepsy (DRE) is still unknown. Neural tetraploidy has been associated with neuropathology. The aim of this study was to assess the presence of tetraploid neurons and astrocytes in DRE.

Materials & methods

Cortex, hippocampus and amygdala samples were obtained from patients subjected to surgical resection of the epileptogenic zone. Postmortem brain tissue of subjects without previous records of neurological, neurodegenerative or psychiatric diseases were used as controls. The percentage of tetraploid cells was measured by immunostaining of neurons (NeuN) or astrocytes (S100β) followed by flow cytometry analysis. Results were confirmed by image cytometry (ImageStream X Amnis System Cytometer) and with an alternative astrocyte biomarker (NDRG2). Statistical comparison was performed using univariate tests.

Results

A total of 22 patients and 10 controls were included. Tetraploid neurons and astrocytes were found both in healthy individuals and DRE patients in the three brain areas analyzed: cortex, hippocampus and amygdala. DRE patients presented a higher number of tetraploid neurons (p=0.020) and astrocytes (p=0.002) in the hippocampus compared to controls. These results were validated by image cytometry.

Conclusion

We demonstrated the presence of both tetraploid neurons and astrocytes in healthy subjects and increased levels of both cell types in DRE patients. This is the first time that tetraploid astrocytes are described in healthy subjects. Furthermore, these results provide new insights into epilepsy, opening new avenues for future treatment.
Fusing spheroids to aligned μ-tissues in a Heart-on-Chip featuring oxygen sensing and electrical pacing capabilities
Authors: Schneider Oliver , Moruzzi Alessia , Fuchs Stefanie , Grobel Alina , Schulze Henrike S. , Mayr Torsten , Loskill Peter
Published: March 2022

Over the last decade Organ-on-Chip (OOC) emerged as a promising technology for advanced in vitro models, recapitulating key physiological cues. OOC approaches tailored for cardiac tissue engineering resulted in a variety of platforms, some of which integrate stimulation or probing capabilities. Due to manual handling processes, however, a large-scale standardized and robust tissue generation, applicable in an industrial setting, is still out of reach. Here, we present a novel cell injection and tissue generation concept relying on spheroids, which can be produced in large quantities and uniform size from induced pluripotent stem cell-derived human cardiomyocytes. Hydrostatic flow transports and accumulates spheroids in dogbone-shaped cultivation chambers, which subsequently fuse and form aligned, contracting cardiac muscle fibers. Furthermore, we demonstrate electrical stimulation capabilities by utilizing fluidic media connectors as electrodes and provide the blueprint of a low-cost, open-source, scriptable pulse generator. We report on a novel integration strategy of optical O2 sensor spots into resin-based microfluidic systems, enabling in situ determination of O2 partial pressures. Finally, proof-of-concept demonstrating electrical stimulation combined with in situ monitoring of metabolic activity in cardiac tissues is provided. The developed system thus opens the door for advanced OOCs integrating biophysical stimulation as well as probing capabilities and serves as blueprint for the facile and robust generation of high density microtissues in microfluidic modules amenable for scale-up and automation.
The chromatin landscape of Th17 cells reveals mechanisms of diversification of regulatory and pro-inflammatory states
Authors: Thakore Pratiksha I. , Schnell Alexandra , Zhao Maryann , Huang Linglin , Hou Yu , Christian Elena , Zaghouani Sarah , Wang Chao , Singh Vasundhara , Ma Sai , Sankar Venkat , Notarbartolo Samuele , Buenrostro Jason D. , Sallusto Federica , Patsopoulos Nikolaos A. , Rozenblatt-Rosen Orit , Kuchroo Vijay K. , Regev Aviv
Published: February 2022

Th17 cells are a heterogenous cell population consisting of non-pathogenic Th17 cells (npTh17) that contribute to tissue homeostasis and pathogenic Th17 cells (pTh17) that are potent mediators of tissue inflammation. To reveal regulatory mechanisms underlying Th17 heterogeneity, we performed combined ATAC-seq and RNA-seq and discovered substantial differences in the chromatin landscape of npTh17 and pTh17 cells bothin vitroandin vivo. Compared to other CD4+T cell subsets, npTh17 cells share accessible chromatin programs with Tregs, and pTh17 cells have an intermediate profile spanning features of npTh17 cells and Th1 cells. Integrating single-cell ATAC-seq and single-cell RNA-seq, we inferred self-reinforcing and mutually exclusive regulatory networks controlling the different cell states and predicted transcription factors (TFs) shaping the chromatin landscape of Th17 cell pathogenicity. We validated one novel TF, BACH2, which promotes immunomodulatory npTh17 programs and restrains pro-inflammatory Th1-like programs in Th17 cells and showed genetic evidence for protective variants in the human BACH2 locus associated with multiple sclerosis. Our work uncovered mechanisms that regulate Th17 heterogeneity, revealed shared regulatory programs with other CD4+T cell subsets, and identified novel drivers of Th17 pathogenicity as potential targets to mitigate autoimmunity.
General theory of specific binding: insights from a genetic-mechano-chemical protein model
Authors: McBride John M. , Eckmann Jean-Pierre , Tlusty Tsvi
Published: February 2022

Proteins need to selectively interact with specific targets among a multitude of similar molecules in the cell. But despite a firm physical understanding of binding interactions, we lack a general theory of how proteins evolve high specificity. Here, we present such a model that combines chemistry, mechanics and genetics, and explains how their interplay governs the evolution of specific protein-ligand interactions. The model shows that there are many routes to achieving molecular discrimination – by varying degrees of flexibility and shape/chemistry complementarity – but the key ingredient is precision. Harder discrimination tasks require more collective and precise coaction of structure, forces and movements. Proteins can achieve this through correlated mutations extending far from a binding site, which fine-tune the localized interaction with the ligand. Thus, the solution of more complicated tasks is enabled by increasing the protein size, and proteins become more evolvable and robust when they are larger than the bare minimum required for discrimination. The model makes testable, specific predictions about the role of flexibility and shape mismatch in discrimination, and how evolution can independently tune affinity and specificity. Thus, the proposed theory of specific binding addresses the natural question of “why are proteins so big?”. A possible answer is that molecular discrimination is often a hard task best performed by adding more layers to the protein.
Distinct chromosomal “niches” in the genome ofS. cerevisiaeprovide the background for genomic innovation and shape the fate of gene duplicates
Authors: Stavropoulou Athanasia , Tassios Aimilios , Kalyva Maria , Georgoulopoulos Michalis , Vakirlis Nikolaos , Iliopoulos Ioannis , Nikolaou Christoforos
Published: February 2022

Gene duplication is a major source of genomic innovation in all eukaryotes, with large proportions of genes being the result of either small-scale (SSD) or genome-wide duplication (WGD) events. In the model unicellular eukaryoteSaccharomyces cerevisiae, of which nearly one third of the genome corresponds to gene duplicates, the two modes of duplication have been shown to follow different evolutionary fates, with SSD genes being more prone to acquire novel functionalities (neofunctionalization) and WGD more likely to retain different parts of the original ancestral function (subfunctionalization). Having previously described aspects of functional compartmentalization for the genes ofS. cerevisiae, in this work we set out to investigate the existence of positional preferences of gene duplicates. We found that SSD and WGD genes are organized in distinct gene clusters that are, furthermore, segregated, occupying different regions, with SSD being more peripheral and WGD more centrally positioned close to centromeric chromatin.

Duplicate gene clusters differ from the rest of the genome in terms of gene size and spacing, gene expression variability and regulatory complexity. What is more interesting, some of these properties are also shared by singleton genes residing in duplicate-rich regions in a position-dependent manner. Our analysis further reveals particular chromatin architectures in the promoters of duplicate genes, which are generally longer, with less pronounced nucleosome-free regions, strong structural constraints and a larger number of regulatory elements. Such structural features appear to be important for gene evolution as we find SSD gene-pair co-expression to be strongly associated with the similarity of nucleosome positioning patterns.

We propose that specific regions of the yeast genome provide a favourable environment for the generation and maintenance of small-scale gene duplicates. The existence of such genomic “niches” is supported by the enrichment of these regions in singleton genes bearing similarities with gene “relics”, remnants of recent duplications that have reverted to single gene status. Our findings provide a valuable framework for the study of genomic innovation and suggest taking into account positional preferences in the study of gene emergence and fixation in experimentally and naturally evolving populations.
Numerical discrimination in Drosophila melanogaster
Authors: Bengochea Mercedes , Sitt Jacobo D. , Preat Thomas , Izard Veronique , Cohen Laurent , Hassan Bassem A.
Published: March 2022

Sensitivity to numbers is a crucial and evolutionarily conserved cognitive ability. The lack of experimental models amenable to systematic genetic and neural manipulation has precluded discovering circuits required for numerical cognition. Here, we demonstrate that in a two-choice task Drosophila fruit flies spontaneously prefer sets containing more objects. This preference is determined by the ratio between the two numerical quantities tested, a characteristic signature of numerical cognition across species. Individual flies maintained their numerical choice over consecutive days. Using a numerical visual conditioning paradigm, we found that flies are capable of associating sucrose with numerical quantities and can be trained to reverse their spontaneous preference for large quantities. Finally, we show that silencing LC11 neurons reduces the preference for more objects, thus identifying a neuronal substrate for numerical cognition in invertebrates. This discovery paves the way for the systematic analysis of the behavioral and neural mechanisms underlying sensitivity to numerosity.
Constructive Connectomics: how neuronal axons get from here to there using gene-expression maps derived from their family trees
Authors: Kerstjens Stan , Michel Gabriela , Douglas Rodney J.
Published: February 2022

During brain development, billions of axons must navigate over multiple spatial scales to reach specific neuronal targets, and so build the processing circuits that generate the intelligent behavior of animals. However, the limited information capacity of the zygotic genome puts a strong constraint on how, and which, axonal routes can be encoded. We propose and validate a mechanism of development that can provide an efficient encoding of this global wiring task. The key principle, confirmed through simulation, is that basic constraints on mitoses of neural stem cells—that mitotic daughters have similar gene expression to their parent and do not stray far from one another—induce a global hierarchical map of nested regions, each marked by the expression profile of its common progenitor population. Thus, a traversal of the lineal hierarchy generates a systematic sequence of expression profiles that traces a staged route, which growth cones can follow to their remote targets. We have analyzed gene expression data of developing and adult mouse brains published by the Allen Institute for Brain Science, and found them consistent with our simulations: gene expression indeed partitions the brain into a global spatial hierarchy of nested contiguous regions that is stable at least from embryonic day 11.5 to postnatal day 56. We use this experimental data to demonstrate that our axonal guidance algorithm is able to robustly extend arbors over long distances to specific targets, and that these connections result in a qualitatively plausible connectome. We conclude that, paradoxically, cell division may be the key to uniting the neurons of the brain.

Author Summary

The embryological development of each brain installs an essentially identical communication network between its cells that is roughly as complex as that between the billions of people living on Earth. Although vast scientific resources are currently applied to identifying the final pattern of connections, the connectome, there has until now been relatively little effort to answer the fundamental question of how this complex network across billions of neurons realized through the mitotic elaboration of the initial embryonic cell. The problem is sharpened by the constraints that construction of the network is limited by the information budget of the initial genome, and that it has no pre-existing address space for placing neurons and guiding axons. We explain how Biology can solve this problem by using the family tree of neurons to install a global space of molecular addresses, which axons can use to navigate from their source neuron to its relatives. We provide experimental evidence for this familial address space in gene expression patterns of the developing mouse brain, and demonstrate through simulation that the experimentally observed address space indeed supports global navigation to produce a qualitatively plausible default connectome.
RNA fluorescence in situ hybridization (FISH) to visualize microbial colonization and infection in the Caenorhabditis elegans intestines
Authors: Rivera Dalaena E , Lažetić Vladimir , Troemel Emily R , Luallen Robert J
Published: March 2022

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools.

This protocol introduces RNA fluorescence in situ hybridization (FISH) as a tool used for the identification, visualization, and quantification of the microbes within the intestines of C. elegans. FISH probes that label the highly abundant small subunit ribosomal RNA can produce a bright signal for bacteria and microsporidian cells, and similar probes can be used to label viral RNA. FISH probes can be ordered from a commercial source as single-stranded DNA end-labeled with fluorophores. One limitation is that FISH may not provide robust signal against low copy targets, although signal can be boosted by using multiple probes (so-called ‘single-molecule FISH’). FISH staining involves collecting colonized or infected animals, washing to eliminate external contamination, followed by fixation in either paraformaldehyde or acetone. After fixation, FISH probes are incubated with samples to allow for the hybridization of probes to the desired target. To remove excess background, the animals are washed again, and then examined on microscope slides or using automated approaches.

Overall, this protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

Gut microbiome bacteria and intestinal intracellular pathogens, like the Orsay virus and microsporidia, are often found associated with wild Caenorhabditis nematodes. This protocol presents RNA FISH as a method for the detection, quantification, and identification of colonizing or infectious microbes within the context of intact C. elegans nematodes.
Sound-evoked responses of distinct neuron classes from the tail of the striatum
Authors: Nardoci Matthew B. , Lakunina Anna A. , Henderling Devin C. , Pedregon Jewlyssa C. , Jaramillo Santiago
Published: February 2022

Given its inputs from auditory structures and neuromodulatory systems, the posterior tail of the striatum is ideally positioned to influence behavioral responses to acoustic stimuli according to context and previous rewards. Results from previous studies indicate that neurons in this striatal region display selective responses to sounds. However, it is not clear whether different striatal cell classes code for distinct features of sounds, nor how different striatal output pathways may use acoustic information to guide behavior. Here we compared the sound-evoked responses of posterior striatal neurons that form the striatal direct pathway (and express the dopamine receptor D1) to the responses of neighboring neurons in naive mice. We achieved this via optogenetic photo-identification of D1-expressing neurons during extracellular electrophysiological recordings in awake head-fixed mice of both sexes. We found that the frequency tuning of sound-responsive direct-pathway striatal neurons is comparable to that of their sound-responsive neighbors. Moreover, we found that both populations encode amplitude modulated sounds in a similar fashion. These results suggest that different classes of neurons in the posterior striatum of naive animals have similar access to acoustic features conveyed by the auditory system even outside the context of an auditory task.
The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization
Authors: Karger Amy B. , Brien James D. , Christen Jayne M. , Dhakal Santosh , Kemp Troy J. , Klein Sabra L. , Pinto Ligia A. , Premkumar Lakshmanane , Roback John D. , Binder Raquel A. , Boehme Karl W. , Boppana Suresh , Cordon-Cardo Carlos , Crawford James M. , Daiss John L. , Dupuis Alan P. , Espino Ana M. , Firpo-Betancourt Adolfo , Forconi Catherine , Forrest J. Craig , Girardin Roxie C. , Granger Douglas A. , Granger Steve W. , Haddad Natalie S. , Heaney Christopher D. , Hunt Danielle T. , Kennedy Joshua L. , King Christopher L. , Krammer Florian , Kruczynski Kate , LaBaer Joshua , Lee F. Eun-Hyung , Lee William T. , Liu Shan-Lu , Lozanski Gerard , Lucas Todd , Mendu Damodara Rao , Moormann Ann M. , Murugan Vel , Okoye Nkemakonam C. , Pantoja Petraleigh , Payne Anne F. , Park Jin , Pinninti Swetha , Pinto Amelia K. , Pisanic Nora , Qiu Ji , Sariol Carlos A. , Simon Viviana , Song Lusheng , Steffen Tara L. , Stone E. Taylor , Styer Linda M. , Suthar Mehul S. , Thomas Stefani N. , Thyagarajan Bharat , Wajnberg Ania , Yates Jennifer L. , Sobhani Kimia
Published: March 2022

Background

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and “to develop, validate, improve, and implement serological testing and associated technologies.” SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization.

Methods

To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data.

Results

SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards.

Conclusions

SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.
Clinically oriented prediction of patient response to targeted and immunotherapies from the tumor transcriptome
Authors: Dinstag Gal , Shulman Eldad D. , Elis Efrat , Ben-Zvi Doreen S. , Tirosh Omer , Maimon Eden , Meilijson Isaac , Elalouf Emmanuel , Temkin Boris , Vitkovsky Philipp , Schiff Eyal , Hoang Danh-Tai , Sinha Sanju , Nair Nishanth Ulhas , Sang Lee Joo , Schäffer Alejandro A. , Ronai Ze’ev , Juric Dejan , Apolo Andrea B. , Dahut William L. , Lipkowitz Stanley , Berger Raanan , Kurzrock Razelle , Papanicolau-Sengos Antonios , Karzai Fatima , Gilbert Mark R. , Aldape Kenneth , Rajagopal Padma S. , Beker Tuvik , Ruppin Eytan , Aharonov Ranit
Published: March 2022

Background

Precision oncology is gradually advancing into mainstream clinical practice, demonstrating significant survival benefits. However, eligibility and response rates remain limited in many cases, calling for better predictive biomarkers.

Methods

We present ENLIGHT, a transcriptomics-based computational approach that identifies clinically relevant genetic interactions and uses them to predict a patient’s response to a variety of therapies in multiple cancer types, without training on previous treatment response data. We study ENLIGHT in two translationally oriented scenarios:Personalized Oncology (PO), aimed at prioritizing treatments for a single patient, andClinical Trial Design (CTD), selecting the most likely responders in a patient cohort.

Findings

Evaluating ENLIGHT’s performance on 21 blinded clinical trial datasets in the PO setting, we show that it can effectively predict a patient’s treatment response across multiple therapies and cancer types. Its prediction accuracy is better than previously published transcriptomics-based signatures and is comparable to that of supervised predictors developed for specific indications and drugs. In combination with the IFN-γsignature, ENLIGHT achieves an odds ratio larger than 4 in predicting response to immune checkpoint therapy. In the CTD scenario, ENLIGHT can potentially enhance clinical trial success for immunotherapies and other monoclonal antibodies by excluding non-responders, while overall achieving more than 90% of the response rate attainable under an optimal exclusion strategy.

Conclusion

ENLIGHT demonstrably enhances the ability to predict therapeutic response across multiple cancer types from the bulk tumor transcriptome.

Funding

This research was supported in part by the Intramural Research Program, NIH and by the Israeli Innovation Authority.
Aging-regulated TUG1 is dispensable for endothelial cell function
Authors: Gimbel Anna Theresa , Koziarek Susanne , Theodorou Kosta , Schulz Jana Felicitas , Stanicek Laura , Ali Tamer , Günther Stefan , Kumar Sandeep , Jo Hanjoong , Hübner Norbert , Maegdefessel Lars , Dimmeler Stefanie , van Heesch Sebastiaan , Boon Reinier A.
Published: March 2022

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function.
Genetic Examination of Hematological Parameters in SARS-CoV-2 Infection and COVID-19
Authors: Rowland Bryce , Sun Quan , Wang Wanjiang , Miller-Fleming Tyne , Cox Nancy , Graff Misa , Faucon Annika , Shuey Megan M. , Blue Elizabeth E. , Auer Paul , Li Yun , Sankaran Vijay G. , Reiner Alexander P. , Raffield Laura M.
Published: March 2022

Background

People hospitalized with COVID-19 often exhibit hematological alterations, such as lower lymphocyte and platelet counts, which have been reported to associate with disease prognosis. It is unclear whether inter-individual variability in baseline hematological parameters prior to acute infection influences risk of SARS-CoV-2 infection and progression to severe COVID-19.

Methods

We assessed the association of blood cell counts and indices with incident SARS-CoV-2 infection and severe COVID-19 in UK Biobank and the Vanderbilt University Medical Center Synthetic Derivative (VUMC SD). Since genetically determined blood cell measures better represent cell abundance across the lifecourse, we used summary statistics from genome-wide association studies to assess the shared genetic architecture of baseline blood cell counts and indices on COVID-19 outcomes.

Results

We observed inconsistent associations between measured blood cell indices and both SARS-CoV-2 infection and COVID-19 hospitalization in UK Biobank and VUMC SD. In Mendelian randomization analyses using genetic summary statistics, no putative causal relationships were identified between COVID-19 related outcomes and hematological indices after adjusting for multiple testing. We observed overlapping genetic association signals between hematological parameters and COVID-19 traits. For example, we observed overlap between infection susceptibility-associated variants atPPP1R15Aand red blood cell parameters, and between disease severity-associated variants atTYK2and lymphocyte and platelet phenotypes.

Conclusions

We did not find convincing evidence of a relationship between baseline hematological parameters and susceptibility to SARS-CoV-2 infection or COVID-19 severity, though this relationship should be re-examined as larger and better-powered genetic analyses of SARS-CoV-2 infection and severe COVID-19 become available.
Impact of the representation of contact data on the evaluation of interventions in infectious diseases simulations
Authors: Contreras Diego Andrés , Colosi Elisabetta , Bassignana Giulia , Colizza Vittoria , Barrat Alain
Published: March 2022

Computational models offer a unique setting to test strategies to mitigate infectious diseases’ spread, providing useful insights to applied public health. To be actionable, models need to be informed by data, which can be available at different levels of detail. While high resolution data describing contacts between individuals are increasingly available, data gathering remains challenging, especially during a health emergency: many models thus use synthetic data or coarse information to evaluate intervention protocols. Here, we evaluate how the representation of contact data might affect the impact of various strategies in models, in the realm of COVID-19 transmission in educational and work contexts. Starting from high resolution contact data, we use data representations ranging from very detailed to very coarse to inform a model for the spread of SARS-CoV-2 and simulate several mitigation strategies. We find that coarse data representations underestimate the risk of super-spreading events. However, the rankings of protocols according to their efficiency or cost remain coherent across representations, ensuring the consistency of model findings to inform public health advice. Caution should be taken, however, on the quantitative estimations of those benefits and costs that may trigger the adoption of protocols, as these may depend on data representation.
Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape
Authors: Hunter Byron , Benoit Matthieu P.M.H. , Asenjo Ana B. , Doubleday Caitlin , Trofimova Daria , Sosa Hernando , Allingham John S.
Published: March 2022

Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 Å) ofCandida albicansKip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unboundCaKip3-ADP crystal structure (2.0 Å). Together with biochemical analyses ofCaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.
Extracellular vesicles and high-density lipoproteins: Exercise and estrogen-responsive small RNA carriers
Authors: Karvinen Sira , Korhonen Tia-Marje , Sievänen Tero , Karppinen Jari E. , Juppi Hanna-Kaarina , Jakoaho Veera , Kujala Urho M. , Laukkanen Jari A. , Lehti Maarit , Laakkonen Eija K.
Published: March 2022

Decreased systemic estrogen levels (i.e., menopause) affect metabolic health. However, the detailed mechanisms underlying this process remain unclear. Both estrogens and exercise have been shown to improve metabolic health, which may be partly mediated by circulating microRNA (c-miR) signaling. In recent years, extracellular vesicles (EV) have increased interest in the field of tissue crosstalk. However, in many studies on EV-carried miRs, the co-isolation of high-density lipoprotein (HDL) particles with EVs has not been considered, potentially affecting the results. Here, we demonstrate that EV and HDL particles have distinct small RNA (sRNA) content, including both host and nonhost sRNAs. Exercise caused an acute increase in relative miR abundancy in EVs, whereas in HDL particles, it caused an increase in transfer RNA-derived sRNA. Furthermore, we demonstrate that estrogen deficiency caused by menopause blunts acute exercise-induced systemic miR-response in both EV and HDL particles.

HIGHLIGHTS

Extracellular vesicles and HDL particles have a distinct sRNA content

Extracellular vesicles and HDL particles carry both host and nonhost sRNA cargo

Estrogen deficiency blunts the c-miR-response induced by acute exercise

Exercise responsive miRs in HT users may regulate the choice of energy substrate