bluuebunny/crossref_metadata_2025 huggingface dataset:
Representational Connectivity Analysis: Identifying Networks of Shared Changes in Representational Strength through Jackknife Resampling
Authors: Coutanche Marc N. , Akpan Essang , Buckser Rae R.
Published: May 2020

The structure of information in the brain is crucial to cognitive function. The representational space of a brain region can be identified through Representational Similarity Analysis (RSA) applied to functional magnetic resonance imaging (fMRI) data. In its classic form, RSA collapses the time-series of each condition, eliminating fluctuations in similarity over time. We propose a method for identifying representational connectivity (RC) networks, which share fluctuations in representational strength, in an analogous manner to functional connectivity (FC), which tracks fluctuations in BOLD signal, and informational connectivity, which tracks fluctuations in pattern discriminability. We utilize jackknife resampling, a statistical technique in which observations are removed in turn to determine their influence. We applied the jackknife technique to an existing fMRI dataset collected as participants viewed videos of animals (Nastase et al., 2017). We used ventral temporal cortex (VT) as a seed region, and compared the resulting network to a second-order RSA, in which brain regions’ representational spaces are compared, and to the network identified through FC. The novel representational connectivity analysis identified a network comprising regions associated with lower-level visual processing, spatial cognition, perceptual-motor integration, and visual attention, indicating that these regions shared fluctuations in representational similarity strength with VT. RC, second-order RSA and FC identified areas unique to each method, indicating that analyzing shared fluctuations in the strength of representational similarity reveals previously undetectable networks of regions. The RC analysis thus offers a new way to understand representational similarity at the network level.
Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing
Authors: Thorarinn Blondal , Cristina Gamba , Møller Jagd Lea , Ling Su , Dimiter Demirov , Shuang Guo , Johnston Camille M. , Riising Eva M. , Xiaolin Wu , Mikkelsen Marie J. , Ludmila Szabova , Peter Mouritzen
Published: May 2020

Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the gene editing site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~ 100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene edited region in 4 cell lines on long- and short -read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kb large insertions in 3 of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.
Computational modeling reveals cell-cycle dependent kinetics of H4K20 methylation states during Xenopus embryogenesis
Authors: Schuh Lea , Loos Carolin , Pokrovsky Daniil , Imhof Axel , Rupp Ralph , Marr Carsten
Published: May 2020

Histone modifications regulate chromatin architecture and thereby control gene expression. Rapid cell divisions and DNA replication however lead to a dilution of histone modifications and can thus affect chromatin mediated gene regulation So how does the cell-cycle shape the histone modification landscape, in particular during embryogenesis when a fast and precise control of cell-specific gene expression is required?

We addressed this question in vivo by manipulating the cell-cycle during early Xenopus laevis embryogenesis. The global distribution of un-, mono- di- and tri-methylated histone H4K20 was measured by mass spectrometry in normal and cell-cycle arrested embryos over time. Using multi-start maximum likelihood optimization and quantitative model selection, we found that three specific methylation rate constants were required to explain the measured H4K20 methylation state kinetics. Interestingly, demethylation was found to be redundant in the cycling embryos but essential in the cell-cycle arrested embryos.

Together, we present the first quantitative analysis of in vivo histone H4K20 methylation kinetics. Our computational model shows that demethylation is only essential for regulating H4K20 methylation kinetics in non-cycling cells. In rapidly dividing cells of early embryos, we predict that demethylation is dispensable, suggesting that cell-cycle mediated dilution of chromatin marks is an essential regulatory component for shaping the epigenetic landscape during early embryonic development.
Evidence for an Active Role of Inferior Frontal Cortex in Conscious Experience
Authors: Weilnhammer Veith , Fritsch Merve , Chikermane Meera , Eckert Anna-Lena , Kanthak Katharina , Stuke Heiner , Kaminski Jakob , Sterzer Philipp
Published: May 2020

2

In the search for the neural correlates of consciousness, it has remained controversial whether prefrontal cortex determines what is consciously experienced or, alternatively, serves only complementary functions such as introspection or action.

Here, we provide converging evidence from computational modeling and two functional magnetic resonance imaging experiments for a key role of inferior frontal cortex in detecting perceptual conflicts that emerge from ambiguous sensory information. Crucially, the detection of perceptual conflicts by prefrontal cortex turned out to be critical in the process of transforming ambiguous sensory information into unambiguous conscious experiences: In a third experiment, disruption of neural activity in inferior frontal cortex through transcranial magnetic stimulation slowed down the updating of conscious experience that occurs in response to perceptual conflicts.

These findings show that inferior frontal cortex actively contributes to the resolution of perceptual ambiguities. Prefrontal cortex is thus causally involved in determining the contents of conscious experience.

3

One-sentence Summary

Inferior frontal cortex detects and resolves perceptual conflict during bistable perception.
The genome of a nonphotosynthetic diatom provides insights into the metabolic shift to heterotrophy and constraints on the loss of photosynthesis
Authors: Pendergrass Anastasiia , Roberts Wade R. , Ruck Elizabeth C. , Lewis Jeffrey A. , Alverson Andrew J.
Published: May 2020

Although most of the tens of thousands of diatom species are obligate photoautotrophs, many mixotrophic species can also use extracellular organic carbon for growth, and a small number of obligate heterotrophs have lost photosynthesis entirely. We sequenced the genome of a nonphotosynthetic diatom,Nitzschiasp. strain Nitz4, to determine how carbon metabolism was altered in the wake of this rare and radical trophic shift in diatoms. Like other groups that have lost photosynthesis, the genomic consequences were most evident in the plastid genome, which is exceptionally AT-rich and missing photosynthesis-related genes. The relatively small (27 Mb) nuclear genome did not differ dramatically from photosynthetic diatoms in gene or intron density. Genome-based models suggest that central carbon metabolism, including a central role for the plastid, remains relatively intact in the absence of photosynthesis. All diatom plastids lack an oxidative pentose phosphate pathway (PPP), leaving photosynthesis as the main source of plastid NADPH. Consequently, nonphotosynthetic diatoms lack the primary source of NADPH required for essential biosynthetic pathways that remain in the plastid. Genomic models highlighted similarities between nonphotosynthetic diatoms and apicomplexan parasites for provisioning NADPH in their plastids. The ancestral absence of a plastid PPP might constrain loss of photosynthesis in diatoms compared to Archaeplastida, whose plastid PPP continues to produce reducing cofactors following loss of photosynthesis. Finally,Nitzschiapossesses a complete β-ketoadipate pathway. Previously known only from fungi and bacteria, this pathway may allow mixotrophic and heterotrophic diatoms to obtain energy through the degradation of abundant plant-derived aromatic compounds.
Deterioration of Glutaraldehyde Crosslinked Heterograft Biomaterials due to Advanced Glycation End Product Formation and Serum Albumin Infiltration
Authors: Rock Christopher A. , Keeney Samuel , Zakharchenko Andrey , Takano Hajime , Spiegel David A. , Krieger Abba M. , Ferrari Giovanni , Levy Robert J.
Published: May 2020

Bioprosthetic heart valves (BHV) are fabricated from glutaraldehyde cross-linked heterograft tissue, such as bovine pericardium (BP) or porcine aortic valves. BHV develop structural valve degeneration (SVD), often with calcification, requiring BHV replacement. Advanced glycation end products (AGE) are post-translational, non-enzymatic carbohydrate protein modifications. AGE are present in SVD-BHV clinical explants and not detectable in unimplanted BHV. Here, we studied the hypothesis that BHV susceptibility to AGE formation and serum protein infiltration results in deterioration of both leaflet collagen structure and mechanical properties. In vitro experiments studied BP and porcine collagen sponges (CS) for susceptibility to AGE formation using14C-glucose and14C-glyoxal with and without bovine serum albumin (BSA), as a model serum protein. The results showed AGE formation is a rapid and progressive process. BSA co-incubations reduced glyoxal and glucose uptake by BP and CS. Incubating BP in BSA caused a substantial increase in BP mass, enhanced by glyoxal co-incubation. Per two-photon microscopy, BP with AGE formation and BSA infiltration each induced significant disruption in collagen microarchitecture, with loss of collagen alignment and crimp. These effects are cumulative with the greatest disruption occurring when there was both AGE formation and BSA infiltration. Uniaxial testing of CS demonstrated that AGE formation, together with BSA uptake compared to controls, caused a significant deterioration in mechanical properties with a loss of viscoelastic relaxation and increased stiffness. It is concluded that AGE-BSA associated collagen structural disruption and deterioration of mechanical properties contribute to SVD.
Photoreceptor complexity accompanies adaptation to challenging marine environments in Anthozoa
Authors: Gornik Sebastian G. , Bergheim B. Gideon , Foulkes Nicholas S. , Guse Annika
Published: May 2020

Light represents a key environmental factor, which shapes the physiology and evolution of most organisms. Notable illustrations of this are reef-building corals (Anthozoa), which adapted to shallow, oligotrophic, tropical oceans by exploiting light from the sun and the moon to regulate various aspects of physiology including sexual reproduction, phototaxis and photosymbiosis. Together with the Medusozoa, (including jellyfish), the Anthozoa constitute the ancestral metazoan phylum cnidaria. While light perception in Medusozoa has received attention, the mechanisms of light sensing in Anthozoa remain largely unknown. Cnidaria express two principle groups of light-sensing proteins: opsins and photolyases/cryptochromes. By inspecting the genomic loci encoding these photoreceptors in over 35 cnidarian species, we reveal that Anthozoa have substantially expanded and diversified their photoreceptor repertoire. We confirm that, in contrast to Medusozoa, which retained one opsin class, anthozoans possess all three urmetazoan opsin classes. We show that anthozoans also evolved an extra sub-group (actinarian ASO-IIs). Strikingly, we reveal that cryptochromes including CRY-IIs are absent in Medusozoa, while the Anthozoa retained these and evolved an additional, novel cryptochrome class (AnthoCRYs), which contain unique tandem duplications of up to 6 copies of the PHR region. We explored the functionality of these photoreceptor groups by structure-function and gene expression analysis in the anthozoan model speciesExaiptasia pallida(Aiptasia), which recapitulates key photo-behaviors of corals. We identified an array of features that we speculate reflect adaptations to shallow aquatic environments, moonlight-induced spawning synchronization and photosymbiosis. We further propose that photoreceptor complexity and diversity in Anthozoa reflects adaptation to challenging habitats.
An enhanced isothermal amplification assay for viral detection
Authors: Qian Jason , Boswell Sarah A. , Chidley Christopher , Lu Zhi-xiang , Pettit Mary E. , Gaudio Benjamin L. , Fajnzylber Jesse M. , Ingram Ryan T. , Ward Rebecca H. , Li Jonathan Z. , Springer Michael
Published: May 2020

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS-CoV-2, FIND (Fast Isothermal Nucleic acid Detection), based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT-qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross-react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available.

One sentence summary

Sensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS-CoV-2 from patient samples.
Torix groupRickettsiaare widespread in New Zealand freshwater amphipods: using blocking primers to rescue host COI sequences
Authors: Park Eunji , Poulin Robert
Published: May 2020

Endosymbionts and intracellular parasites are common in arthropods and other invertebrate hosts. As a consequence, (co)amplification of untargeted bacterial sequences has been occasionally reported as a common problem in DNA barcoding. The bacterial genusRickettsiabelongs to the order Rickettsiales and consists of two lineages: one including diverse pathogens infecting arthropod hosts, the other consisting of non-pathogenic species with a broader host taxonomic range. While discriminating among amphipod species with universal primers for the COI region, we unexpectedly detected rickettsial endosymbionts belonging to the Torix group. To map the distribution and diversity ofRickettsiaamong amphipods hosts, we conducted a nationwide molecular screening of seven families of freshwater amphipods collected throughout New Zealand. In addition to uncovering a diversity of Torix groupRickettsiaacross multiple amphipod populations from three different families, our research indicates that 1) detecting TorixRickettsiawith universal primers is not uncommon, 2) obtaining ‘RickettsiaCOI sequences’ from many host individuals is highly likely when a population is infected, and 3) obtaining ‘host COI’ may not be possible with a conventional PCR if an individual is infected. BecauseRickettsiaCOI is highly conserved across diverse host taxa, we were able to design blocking primers that can be used in a wide range of host species infected with TorixRickettsia. We propose the use of blocking primers to circumvent problems caused by unwanted amplification ofRickettsiaand to obtain targeted host COI sequences for DNA barcoding, population genetics, and phylogeographic studies.
Severe dysregulation of TNF expression leads to multiple inflammatory diseases and embryonic death
Authors: Clayer Elise , Dalseno Destiny , Kueh Andrew , Lacey Derek , Tsai Minhsuang , Carr Elysa , Wimmer Verena C. , Bouillet Philippe
Published: May 2020

Post-transcriptional regulation mechanisms regulate mRNA stability or translational efficiency via ribosomes and recent evidence indicates that it is a major determinant of the accurate levels of cytokine mRNAs. While transcriptional regulation ofTnfhas been well studied and found to be important for the rapid induction ofTnfmRNA and regulation of the acute phase of inflammation, study of its post-transcriptional regulation has been largely limited to the role of the AU-rich element (ARE), and to a lesser extent, that of the constitutive decay element (CDE). We have identified a new regulatory element (NRE) in the 3’ untranslated region (3’UTR) ofTnf, and demonstrate that ARE, CDE and NRE cooperate to efficiently down regulateTnfexpression and prevent autoimmune inflammatory diseases. We also show for the first time that excessive TNF may lead to embryonic death.
Agile workflow for interactive analysis of mass cytometry data
Authors: Casado Julia , Lehtonen Oskari , Rantanen Ville , Kaipio Katja , Pasquini Luca , Häkkinen Antti , Petrucci Elenora , Carpén Olli , Biffoni Mauro , Färkkilä Anniina , Hautaniemi Sampsa
Published: May 2020

Motivation

Single-cell proteomics technologies, such as mass cytometry, have enabled characterization of cell-to-cell variation and cell populations at a single cell resolution. These large amounts of data, however, require dedicated, interactive tools for translating the data into knowledge.

Results

We present a comprehensive, interactive method calledCytoto streamline analysis of large-scale cytometry data.Cytois a workflow-based open-source solution that automatizes the use of of state-of-the-art single-cell analysis methods with interactive visualization. We show the utility ofCytoby applying it to mass cytometry data from peripheral blood and high-grade serous ovarian cancer (HGSOC) samples. Our results show that Cyto is able to reliably capture the immune cell sub-populations from peripheral blood as well as cellular compositions of unique immune- and cancer cell subpopulations in HGSOC tumor and ascites samples.

Availability

The method is available as a Docker container athttps://hub.docker.com/r/anduril/cytoand the user guide and source code are available athttps://bitbucket.org/anduril-dev/cyto

Contact

sampsa.hautaniemi@helsinki.fi

Supplementary information

Supplementary material is available and FCS files are hosted at flowrepository.org/id/FR-FCM-Z2LW
Heterogeneous somatostatin-expressing neuron population in mouse ventral tegmental area
Authors: Nagaeva Elina , Zubarev Ivan , Gonzales Carolina Bengtsson , Forss Mikko , Nikouei Kasra , de Miguel Elena , Elsilä Lauri , Linden Anni-Maija , Hjerling-Leffler Jens , Augustine George J. , Korpi Esa R.
Published: May 2020

The cellular architecture of the ventral tegmental area (VTA), the main hub of the brain reward system, remains only partially characterized. To extend the characterization to inhibitory neurons, we have identified three distinct subtypes of somatostatin (Sst)-expressing neurons in the mouse VTA. These neurons differ in their electrophysiological and morphological properties, anatomical localization, as well as mRNA expression profiles. Importantly, similar to cortical Sst-containing interneurons, most VTA Sst neurons express GABAergic inhibitory markers, but some of them also express glutamatergic excitatory markers and a subpopulation even express dopaminergic markers. Furthermore, only some of the proposed marker genes for cortical Sst neurons were expressed in the VTA Sst neurons. Physiologically, one of the VTA Sst neuron subtypes locally inhibited neighboring dopamine neurons. Overall, our results demonstrate the remarkable complexity and heterogeneity of VTA Sst neurons and suggest that these cells are multifunctional players in the midbrain reward circuitry.
The anterior insular cortex in the rat exerts an inhibitory influence over the loss of control of heroin intake and subsequent propensity to relapse
Authors: Joshi Dhaval D. , Puaud Mickaël , Fouyssac Maxime , Belin-Rauscent Aude , Everitt Barry , Belin David
Published: May 2020

The anterior insular cortex (AIC) has been implicated in addictive behaviour, including the loss of control over drug intake, craving and the propensity to relapse. Evidence suggests that the influence of the AIC on drug-related behaviours is complex since in rats exposed to extended access to cocaine self-administration, the AIC was shown to exert a state-dependent, bidirectional influence on the development and expression of loss of control over drug intake, facilitating the latter but impairing the former. However, it is unclear whether this influence of the AIC is confined to stimulant drugs that have marked peripheral sympathomimetic and anxiogenic effects or whether it extends to other addictive drugs, such as opiates, that lack overt acute aversive peripheral effects. Thus, we investigated in outbred rats the effects of bilateral excitotoxic lesions of AIC, induced both prior to or after long-term exposure to extended access heroin self-administration, on the development and maintenance of escalated heroin intake and the subsequent vulnerability to relapse following abstinence. Compared to sham-surgeries, pre-exposure AIC lesions had no effect on the development of loss of control over heroin intake, but lesions made after a history of escalated heroin intake potentiated escalation and also enhanced responding at relapse. These data show that the AIC inhibits or limits the loss of control over heroin intake and propensity to relapse, in marked contrast to its influence on the loss of control over cocaine intake.
Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish
Authors: Buono Lorena , Naranjo Silvia , Moreno-Marmol Tania , de la Cerda Berta , Polvillo Rocío , Díaz-Corrales Francisco-Javier , Bogdanovic Ozren , Bovolenta Paola , Martínez-Morales Juan-Ramón
Published: May 2020

Sight depends on the tight cooperation between photoreceptors and pigmented cells. Both derive from common progenitors in which a single gene regulatory network (GRN) bifurcates into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics (RNA-seq) and chromatin accessibility (ATAC-seq) in segregating NR/RPE populations in zebrafish. Analysis of active cis-regulatory modules and enriched transcription factor (TF) motives suggest extensive network redundancy and context-dependent TF activity. Downstream targets identification highlights an early recruitment of desmosomal genes in the flattening RPE, revealing Tead factors as upstream regulators. Investigation of GRNs dynamics uncovers an unexpected sequence of TF recruitment during RPE specification, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counselling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.
Overlapping connectivity gradients in the anterior temporal lobe underlie semantic cognition
Authors: Faber Myrthe , Przeździk Izabela , Fernández Guillén , Haak Koen V. , Beckmann Christian F.
Published: May 2020

Convergent evidence from neuroimaging, computational, and clinical research has shown that the anterior temporal lobe (ATL) is critically involved in two key aspects of semantic cognition: the representation of semantic knowledge, and the executive regulation of this knowledge. Both are necessary for integrating features to understand concepts, and to integrate concepts to understand discourse. Here, we tested the hypothesis that these differential aspects of integration map onto different patterns of ATL connectivity. Specifically, we hypothesized that there are two overlapping modes of functional connectivity of the ATL that each predict distinct aspects of semantic cognition on an individual level. We used a novel analytical approach (connectopic mapping) to identify the first two dominant modes connection topographies (i.e. maps of spatially varying connectivity) in the ATL in 766 participants (Human Connectome Project), and summarized these into 16 parameters that reflect inter-individual differences in their functional organization. If these connection topographies reflect the ATL’s functional multiplicity, then we would expect to find a dissociation where one mode (but not the other) correlates with cross-modal matching of verbal and visual information (picture vocabulary naming), and the other (but not the former) correlates with how quickly and accurately relevant semantic information is retrieved (story comprehension). Our analysis revealed a gradient of spatially varying connectivity along the inferior-superior axis, and secondly, an anterior to posterior gradient. Multiple regression analyses revealed a double dissociation such that individual differences in the inferior-superior gradient are predictive of differences in story comprehension, whereas the anterior-posterior gradient maps onto differences in picture vocabulary naming, but not vice versa. These findings indicate that overlapping gradients of functional connectivity in the ATL are related to differential behaviors, which is important for understanding how its functional organization underlies its multiple functions.
The Atypical Fibrin Fibre Network in Rheumatoid Arthritis and its Relation to Autoimmunity, Inflammation and Thrombosis
Authors: Bezuidenhout Johannes A. , Venter Chantelle , Roberts Timothy , Tarr Gareth , Kell Douglas B. , Pretorius Etheresia
Published: May 2020

Objective

The risk of cardiovascular events in patients with RA is disproportionately heightened as a result of systemic inflammation. The relative effect of autoimmune-associated citrullination on the structure and thrombotic potential of fibrin(ogen) remains unknown. We therefore compared indices of vascular function, inflammation, coagulation and fibrin clot composition in RA patients with healthy controls and evaluated inter-parameter relationships.

Methods

Blood samples were collected from 30 RA patients and 25 age- and gender-matched healthy volunteers. Levels of SAA, CRP, ICAM-1 and VCAM-1 was measured using a sandwich immunoassay. Whole blood coagulation was assessed using Thromboelastography. Fibrin clot networks and fiber structure was investigated using Scanning Electron Microscopy. The detection and quantification of citrullination in formed fibrin clots were performed using a fluorescently labeled Citrulline monoclonal antibody with Confocal Microscopy.

Results

Concentrations of SAA, CRP and ICAM-1 were significantly elevated in RA patients compared to controls. TEG parameters relating to coagulation initiation (R and K), rate of fibrin cross-linking (α-Angle), and time to reach maximum thrombus generation (TMRTG) were attenuated in RA patients. Parameters relating to clot strength (MA, MRTG, TGG) did not statistically differ between RA and controls. Logistic regression modelling revealed stronger association between acute phase reactants (CRP, SAA) with TEG parameters than endothelial function markers. Microscopic analysis revealed denser networks of thicker fibrin fibers in RA patients compared to controls [median (interquartile range) 214 (170-285)vs120 (100-144) nm respectively, p<0.0001, Odds ratio=22.7). Detection of multiple citrullinated regions within fibrin clot structures in RA patients, which was less prevalent in control samples (p<0.05, OR=2.2).

Conclusion

Patients with active RA display a coagulation profile that is dissimilar to general findings associated with other inflammatory conditions. The alteration of protein structures by autoimmune linked citrullination could play a role in determining the structure of fibrin and the potential of conferring a heightened thrombotic risk in RA patients.
Scaffolding protein IQ motif containing GTPase activating protein 2 (IQGAP2) regulates glycogen metabolism
Authors: Sen Anushna , Youssef Sara , Wendt Karen , Anakk Sayeepriyadarshini
Published: May 2020

The liver is critical in maintaining metabolic homeostasis, regulating both anabolic and catabolic processes of fats, proteins, and carbohydrates. The IQ motif containing GTPase activating protein 2 (IQGAP2) is a member of the IQGAP family. Of the three homologous isoforms, the IQGAP2 scaffolding protein is predominantly found in the liver. To characterize its role in regulating metabolism, Iqgap2−/− female and male mice, and their WT controls, were fed ad libitum or fasted for 24 hours. Hepatic gene expression, protein levels, and the metabolic response were compared between WT and Iqgap2−/− mice, using RT-qPCR, western blot analysis, and histological stains. We found that loss of IQGAP2 alters the phosphorylation of active glycogen synthase kinase 3 (GSK3) expression, a known regulator of glycogen synthesis and lipogenesis. Consistent with this result, Iqgap2−/− female mice displayed depletion of periportal glycogen even in the fed state. We also observed the blunted expression of genes involved in glycogenesis and lipogenesis when IQGAP2 was deleted. Since GSK3 is known to regulate the activity of β-catenin, we examined and found it to be reduced in Iqgap2−/− mice. Our findings demonstrate that IQGAP2 plays an important role in regulating glycogen synthesis.
Structure-function relationship ofGossypium hirsutumNAC transcription factor, GhNAC4 with regard to ABA and abiotic stress responses
Authors: Trishla Vikas Shalibhadra , Kirti Pulugurtha Bharadwaja
Published: May 2020

Our previous study demonstrated that the expression ofGhNAC4, a NAC transcription factor from cotton, was induced by abiotic stresses and abscisic acid (ABA) treatment. In the present study, we investigated the molecular mechanisms underlying ABA and stress response of GhNAC4. Over-expression ofGhNAC4in transgenic tobacco conferred tolerance to salinity and drought treatments with associated enhanced expression of several stress-responsive marker genes. GhNAC4 is a nuclear protein that exhibits transcriptional activation property, and the C-terminal transcriptional regulatory (TR) domain is responsible for it. GhNAC4 also forms homo-dimers and the N-terminal NAC-domain is essential for this activity. The structure-function relationship of NAC transcription factors, particularly with respect to abiotic stress tolerance remains largely unclear. In this study, we investigated the domains essential for the biochemical functions of GhNAC4. We developed transgenic tobacco plants overexpressing the GhNAC4 NAC-domain and the TR-domain separately. NAC-domain transgenics showed hypersensitivity to exogenous ABA while TR-domain transgenics exhibited reduced sensitivity. Abiotic stress assays indicated that transgenic plants expressing both the domains separately were more tolerant than wild-type plants with the NAC-domain transgenics showing increased tolerance as compared to TR-domain transgenics. Expression analysis revealed that various stress-responsive genes were upregulated in both NAC-domain and TR-domain transgenics as compared to wild-type under salinity and drought treatments. These results suggest that the stress tolerance ability of GhNAC4 is associated with both the component domains while the ABA responsiveness is largely associated with N-terminal NAC-domain.

Key Message

NAC and transcriptional regulatory domains are responsible for the abiotic stress tolerance ability of the cotton NAC transcription factor GhNAC4 while the ABA-responsiveness is largely associated with the NAC-domain.
Quantifying multi-species microbial interactions in the larval zebrafish gut
Authors: Sundarraman Deepika , Hay Edouard A. , Martins Dylan M. , Shields Drew S. , Pettinari Noah L. , Parthasarathy Raghuveer
Published: May 2020

The microbial communities resident in animal intestines are composed of multiple species that together play important roles in host development, health and disease. Due to the complexity of these communities and the difficulty of characterizing them in situ, the determinants of microbial composition remain largely unknown. Further, it is unclear for many multi-species consortia whether their species-level makeup can be predicted based on an understanding of pairwise species interactions, or whether higher-order interactions are needed to explain emergent compositions. To address this, we examine commensal intestinal microbes in larval zebrafish, initially raised germ-free to allow introduction of controlled combinations of bacterial species. Using a dissection and plating assay, we demonstrate the construction of communities of one to five bacterial species and show that the outcomes from the two-species competitions fail to predict species abundances in more complex communities. With multiple species present, inter-bacterial interactions become weaker and more cooperative, suggesting that higher-order interactions in the vertebrate gut may stabilize complex communities.
Sonic Hedgehog is not a limb morphogen but acts as a trigger to specify all digits
Authors: Zhu Jianjian , Patel Rashmi , Trofka Anna , Harfe Brian D. , Mackem Susan
Published: May 2020

Limb patterning by Sonic hedgehog (Shh) has served as a paradigm of “morphogen” function, acting either by graded spatial or temporal signal integration. Yet how Shh instructs distinct digit identities remains controversial. Here, we bypassed the requirement for Shh in cell survival during limb bud outgrowth and demonstrate that a transient, early Shh pulse is both necessary and sufficient for normal limb development. Our analysis of Shh-responding cells shows signaling acts at only short-range during this time window and that Shh patterns digits indirectly, via a relay mechanism, rather than by direct spatial or temporal signal integration. Using a genetic assay for relay signaling, we unexpectedly discovered Shh also specifies digit 1 (the thumb; previously thought to be exclusively Shh-independent) indirectly, thus implicating Shh in a unique regulatory hierarchy for digit 1 evolutionary adaptations, such as opposable thumbs. This study illuminates Shh as a trigger for a relay network that becomes rapidly self-sustaining, with mechanistic relevance for limb development, regeneration, and evolution.
An atlas of robust microbiome associations with phenotypic traits based on large-scale cohorts from two continents
Authors: Rothschild Daphna , Leviatan Sigal , Hanemann Ariel , Cohen Yossi , Weissbrod Omer , Segal Eran
Published: May 2020

Numerous human conditions are associated with the microbiome, yet studies are inconsistent as to the magnitude of the associations and the bacteria involved, likely reflecting insufficiently employed sample sizes. Here, we collected diverse phenotypes and gut microbiota from 34,057 individuals from Israel and the U.S.. Analyzing these data using a much-expanded microbial genomes set, we derive an atlas of robust and numerous unreported associations between bacteria and physiological human traits, which we show to replicate in cohorts from both continents. Using machine learning models trained on microbiome data, we predict human traits with high accuracy across continents. Subsampling our cohort to smaller cohort sizes yielded highly variable models and thus sensitivity to the selected cohort, underscoring the utility of large cohorts and possibly explaining the source of discrepancies across studies. Finally, many of our prediction models saturate at these numbers of individuals, suggesting that similar analyses on larger cohorts may not further improve these predictions.
A deep intronic variant activates a pseudoexon in theMTM1gene in a family with X-linked myotubular myopathy
Authors: Fitzgerald Jamie , Feist Cori , Dietz Paula , Moore Stephen , Basel Donald
Published: May 2020

We report a novel intronic variant in theMTM1gene in four males in a family with severe X-linked myotubular myopathy. The A>G variant in deep intronic space activates a cryptic 5’ donor splice site resulting in the inclusion of a 48bp pseudoexon into the matureMTM1mRNA. The variant is present in all affected males, absent in unaffected males and heterozygous in the mother of the affected males. The included intronic sequence contains a premature stop codon and experiments using a translational inhibitor indicate that the mutant mRNAs undergo nonsense-mediate decay. We conclude that affected males produce no, or low, levels of myotubularin-1 protein leading to a severe neonatal myopathy. The study highlights the need to consider non-coding variants in genomic screening in families with X-linked myotubular myopathy.
Natural selection in the evolution of SARS-CoV-2 in bats, not humans, created a highly capable human pathogen
Authors: MacLean Oscar A. , Lytras Spyros , Weaver Steven , Singer Joshua B. , Boni Maciej F. , Lemey Philippe , Kosakovsky Pond Sergei L. , Robertson David L.
Published: May 2020

RNA viruses are proficient at switching host species, and evolving adaptations to exploit the new host’s cells efficiently. Surprisingly, SARS-CoV-2 has apparently required no significant adaptation to humans since the start of the COVID-19 pandemic, with no observed selective sweeps since genome sampling began. Here we assess the types of natural selection taking place inSarbecovirusesin horseshoe bats versus SARS-CoV-2 evolution in humans. While there is moderate evidence of diversifying positive selection in SARS-CoV-2 in humans, it is limited to the early phase of the pandemic, and purifying selection is much weaker in SARS-CoV-2 than in related batSarbecoviruses. In contrast, our analysis detects significant positive episodic diversifying selection acting on the bat virus lineage SARS-CoV-2 emerged from, accompanied by an adaptive depletion in CpG composition presumed to be linked to the action of antiviral mechanisms in ancestral hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor ∼1976), is a recombinant with a structure that includes differential CpG content in Spike; clear evidence of coinfection and evolution in bats without involvement of other species. Collectively our results demonstrate the progenitor of SARS-CoV-2 was capable of near immediate human-human transmission as a consequence of its adaptive evolutionary history in bats, not humans.
Single-molecule long-read sequencing reveals a conserved selection mechanism determining intact long RNA and miRNA profiles in sperm
Authors: Sun Yu H. , Wang Anqi , Song Chi , Srivastava Rajesh K. , Au Kin Fai , Li Xin Zhiguo
Published: May 2020

Sperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to be shaped by paternal environments and impact offspring phenotypes, we know little about long RNAs in sperm, including mRNAs and long non-coding RNAs. Here, by integrating PacBio single-molecule long reads with Illumina short reads, we found 2,778 sperm intact long transcript (SpILT) species in mouse. The SpILTs profile is evolutionarily conserved between rodents and primates. mRNAs encoding ribosomal proteins are enriched in SpILTs, and in mice they are sensitive to early trauma. Mouse and human SpILT profiles are determined by a post-transcriptional selection process during spermiogenesis, and are co-retained in sperm with base pair-complementary miRNAs. In sum, we have developed a bioinformatics pipeline to define intact transcripts, added SplLTs into the “sperm RNA code” for use in future research and potential diagnosis, and uncovered selection mechanism(s) controlling sperm RNA profiles.
Convergent evolution between PALI1 and JARID2 for the allosteric activation of PRC2
Authors: Zhang Qi , Agius Samuel C. , Flanigan Sarena F. , Levina Vitalina , Owen Brady M. , Davidovich Chen
Published: May 2020

The polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identities. JARID2 is the only accessory subunit of PRC2 that known to trigger an allosteric activation of methyltransferase. Yet, this mechanism cannot be generalised to all PRC2 variants as, in vertebrates, JARID2 is mutually exclusive with most of the accessory subunits of PRC2. Here we provide functional and structural evidence that the vertebrate-specific PRC2 accessory subunit PALI1 emerged through a convergent evolution to mimic JARID2 at the molecular level. Mechanistically, PRC2 methylates PALI1 K1241, which then binds to the PRC2-regulatory subunit EED to allosterically activate PRC2. PALI1 K1241 is methylated in mouse and human cell lines and is essential for PALI1-induced allosteric activation of PRC2. High-resolution crystal structures revealed that PALI1 mimics the regulatory interactions formed between JARID2 and EED. Independently, PALI1 also facilitates DNA and nucleosome binding by PRC2. In acute myelogenous leukemia cells, overexpression of PALI1 leads to cell differentiation, with the phenotype abrogated by a separation-of-function PALI1 mutation, defective in allosteric activation and active in DNA binding. Collectively, we show that PALI1 facilitates catalysis and substrate binding by PRC2 and provide evidence that subunit-induced allosteric activation is a general property of holo-PRC2 complexes.
Callose deposition is essential for the completion of cytokinesis in the unicellular alga,Penium margaritaceum
Authors: Davis Destiny J. , Wang Minmin , Sørensen Iben , Rose Jocelyn K.C. , Domozych David S. , Drakakaki Georgia
Published: May 2020

Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose is a β-1,3 glucan that transiently accumulates at later stages of cell plate development and is thought to stabilize the delicate membrane network of the cell plate as it expands. Cytokinetic callose deposition is currently considered specific to multicellular plant species as it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte,Penium margaritaceum. Notably, callose deposition at the division plane ofP. margaritaceumshowed distinct, spatiotemporal patterns that could represent distinct roles of this polymer in cytokinesis and cell wall assembly. Pharmacological inhibition of cytokinetic callose deposition by Endosidin 7 treatment resulted in cytokinesis defects, consistent with the essential role for this polymer inP. margaritaceumcell division. Cell wall deposition and assembly at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly inP. margaritaceum. The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis inP. margaritaceum. The evolutionary implications of cytokinetic callose in this unicellular Zygnematopycean alga is discussed in the context of the conquest of land by plants.

Summary Statement

Evolutionarily conserved callose inPenium margaritaceumis essential for the completion of cytokinesis.
Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding
Authors: Freed Nikki E. , Vlková Markéta , Faisal Muhammad B. , Silander Olin K.
Published: May 2020

Rapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cqvalues between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.
Optimized pseudotyping conditions for the SARS-COV2 Spike glycoprotein
Authors: Johnson Marc C. , Lyddon Terri D. , Suarez Reinier , Salcedo Braxton , LePique Mary , Graham Maddie , Ricana Clifton , Robinson Carolyn , Ritter Detlef G.
Published: May 2020

The SARS-COV2 Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here we characterized the use of a codon-optimized SARS-COV2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and VSV particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modification of a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titer of pseudotyped particles reached almost 106infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals.

Importance

When working with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here we describe a series of modifications that result in the production of relatively high titer SARS-COV2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.