bluuebunny/crossref_metadata_2025 huggingface dataset:
Optical Tissue Clearing Enables Rapid, Precise and Comprehensive Assessment of Three-Dimensional Morphology in Experimental Nerve Regeneration Research
Authors: Daeschler Simeon C. , Zhang Jennifer , Gordon Tessa , Borschel Gregory H.
Published: January 2021

Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections. Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain. To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats. The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 h while preserving tissue architecture and fluorescence. Optical clearing is compatible with conventional laboratory techniques, including retrograde labelling studies, and computational image segmentation, providing fast and precise cell quantitation. Further, optically cleared organs enable three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies, tracing of intramuscular nerve branches and mapping of neurovascular networks. Given their wide-ranging applicability, rapid processing times and low costs, tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies.
Amplicon sequencing of variable 16S rRNA and ITS2 regions reveal honeybee susceptibility to diseases resulted of their dietary preferences under anthropogenic landforms
Authors: Ptaszyńska Aneta A. , Latoch Przemysław , Hurd Paul J. , Polaszek Andrew , Michalska-Madej Joanna , Grochowalski Łukasz , Strapagiel Dominik , Gnat Sebastian , Załuski Daniel , Gancarz Marek , Rusinek Robert , Krutmuang Patcharin , Martín Hernández Raquel , Pascual Mariano Higes , Starosta Agata L.
Published: January 2021

EuropeanApis melliferaand AsianApis ceranahoneybees, are essential crop pollinators. Microbiome studies can provide complex information on health and fitness of these insects in relation to environmental changes, and plant availability. Amplicon sequencing of variable regions of 16S rRNA and internally transcribed spacers (ITSs) allow identification of the metabiome. These methods provide a tool for monitoring otherwise uncultured microbes isolated from the gut of the honeybees. They also help monitor the composition of the gut fungi and, intriguingly, pollens collected by the insect. Here, we present data from amplicon sequencing of the 16S rRNA and ITS2 regions from honeybees collected at various time points from anthropogenic landforms as urban areas in Poland, UK, Spain, Greece, and Thailand. We have analysed microbial composition of honeybee intestine as well as fungi and pollens. We conclude that differences between samples were mainly influenced by the bacteria, plant pollens and fungi, respectively. Moreover, honeybees feeding on a honeydew diet, mainly based on sugars, were more prone to fungal pathogens (Nosema ceranae) and neogregarines. Finally, the period when honeybees switch to the winter generation (longer-lived forager honeybees) is the most sensitive to diet perturbations and hence pathogens attack, for the whole beekeeping season. It is possible that evolutionary adaptation of bees fails to benefit them in the modern anthropomorphised environment.
TRIPLE-NEGATIVE BREAST CANCER CELLS RECRUIT NEUTROPHILS BY SECRETING TGF-β AND CXCR2 LIGANDS
Authors: SenGupta Shuvasree , Hein Lauren E. , Xu Yang , Zhang Jason , Konwerski Jamie , Li Ye , Johnson Craig , Cai Dawen , Smith Janet , Parent Carole A.
Published: January 2021

Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-β cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-β, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-β and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.
A novel allele in theArabidopsis thalianaMACPF protein CAD1 results in deregulated immune signaling
Authors: Holmes Danalyn R. , Bredow Melissa , Thor Kathrin , Pascetta Sydney A. , Sementchoukova Irina , Siegel Kristen R. , Zipfel Cyril , Monaghan Jacqueline
Published: January 2021

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of ‘non-self’) or damaged plant cells (indicative of ‘infected-self’), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show thatcad1-5mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.
Methylation of histone H3 lysine 4 is required for maintenance of beta cell function in adult mice
Authors: Vanderkruk Ben , Maeshima Nina , Pasula Daniel J. , An Meilin , McDonald Cassandra L. , Lynn Francis C. , Luciani Dan S. , Hoffman Brad G.
Published: January 2021

Pancreatic β-cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialized gene expression program which is established during development and then sustained, with limited flexibility, in terminally differentiated β-cells. Dysregulation of this program is seen in type 2 diabetes (T2D) but mechanisms that preserve gene expression or underlie its dysregulation in mature β-cells are not well resolved. Here we show that trithorax group-dependent histone H3 lysine 4 trimethylation (H3K4me3) maintains expression of genes important for insulin biosynthesis and glucose-responsiveness in β-cells. Transcriptional changes in H3K4me3-deficient β-cells lead to severe hyperglycemia in adult mice. We show that H3K4me3 deficiency leads to a less active and more repressed epigenome profile, which locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We then show that H3K4me3 is re-organized in diabeticLeprdb/dbmouse islets in favour of weakly active and disallowed genes at the expense of terminal β-cell markers with broad H3K4me3 peaks. Our results point to key roles of H3K4me3 in maintaining mature β-cell function and establishing a dysfunctional transcriptome in diabetic islets.
Streptococcus pneumoniae,S. pyogenes, andS. agalactiaemembrane phospholipid remodeling in response to human serum
Authors: Joyce Luke. R. , Guan Ziqiang , Palmer Kelli L.
Published: January 2021

Streptococcus pneumoniae,S. pyogenes(Group AStreptococcus; GAS), andS. agalactiae(Group BStreptococcus; GBS) are major etiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization mass spectrometry, we characterized the phospholipids and glycolipids ofS. pneumoniae, GAS, and GBS in routine undefined laboratory medium, streptococcal defined medium, and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported thatS. pneumoniaeutilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodeling by the major pathogenic streptococci in response to metabolites present in human serum.
Adaptations and responses of the common dandelion to low atmospheric pressure in high altitude environments
Authors: Arce Carla C. M. , Bont Zoe , Machado Ricardo A. R. , Cristaldo Paulo F. , Erb Matthias
Published: January 2021

Atmospheric pressure is an important, yet understudied factor that may shape plant ecology and evolution. By growing plants under controlled conditions at different experimental stations in the Swiss alps, we evaluated the impact of ecologically realistic atmospheric pressures between 660 and 950 hPa on the growth and defence of different dandelion populations. Low atmospheric pressure was associated with reduced root growth and defensive sesquiterpene lactone production. Defense suppression only occurred in populations originating from lower altitudes. Populations from higher altitudes constitutively produced less sesquiterpene lactones and did not suffer from suppression under low atmospheric pressure. We conclude that atmospheric pressure modulates root growth and defence traits, and that evolutionary history shapes plant phenotypic responses to atmospheric pressure. Our findings have important implications for our understanding of altitudinal gradients and the future use of plants as a source of food and bioactive metabolites in extraterrestrial habitats.
Heterologous vaccination regimens with self-amplifying RNA and Adenoviral COVID vaccines induce robust immune responses in mice
Authors: Spencer Alexandra J , McKay Paul F , Belij-Rammerstorfer Sandra , Ulaszewska Marta , Bissett Cameron D , Hu Kai , Samnuan Karnyart , Blakney Anna K. , Wright Daniel , Sharpe Hannah R , Gilbride Ciaran , Truby Adam , Allen Elizabeth R , Gilbert Sarah C , Shattock Robin J , Lambe Teresa
Published: January 2021

Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two dose heterologous vaccination regimens than single dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+CD4 T cells which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.
Novel regulators of PrPCbiosynthesis revealed by genome-wide RNA interference
Authors: Heinzer Daniel , Avar Merve , Pease Daniel Patrick , Dhingra Ashutosh , Yin Jiang-An , Schaper Elke , Doğançay Berre , Emmenegger Marc , Spinelli Anna , Maggi Kevin , Chincisan Andra , Mead Simon , Hornemann Simone , Heutink Peter , Aguzzi Adriano
Published: January 2021

The cellular prion protein PrPCis necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPCmay represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPCexpression. We cultured human U251-MG glioblastoma cells in the presence of 64’752 unique siRNAs targeting 21’584 annotated human genes, and measured PrPCusing a one-pot fluorescence-resonance energy transfer immunoassay in 51’128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPCexpression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPCbiosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPCexpression through the degradation ofPRNPmRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPClevels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.
Contribution of climate models and APSIM phenological parameters to uncertainties in spring wheat simulations: application of SUFI-2 algorithm in northeast Australia
Authors: Collins Brian , Najeeb Ullah , Luo Qunying , Tan Daniel K.Y.
Published: January 2021

We used SUFI-2 for the first time to calibrate the phenology module of the APSIM-wheat model for 10 spring wheat cultivars cultivated in northeast Australia (south-eastern Queensland). Calibration resulted in an average RMSE of 5.5 d for developmental stages from stem elongation up to flowering. Projections from 33 climate models under the representative concentration pathway 8.5 were used for simulations at 17 sites. Using adapted sowing times, we simulated significantly shorter crop cycles and grain yield improvements for the period 2036-2065 relative to 1990-2019 for three selected cultivars (Hartog, Scout and Gregory). Photoperiod and vernalisation sensitivities were shown to be the largest and smallest contributors to total uncertainties in the simulated flowering day and grain yield. Uncertainties in climate models had a relatively minor contribution to the total uncertainties in the simulated values of target traits. This contribution significantly increased when climate change impact on the target traits was estimated.
Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM
Authors: Antanasijevic Aleksandar , Sewall Leigh M. , Cottrell Christopher A. , Carnathan Diane G. , Jimenez Luis E. , Ngo Julia T. , Silverman Jennifer B. , Groschel Bettina , Georgeson Erik , Bhiman Jinal , Bastidas Raiza , LaBranche Celia , Allen Joel D. , Copps Jeffrey , Perrett Hailee R. , Rantalainen Kimmo , Cannac Fabien , Yang Yuhe R. , de la Peña Alba Torrents , Rocha Rebeca Froes , Berndsen Zachary T. , Baker David , King Neil P. , Sanders Rogier W. , Moore John P. , Crotty Shane , Crispin Max , Montefiori David C. , Burton Dennis R. , Schief William R. , Silvestri Guido , Ward Andrew B.
Published: January 2021

In Brief

Herein, we evaluated the immunogenicity of several BG505 SOSIP-based HIV Env immunogens in the rhesus macaque animal model using a combination of serology and biophysical approaches. We applied electron cryo-microscopy for high-resolution mapping of elicited polyclonal antibody responses, which provided detailed insights into the binding modes of the most common classes of antibodies elicited by BG505 SOSIP immunogens as well as the critical differences in immunogenicity that can occur as a consequence of engineered stabilizing mutations and partial glycan occupancy at different sites.

Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.

Graphical abstract
Coevolution between primates and venomous snakes revealed by α-neurotoxin susceptibility
Authors: Harris Richard J. , Nekaris K. Anne-Isola , Fry Bryan G.
Published: January 2021

Evidence suggests venomous snakes and primates have evolved certain traits in response to a coevolutionary arms-race. In both clades, evolved traits include an increase in brain size and enhanced vision. Lineage specific traits include in primates an inherent fear of snakes, while cobras have evolved defensive toxins, hooding, aposematism and venom spitting. To strengthen the claims of coevolution between venomous snakes and primates, more evidence of coevolved traits is needed to highlight the importance of this arms-race. We report a significantly reduced susceptibility of snake venom α-neurotoxins toward the α-1 nicotinic acetylcholine receptor orthosteric site within the catarrhine primates. This trait is particularly amplified within the clade Homininae. This relationship is supported by post-synaptic neurotoxic symptoms of envenoming relative to prey species being much lower humans due to weak binding of α-neurotoxins to human nicotinic receptors. Catarrhines are sympatric with many species of large, diurnal, neurotoxically venomous snakes and as such are likely to have had a long history of interaction with them. Conversely, the Lemuriformes and Platyrrhini are highly susceptible to binding of α-neurotoxins, which is consistent with them occupying geographical locations either devoid of venomous snakes or areas with neurotoxic snakes that are small, fossorial, and nocturnal. These data are consistent with the snake detection theory in that they follow a similar pattern of evolved traits within specific primate clades that are sympatric with venomous snakes. These results add new strong evidence in support of snakes and primates coevolving through arms-races that shaped selection pressures for both lineages.

Significance Statement

We have discovered a pattern of primate susceptibility towards α-neurotoxins that supports the theory of a coevolutionary arms-race between venomous snakes and primates.
Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory R region of ABCC transporters
Authors: Bickers Sarah C. , Benlekbir Samir , Rubinstein John L. , Kanelis Voula
Published: January 2021

ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is comprised of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homologue of human MRP1, determined by electron cryomicroscopy (cryo-EM). Comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a 145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggests how post-translational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.

Statement of Significance

The Ycf1p structure provides an atomic model for the TMD0 domain of ABCC transporters and for two segments of the regulatory (R) region that links NBD1 to TMD2. The orientation of TMD0 in Ycf1p differs from that seen in SUR1, the regulatory ABCC protein in KATP channels, demonstrating flexibility in TMD0/ABC core contacts. The structure suggests how post-translational modifications of the R region modulate ABC protein activity and provides a mechanistic understanding of several diseases that occur due to mutation of human homologues of Ycf1p.
The regulatory landscape of cells in the developing mouse cerebellum
Authors: Sarropoulos Ioannis , Sepp Mari , Frömel Robert , Leiss Kevin , Trost Nils , Leushkin Evgeny , Okonechnikov Konstantin , Joshi Piyush , Kutscher Lena M. , Cardoso-Moreira Margarida , Pfister Stefan M. , Kaessmann Henrik
Published: January 2021

Organ development is orchestrated by cell- and time-specific gene regulatory networks. Here we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq profiles for ~90,000 cells spanning eleven stages, we mapped all major cerebellar cell types and identified candidatecis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and characterized the regulatory programs underlying the differentiation of cerebellar neurons. Although CRE activity is predominantly cell type- and time-specific, periods of greater regulatory change are shared across cell types. There is a universal decrease in CRE conservation and pleiotropy during development and differentiation, but the degree of evolutionary constraint differs between cerebellar cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides general insights into mammalian organ development.
Natural loss of function of ephrin-B3 shapes spinal flight circuitry in birds
Authors: Haimson Baruch , Meir Oren , Sudakevitz-Merzbach Reut , Elberg Gerard , Friedrich Samantha , Lovell Peter V. , Paixão Sónia , Klein Rüdiger , Mello Claudio V. , Klar Avihu
Published: January 2021

Flight in birds evolved through patterning of the wings from forelimbs and transition from alternating gait to synchronous flapping. In mammals, the spinal midline guidance molecule ephrin-B3 instructs the wiring that enables limb alternation, and its deletion leads to synchronous hopping gait. Here we show that the ephrin-B3 protein in birds lacks several motifs present in other vertebrates, diminishing its affinity for the EphA4 receptor. The avianephrin-B3gene lacks an enhancer that drives midline expression, and is missing in Galliformes. The morphology and wiring at brachial levels of the chick spinal cord resemble those ofephrin-B3null mice. Importantly, dorsal midline decussation, evident in the mutant mouse, is apparent at the chick brachial level, and is prevented by expression of exogenousephrin-B3at the roof plate. Our findings support a role for loss of ephrin-B3 function in shaping the avian brachial spinal cord circuitry and facilitating synchronous wing flapping.

Teaser

Walking vs flying: Deciphering the organization and evolution of the neuronal network that controls wing flapping in birds.
The evolutionary making of SARS-CoV-2
Authors: Iruegas Ruben , Dosch Julian , Sikora Mateusz , Hummer Gerhard , Covino Roberto , Ebersberger Ingo
Published: January 2021

A mechanistic understanding of how SARS-CoV-2 (sarbecovirus, betacoronavirus) infects human cells is emerging, but the evolutionary trajectory that gave rise to this pathogen is poorly understood. Here we scan SARS-CoV-2 protein sequences in-silico for innovations along the evolutionary lineage starting with the last common ancestor of coronaviruses. SARS-CoV-2 substantially differs from viruses outside sarbecovirus both in its set of encoded proteins and in their domain architectures, indicating divergent functional demands. Within sarbecoviruses, sub-domain level profiling using predicted linear epitopes reveals how the primary interface between host cell and virus, the spike, was gradually reshaped. The only epitope that is private to SARS-CoV-2 overlaps with the furin cleavage site, a “switch” that modulates spike’s conformational landscape in response to host-cell interaction. This cleavage site has fundamental relevance for both immune evasion and cell infection, and the apparently ongoing evolutionary fine-tuning of its use by SARS-CoV-2 should be monitored.
Crystal structure and Hirshfeld surface analysis of 3-amino-1-oxo-2,6,8-triphenyl-1,2,7,8-tetrahydroisoquinoline-4-carbonitrile
Authors: Naghiyev Farid N. , Grishina Maria M. , Khrustalev Victor N. , Khalilov Ali N. , Akkurt Mehmet , Akobirshoeva Anzurat A. , Mamedov İbrahim G.
Published: January 2021

In the title compound, C28H21N3O, the 1,2-dihydropyridine ring of the 1,2,7,8-tetrahydroisoquinoline ring system is planar as expected, while the cyclohexa-1,3-diene ring has a twist-boat conformation, with Cremer–Pople parameters Q
T = 0.367 (2) A, θ = 117.3 (3)° and φ = 327.3 (4)°. The dihedral angles between the best planes through the isoquinoline ring system and the three phenyl rings are 81.69 (12), 82.45 (11) and 47.36 (10)°. In the crystal, molecules are linked via N—H...O and C—H...N hydrogen bonds, forming a three-dimensional network. Furthermore, the crystal packing is dominated by C—H...π bonds with a strong interaction involving the phenyl H atoms. The role of the intermolecular interactions in the crystal packing was clarified using Hirshfeld surface analysis, and two-dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H...H (46.0%), C...H/H...C (35.1%) and N...H/H...N (10.5%) contacts.
Crystal structure of 3,14-diethyl-2,6,13,17-tetraazoniatricyclo[16.4.0.07,12]docosane tetrachloride tetrahydrate from synchrotron X-ray data
Authors: Moon Dohyun , Choi Jong-Ha
Published: January 2021

The crystal structure of the hydrated title salt, C22H48N4
4+·4Cl−·4H2O (C22H48N4 = H4
L = 3,14-diethyl-2,6,13,17-tetraazoniatricyclo[16.4.0.07,12]docosane), has been determined using synchrotron radiation at 220 K. The structure determination reveals that protonation has occurred at all four amine N atoms. The asymmetric unit comprises one half of the macrocyclic cation (completed by crystallographic inversion symmetry), two chloride anions and two water molecules. The macrocyclic ring of the tetracation adopts an exodentate (3,4,3,4)-D conformation. The crystal structure is stabilized by intermolecular hydrogen bonds involving the macrocycle N—H groups and water O—H groups as donors, and the O atoms of the water molecules and chloride anions as acceptors, giving rise to a three-dimensional network.
Temperature reduces fish dispersal as larvae grow faster to their settlement size
Authors: Raventos Nuria , Torrado Héctor , Arthur Rohan , Alcoverro Teresa , Macpherson Enrique
Published: January 2021

As species struggle to cope with rising ocean temperatures, temperate marine assemblages are facing major reorganization. Many benthic species have a brief but critical period dispersing through the plankton, when they are particularly susceptible to variations in temperature. Impacts of rising temperatures can thus ripple through the population with community‐wide consequences. However, responses are highly species‐specific, making it difficult to discern assemblage‐wide patterns in the life histories of different fish species.

Here, we evaluate the responses to temperature in the early life histories of several fish species using otolith reconstructive techniques. We also assess the consequences of future warming scenarios to this assemblage.

We sampled recent settlers of nine common species across a temperature gradient in the Mediterranean Sea and obtained environmental data for each individual. Using otolith microstructure, we measured early life traits including pelagic larval duration (PLD), growth rate, settlement size, hatching and settlement dates. We used a GLM framework to examine how environmental variables influenced early life‐history parameters.

We show that increasing temperature results in considerable reduction in the dispersal potential of temperate fish. We find a nearly universal, assemblage‐wide decline in pelagic larval duration (PLD) of between 10% and 25%. This was because, with increasing temperature, larvae grew quicker to their settlement size. Settlement size itself was less affected by temperature and appears to be an ontogenetically fixed process.

Given current estimates of ocean warming, there could be an assemblage‐wide reduction in larval dispersal of up to 50 km across the Mediterranean, reducing connectivity and potentially isolating populations as waters warm.
Combining conservation status and species distribution models for planning assisted colonisation under climate change
Authors: Casazza Gabriele , Abeli Thomas , Bacchetta Gianluigi , Dagnino Davide , Fenu Giuseppe , Gargano Domenico , Minuto Luigi , Montagnani Chiara , Orsenigo Simone , Peruzzi Lorenzo , Varaldo Lucia , Rossi Graziano
Published: January 2021

Effects of climate change are particularly important in the Mediterranean Biodiversity hotspot where rising temperatures and drought are negatively affecting several plant taxa, including endemic species. Assisted colonisation (AC) represents a useful tool for reducing the effect of climate change on endemic plant species threatened by climate change.

We combined species distribution models (SDMs) for 188 taxa endemic to Italy with the IUCN red listing range loss threshold under criterion A (30%) to define: (a) the number of AC (measured as 2 × 2 km grid cells that should be occupied by new populations, i.e. grid cells = new populations) required to fully compensate for predicted range loss and to halt the decline below the 30% of range loss; (b) The number of cells necessary to compensate for range loss was calculated as the number of currently occupied cells lost under future climate due to unsuitable conditions. We used two representative concentration pathways, +2.6 and +8.5 W/m2, optimistic and pessimistic scenarios respectively. Availability of suitable areas for AC was also assessed within the current species distribution and within protected areas.

Under the optimistic scenario, no taxa would lose more than 30% of their range and AC would not be required. Under the pessimistic scenario, roughly the 90% of taxa showed a cell loss higher than 30%. Eight taxa were predicted to lose >95% of their range. For these species, AC was required from 13 to 16 new populations (=13–16 grid cells) per taxon to cap the range loss at 30%. For currently VU or EN species, an average number of 32–35 AC attempts would be necessary to fully compensate their range loss under a pessimistic scenario. Suitable recipient sites within protected areas falling in their projected range were identified, allowing for short‐distance AC.

Synthesis. Combining species distribution models and red listing thresholds under Criterion A has enabled the strategic planning of multiple species assisted colonisation minimising the effort in terms of new populations to be created and maximising the conservation benefit in terms of range loss compensation.
A comparison on the detection accuracy of ante mortem computed tomography vs. autopsy for the diagnosis of pelvic ring injury in legal medicine
Authors: Höch Andreas , Özkurtul Orkun , Hammer Niels , Heinemann Axel , Tse Rexson , Zwirner Johann , Henkelmann Jeanette , Fakler Johannes , Ondruschka Benjamin
Published: January 2021

A detailed knowledge on the exact morphology of pelvic injuries provided crucial information in understanding the mechanisms of injury and has influence on the natural course and subsequent mortality. However, forensic medical literature investigating pelvic fractures in detail is scarce to date. This case series aims to compare the accuracy in detecting pelvic injuries using autopsy and ante mortem computed tomography (CT). Nineteen deceased patients with CT scans of pelvic fractures were included retrospectively. Pelvic injuries were independently assessed by a board‐certified radiologist (R) and a board‐certified trauma surgeon (T), both using the ante mortem CT scans, and by a board‐certified forensic pathologist using autopsy (A) results without knowledge of the CT scan findings. No patient had died causatively from a pelvic fracture. Most injuries of the pelvis were present in the pubic rami (16/18) and sacral bone (13/18), followed by the sacroiliac joint (9/18) and iliac bone fractures (8/18). Ilium fractures (A:100%;R:67%;T:67%) and injuries of the sacroiliac joint (A:83%;R:50%;T:42%) were best detected via autopsy. The diagnosis of sacral fractures (A:19%;R:94%;T:88%) and fractures of the pubic rami (A:67%;R:96%;T:96%) were most often missed in autopsy. The results show deficits in the assessment of the pelvic injury for both CT and autopsy. Autopsy was superior in detecting injuries of the sacroiliac joint, but inferior in detecting sacral and pubic bone fractures. For an encompassing evaluation of ligamento‐skeletal pelvic injuries, the complementary use of both CT and autopsy is recommended.
Predicting a target exercise heart rate that reflects the anaerobic threshold in nonbeta‐blocked hemodialysis patients: The Karvonen and heart rate reserve formulas
Authors: Yabe Hiroki , Kono Kenichi , Onoyama Ayaka , Kiyota Akiho , Moriyama Yoshifumi , Okada Keiko , Kasuga Hirotake
Published: January 2021

The aim of this study was to evaluate constants of the Karvonen (k) and heart rate reserve (HRR) (α) formulas that correspond to the anaerobic threshold (AT) to conveniently estimate the intensity of exercise therapy in nonbeta‐blocked patients undergoing hemodialysis. Twenty‐three patients undergoing hemodialysis performed cardiopulmonary exercise testing (CPX) and their HR at AT was measured. The predictor coefficients for a target HR corresponding to AT were calculated for each patient based on CPX. Interclass correlation coefficients (ICC) and Bland–Altman analysis were used to evaluate the reliability of the formulas. Mean values of coefficient k of the Karvonen formula and α of the HRR formula were 0.24 ± 0.11 and 17.4 ± 8, respectively. The target HR calculated with k = 0.24 and α = 17 had significant ICC between HR at AT (0.74 and 0.77, respectively; P < 0.05). Using the Karvonen and HRR formulas to determine a target HR corresponding to AT is a simple and easy method that can be used to develop exercise programs for hemodialysis patients.
Removing the bad apples: A simple bioinformatic method to improve loci‐recovery in de novo RADseq data for non‐model organisms
Authors: Cerca José , Maurstad Marius F. , Rochette Nicolas C. , Rivera‐Colón Angel G. , Rayamajhi Niraj , Catchen Julian M. , Struck Torsten H.
Published: January 2021

The restriction site‐associated DNA (RADseq) family of protocols involves digesting DNA and sequencing the region flanking the cut site, thus providing a cost and time‐efficient way for obtaining thousands of genomic markers. However, when working with non‐model taxa with few genomic resources, optimization of RADseq wet‐lab and bioinformatic tools may be challenging, often resulting in allele dropout—that is when a given RADseq locus is not sequenced in one or more individuals resulting in missing data. Additionally, as datasets include divergent taxa, rates of dropout will increase since restriction sites may be lost due to mutation. Mitigating the impacts of allele dropout is crucial, as missing data may lead to incorrect inferences in population genetics and phylogenetics.

Here, we demonstrate a simple pipeline for the optimization of RADseq datasets which involves partitioning datasets into subgroups, namely by reducing and analysing the dataset at a population or species level. By running the software Stacks at a subgroup level, we were able to reliably identify and remove individuals with high levels of missing data (bad apples) likely stemming from artefacts in library preparation, DNA quality or sequencing artefacts.

Removal of the bad apples generally led to an increase in loci and decrease in missing data in the final datasets.

The biological interpretability of the data, as measured by the number of retrieved loci and missing data, was considerably increased.
From small forest samples to generalised uni‐ and bimodal stand descriptions
Authors: Mey Reinhard , Stadelmann Golo , Thürig Esther , Bugmann Harald , Zell Jürgen
Published: January 2021

Forests provide multiple services, and in the face of global change adaptive management strategies are needed, which inevitably must be based on models. However, most locally accurate forest models are tied to the stand scale and cannot readily be applied across large areas. Empirical data for model initialisation are often not available at large spatial scales. National Forest Inventories (NFIs) provide spatially representative tree and stand samples, but their samples are typically small, that is, only a few trees are measured per plot, and they are truncated, that is, not each tree has the same probability of being observed. To overcome these issues, we develop and apply a methodology to derive stand descriptions from small sample data, taking the Swiss NFI as a case study.

We extended the traditional Weibull function to (multi‐)truncated unimodal and bimodal forms that are suitable for the representation of samples from survey designs with multiple callipering thresholds. Subsequently, we applied these functions in an extended parameter prediction method to derive stand diameter distributions from representative samples. Additionally, we predicted species compositions using a multinomial logistic regression model and assigned them to the diameter distributions of the stands.

The diameter distribution of 9.1% of the Swiss NFI samples was better described by a bimodal than a unimodal Weibull function. The uni‐ and bimodal diameter model in combination with the model to determine species composition can be used to predict stand descriptions from single small samples or entire forest types in the target area. Thereby, the bimodal form is suitable for capturing stand structures with distinct under‐ and overstorey. In Switzerland, the diameter distributions of stands are typically positively skewed.

Our method can be applied to any large‐scale dataset (e.g. NFI) and allows to generate initial conditions in terms of spatially representative stands. These, in turn, are suitable for forest stand simulators, which allows for developing adaptive forest management strategies at large scales, by simulating realistic and site‐specific stand development while still reflecting detailed management measures. Furthermore, stand descriptions can be used to assess tree species diversity, regeneration and harvest potentials.
Multilocus sequence typing and phylogenetic analysis revealed two distinct almond witches'‐broom phytoplasma subpopulations in Iran
Authors: Mosayyebi Niloofar , Mehraban Zahra , Siampour Majid , Babaei Ghobad , Quaglino Fabio
Published: January 2021

During a field survey conducted in different geographical regions of Iran, phytoplasma‐like symptoms were observed in peach, almond, wild almond and GF‐677 rootstock. Based on 16S rDNA amplification, followed by nucleotide sequence and phylogenetic analyses, almond witches'‐broom (AlmWB) phytoplasma strains were found in association with symptomatic plants. Based on the single‐nucleotide polymorphisms within 16S rDNA sequence, most of the Iranian AlmWB phytoplasma strains were distinguished from the Lebanese strains. Further molecular typing, based on sequence and phylogenetic analyses of rplV‐rpsC (ribosomal proteins) and imp (immunodominant membrane protein) genes, indicated that AlmWB phytoplasma strains can be differentiated in two subpopulations: Ir, including exclusively Iranian strains; Lb, comprising strains from Lebanon and Iran. Moreover, the selection pressure analysis of imp gene sequences suggested that these two AlmWB phytoplasma subpopulations had some distinct biological or ecological properties. Finally, results of the sequence and phylogenetic analyses performed on rplV‐rpsC and imp genes showed a considerably high genetic distance between AlmWB phytoplasma strains (16SrIX‐B and variants) and phytoplasmas belonging to other 16SrIX subgroups. The significance of genetic variation in phytoplasmas of 16SrIX group is discussed in relation to their biological characteristics and geographical distinction.
Resilience to Hazardous Drinking Among Never‐Deployed Male United States Army Reserve and National Guard Soldiers
Authors: Hoopsick Rachel A. , Homish D. Lynn , Vest Bonnie M. , Bartone Paul T. , Homish Gregory G.
Published: January 2021

Background

Negative emotions related to never having been deployed to active duty are associated with an increased risk of hazardous drinking among United States Army Reserve/National Guard (USAR/NG) soldiers. Resiliency factors are known to buffer the effects of combat on hazardous drinking among service members who have been deployed, but it is not known whether these factors are protective for never‐deployed service members, or which domains of hazardous drinking might be affected. Therefore, we examined the effects of a range of resiliency factors (i.e., marital satisfaction, psychological hardiness, intrinsic religiosity) on the relation between nondeployment emotions (NDE) and domains of hazardous drinking.

Methods

We drew a subset of data from Operation: Soldiers and Families Excelling Through the Years (N = 112 never‐deployed male soldiers), an ongoing study of USAR/NG soldiers. Regression models examined the main effects of NDE on each of the domains of hazardous drinking (i.e., total Alcohol Use Disorders Identification Test [AUDIT] score, consumption subscale, dependence subscale, alcohol‐related problems subscale) and effect modification of each of the resiliency factors on the relations between NDE and the domains of hazardous drinking, separately. Final models controlled for years of military service, rank (enlisted vs. officer), number of military friends in the social network, and depression.

Results

Greater NDE were associated with a higher total AUDIT score, alcohol consumption, and alcohol dependence (ps < 0.05), but not alcohol‐related problems (p > 0.05). Marital satisfaction and psychological hardiness buffered the effects of NDE on total AUDIT score and alcohol dependence (p < 0.05). Intrinsic religiosity only modified the effect of NDE on total AUDIT score. None of the resiliency factors modified the effects of NDE on alcohol consumption or alcohol‐related problems.

Conclusions

Soldiers with greater NDE had a greater risk of hazardous drinking in the presence of low resilience. Interventions to promote resiliency are an important consideration for protecting USAR/NG soldiers from hazardous drinking, regardless of their deployment history.
Repeated exposure to cocaine during adolescence enhances the rewarding threshold for cocaine‐conditioned place preference in adulthood
Authors: Caffino Lucia , Moro Federico , Mottarlini Francesca , Targa Giorgia , Di Clemente Angelo , Toia Marianna , Orrù Alessandro , Giannotti Giuseppe , Fumagalli Fabio , Cervo Luigi
Published: January 2021

Previous studies have shown that adolescent exposure to cocaine increases drug use in adulthood, albeit incubation of cocaine seeking was found to be attenuated in rats trained to self‐administer cocaine during adolescence. We here hypothesize that adolescent exposure to cocaine could alter the rewarding properties of the psychostimulant in adulthood. By employing two of the most widely used animal‐experimental‐preclinical models to investigate drug addiction, we evaluated whether contingent versus non‐contingent cocaine self‐administration during adolescence modulates its rewarding threshold in adulthood evaluated by conditioned place preference (CPP). Cocaine self‐administration during adolescence increases the rewarding threshold in adulthood; CPP for cocaine was observed at the higher (20 mg/kg), but not at the lower (10 mg/kg), dose employed. Rats exposed to either contingent or non‐contingent cocaine during adolescence exhibited the same behavior in the CPP paradigm suggesting that, under our experimental conditions, cocaine rewarding properties are shaped by the psychostimulant itself and not by its motivational effects. From a mechanistic standpoint, the preference for the 20 mg/kg cocaine‐paired side in a CPP paradigm appears to depend, at least partially, upon the formation of GluA2‐lacking Ca2+‐permeable AMPA receptors and the consequent increase of αCaMKII activity in the NAc, both of which are instead reduced when the 10 mg/kg dose was used. In conclusion, contingent or non‐contingent cocaine exposure during adolescence desensitizes adult animals to a rewarding dose of cocaine (10 mg/kg) elevating the rewarding threshold necessary (20 mg/kg) to drive conditioned place preference, an effect that may predispose to higher consumption of cocaine during adulthood.