bluuebunny/crossref_metadata_2025 huggingface dataset:
Decoding gene regulation in the fly brain
Authors: Janssens Jasper , Aibar Sara , Taskiran Ibrahim Ihsan , Ismail Joy N. , Spanier Katina I. , Bravo González-Blas Carmen , Quan Xiao Jiang , Papasokrati Dafni , Hulselmans Gert , Makhzami Samira , Waegeneer Maxime De , Christiaens Valerie , Aerts Stein
Published: August 2021

TheDrosophilabrain is a work horse in neuroscience. Single-cell transcriptome analysis1–5,3D morphological classification6, and detailed EM mapping of the connectome7–10have revealed an immense diversity of neuronal and glial cell types that underlie the wide array of functional and behavioral traits in the fruit fly. The identities of these cell types are controlled by – still unknown – gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. To characterize the GRN for each cell type in theDrosophilabrain, we profiled chromatin accessibility of 240,919 single cells spanning nine developmental timepoints, and integrated this data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which around 70,000 are linked to specific developmental trajectories, involving neurogenesis, reprogramming and maturation. For 40 cell types, their uniquely accessible regions could be associated with their expressed transcription factors and downstream target genes, through a combination of motif discovery, network inference techniques, and deep learning. We illustrate how these “enhancer-GRNs” can be used to reveal enhancer architectures leading to a better understanding of neuronal regulatory diversity. Finally, our atlas of regulatory elements can be used to design genetic driver lines for specific cell types at specific timepoints, facilitating the characterization of brain cell types and the manipulation of brain function.
Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe
Authors: Varberg Joseph M. , Unruh Jay R. , Bestul Andrew J. , Khan Azqa A. , Jaspersen Sue L.
Published: August 2021

The number, distribution and composition of nuclear pore complexes (NPCs) in the nuclear envelope (NE) varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization is controlled, we analyzed NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion, which is important for timely mitotic progression. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the NE influences local NPC distribution.
Identification and characterization of distinct cell cycle stages in cardiomyocytes using the FUCCI transgenic system
Authors: Baniol Marion , Murganti Francesca , Smialowska Agata , Panula Joni , Lazar Enikö , Brockman Viveka , Giatrellis Sarantis , Derks Wouter , Bergmann Olaf
Published: August 2021

Understanding the regulatory mechanism by which cardiomyocyte proliferation transitions to endoreplication and cell cycle arrest during the neonatal period is crucial for identifying proproliferative factors and developing regenerative therapies.

We used a transgenic mouse model based on the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to isolate and characterize cycling cardiomyocytes at different cell cycle stages at a single-cell resolution. Single-cell transcriptome analysis of cycling and noncycling cardiomyocytes was performed at postnatal days 0 (P0) and 7 (P7).

The FUCCI system proved to be efficient for the identification of cycling cardiomyocytes with the highest mitotic activity at birth, followed by a gradual decline in the number of cycling and mitotic cardiomyocytes during the neonatal period. Cardiomyocytes showed premature cell cycle exit at G1/S shortly after birth and delayed G1/S progression during endoreplication at P7. Single-cell RNA-seq confirmed previously described signaling pathways involved in cardiomyocyte proliferation (Erbb2 and Hippo/YAP), cardiomyocyte motility, and maturation-related transcriptional changes during postnatal development, including the metabolic switch from glycolysis to fatty acid oxidation in cardiomyocytes. Additionally, we generated transcriptional profiles specific to cell division and endoreplication in cardiomyocytes.

Deciphering transcriptional changes at different developmental stages and in a cell cycle-specific manner may facilitate the identification of genes important for adult cardiomyocyte proliferation and heart regeneration.

Main findings

FUCCI reliably identifies cycling cardiomyocytes at distinct cell cycle stages in neonatal, juvenile, and adult hearts.

Cell cycle activity decreases as the metabolic switch transitions from glycolysis to fatty acid metabolism in postnatal cardiomyocytes.

Cell cycle arrest at G1/S is linked to the DNA damage response in postnatal cardiomyocytes. Distinct gene expression patterns are linked to different cell cycle phases in dividing and endoreplicating postnatal cardiomyocytes.
Early neutrophilia marked by aerobic glycolysis sustains host metabolism and delays cancer cachexia
Authors: Petruzzelli Michele , Ferrer Miriam , Schuijs Martijn , Hall Zoe , Perera David , Raghunathan Shwethaa , Vacca Michele , Gaude Edoardo , Lukey Michael J. , Jodrell Duncan I. , Frezza Christian , Wagner Erwin F. , Janowitz Tobias , Halim Timotheus Y.F. , Venkitaraman Ashok R.
Published: August 2021

An elevated neutrophil-to-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, we show using murine model systems of colorectal and pancreatic cancer that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, alike to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.
Altered sleep intensity upon DBS to hypothalamic sleep-wake centers in rats
Authors: Masneuf S. , Imbach L.L. , Büchele F. , Colacicco G. , Penner M. , Moreira C.G. , Ineichen C. , Jahanshahi A. , Temel Y. , Baumann C.R. , Noain D.
Published: August 2021

Deep brain stimulation (DBS) has been scarcely investigated in the field of sleep research. We hypothesize that DBS onto hypothalamic sleep- and wake-promoting centers will produce significant neuromodulatory effects, and potentially become a therapeutic strategy for patients suffering severe, drug-refractory sleep-wake disturbances. We aimed to investigate whether continuous electrical high-frequency DBS, such as that often implemented in clinical practice, in the ventrolateral preoptic nucleus (VLPO) or the perifornical area of the posterior lateral hypothalamus (PeFLH), significantly modulates sleep-wake characteristics and behavior. We implanted healthy rats with electroencephalographic/electromyographic electrodes and recorded vigilance states in parallel to bilateral bipolar stimulation of VLPO and PeFLH at 125 Hz at 90 μA over 24 h to test the modulating effects of DBS on sleep-wake proportions, stability and spectral power in relation to baseline. We unexpectedly found that VLPO DBS at 125 Hz deepens slow-wave sleep as measured by increased delta power, while sleep proportions and fragmentation remain unaffected. Thus, the intensity, but not the amount of sleep or its stability, is modulated. Similarly, the proportion and stability of vigilance states remained altogether unaltered upon PeFLH DBS but, in contrast to VLPO, 125 Hz stimulation unexpectedly weakened SWS, evidenced by reduced delta power. This study provides novel insights into non-acute functional outputs of major sleep-wake centers in the rat brain in response to electrical high-frequency stimulation, a paradigm frequently used in human DBS. In the conditions assayed, while exerting no major effects on sleep-wake architecture, hypothalamic high-frequency stimulation arises as a provocative sleep intensity-modulating approach.
The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria
Authors: Wang Lawrence T. , Hurlburt Nicholas K. , Schön Arne , Flynn Barbara J. , Pereira Lais S. , Dillon Marlon , Flores-Garcia Yevel , Bonilla Brian , Zavala Fidel , Idris Azza H. , Francica Joseph R. , Pancera Marie , Seder Robert A.
Published: August 2021

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of thePlasmodium falciparumcircumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb’s ontogeny and mechanisms of binding PfCSP to neutralize sporozoites will facilitate vaccine development. Here, we isolated mAbs clonally related to L9 and showed that this B-cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally-related heavy chains resulted in chimeric mAbs that cross-linked two NVDP, cross-reacted with NANP, and more potently neutralized sporozoites compared to their original light chain. Structural analyses revealed that chimeric mAbs bound the minor repeat motif in a type-1 β-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize SPZ and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDP.
Analysis of combinatorial chemokine receptor expression dynamics using multi-receptor reporter mice
Authors: Medina-Ruiz Laura , Bartolini Robin , Dyer Douglas P , Vidler Francesca , Hughes Catherine E , Schuette Fabian , Love Samantha , Fu Jun , Stewart A. Francis , Graham Gerard J
Published: August 2021

Inflammatory chemokines and their receptors are central to the development of inflammatory/immune pathologies. The apparent complexity of this system, coupled with lack of appropriate in vivo models, has limited our understanding of how chemokines orchestrate inflammatory responses and has hampered attempts at targeting this system in inflammatory disease. Novel approaches are therefore needed to provide crucial biological, and therapeutic, insights into the chemokine-chemokine receptor family. Here, we report the generation of transgenic multi-chemokine receptor reporter mice in which spectrally-distinct fluorescent reporters mark expression of CCRs 1, 2, 3 and 5, key receptors for myeloid cell recruitment in inflammation. Analysis of these animals has allowed us to define, for the first time, individual and combinatorial receptor expression patterns on myeloid cells in resting and inflamed conditions. Our results demonstrate that chemokine receptor expression is highly specific, and more selective than previously anticipated.
Dynamic Spatial-Temporal Expression Ratio of X Chromosome to Autosomes but Stable Dosage Compensation in Mammals
Authors: Qian Sheng Hu , Xiong Yu-Li , Chen Lu , Geng Ying-Jie , Tang Xiao-Man , Chen Zhen-Xia
Published: August 2021

In the evolutionary model of dosage compensation, per-allele expression level of the X chromosome was proposed to have two-fold upregulation, compensating for its dose reduction in males (XY) compared to females (XX). However, the upregulation of X chromosome is still in dispute, and comprehensive evaluations are still lacking. By integrating multi-omics datasets in mammals, we investigated the expression ratios and underlying pattern of X to autosomes (X:AA ratio) and X to orthologs (X:XXratio) at the transcriptome, translatome, and proteome layers. The results indicated a dynamic spatial-temporal X:AA ratio during development in human and mouse. Meanwhile, by tracing the evolution of orthologous gene expressions in chicken, platypus, and opossum, we found a constant expression ratio between X-linked genes in human and their autosomal orthologs in other species (X:XX~1) across tissues and developmental stages, demonstrating stable dosage compensation in mammals. We also revealed that different epigenetic regulations could shape the higher tissue- and stage-specificity of X-linked gene expression, and affect X:AA ratios. We conclude that the dynamics of X:AA ratios are attributed to the different gene contents and expression preferences of the X chromosome, instead of the stable dosage compensation.
Essential Loop Dynamics Modulates Catalytic Activity in α-Chymotrypsin
Authors: Biswas Pritam , Pal Uttam , Adhikari Aniruddha , Mondal Susmita , Ghosh Ria , Mukherjee Dipanjan , Saha-Dasgupta Tanusri , Choudhury Sudeshna Shyam , Das Ranjan , Pal Samir Kumar
Published: August 2021

Conformational dynamics of macromolecules including enzymes are essential for their function. The present work reports the role of essential dynamics in alpha-chymotrypsin (CHT) which correlates with its catalytic activity. Detailed optical spectroscopy and classical molecular dynamics (MD) simulation were used to study thermal stability, catalytic activity and dynamical flexibility of the enzyme. The study of the enzyme kinetics reveals an optimum catalytic efficiency at 308K. Polarization gated fluorescence anisotropy with 8-anilino-1-napthelene sulfonate (ANS) have indicated increasing flexibility of the enzyme with an increase in temperature. Examination of the structure of CHT reveal the presence of five loop regions (LRs) around the catalytic S1 pocket. MD simulations have indicated that flexibility increases concurrently with temperature which decreases beyond optimum temperature. Principal component analysis (PCA) of the eigenvectors manifests essential dynamics and gatekeeping role of the five LRs surrounding the catalytic pocket which controls the enzyme activity.
Sub-3 Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly
Authors: Liu Di , Thélot François A. , Piccirilli Joseph A. , Liao Maofu , Yin Peng
Published: August 2021

Many functional RNAs fold into intricate and precise 3D architectures, and high-resolution structures are required to understand their underlying mechanistic principles. However, RNA structural determination is difficult. Herein, we present a nanoarchitectural strategy to enable the efficient single-particle cryogenic electron microscopy (cryo-EM) analysis of RNA-only structures. This strategy, termedRNAoligomerization-enabledcryo-EM via installingkissing-loops (ROCK), involves the engineering of target RNAs by installing kissing-loop sequences onto functionally nonessential stems for the assembly into closed homomeric nanoarchitectures. Assembly with geometric restraints leads to (1) molecular weight multiplication and (2) structural flexibility mitigation, both beneficial for cryo-EM analysis. Together with construct optimization and symmetry-expansion reconstruction, ROCK yields the cryo-EM reconstruction of theTetrahymenagroup I intron at an overall resolution of 2.98 Å (2.85 Å resolution for the core domains), enabling the de novo model building of the complete intron RNA including previously unknown peripheral domains. When applied to smaller RNAs, ROCK readily produces modest-resolution maps, revealing the conformational rearrangement of theAzoarcusgroup I intron and the bound ligand in the FMN riboswitch. Our work unleashes the largely unexplored potential of cryo-EM in RNA structural studies.
Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization
Authors: Acheampong Kofi K. , Schaff Dylan L. , Emert Benjamin L. , Lake Jonathan , Reffsin Sam , Shea Emily K. , Comar Courtney E. , Litzky Leslie A. , Khurram Nigar A. , Linn Rebecca L. , Feldman Michael , Weiss Susan R. , Montone Kathleen T. , Cherry Sara , Shaffer Sydney M.
Published: August 2021

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.
SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host
Authors: Castelli Matteo , Scietti Luigi , Clementi Nicola , Cavallaro Mattia , Faravelli Silvia , Pinnola Alberta , Criscuolo Elena , Diotti Roberta Antonia , Clementi Massimo , Forneris Federico , Mancini Nicasio
Published: August 2021

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 – the closest bat relative – spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.
Role of YAP in early ectodermal specification and a Huntington’s Disease model of human neurulation
Authors: Piccolo Francesco M. , Kastan Nathaniel R. , Haremaki Tomomi , Tian Qingyun , Laundos Tiago L. , De Santis Riccardo , Carroll Thomas S. , Luo Ji-Dung , Gnedeva Ksenia , Etoc Fred , Hudspeth A. J. , Brivanlou Ali H.
Published: August 2021

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAP’s potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and non-neuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntington’s Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAP’s transcriptional activity can partially rescue the HD phenotype.
Genome scale CRISPR Cas9a knockout screen reveals genes that controls glioblastoma susceptibility to the alkylating agent temozolomide
Authors: Awah Chidiebere U , Winter Jan , Ogunwobi Olorunseun O.
Published: August 2021

Glioblastoma is the most fatal of all primary human brain tumors with 14 months survival, at best. The mainstay therapy for this tumor involves temozolomide, surgery, radiotherapy and tumor treating electric field. Cancer resistance to commonly available chemotherapeutics remains a major challenge in glioblastoma patients receiving treatment and unfavorably impact their overall survival and outcome. However, the lack of progress in this area could be attributed to lack of tools to probe unbiasedly at the genome wide level the coding and non-coding elements contribution on a large scale for factors that control resistance to chemotherapeutics. Understanding the mechanisms of resistance to chemotherapeutics will enable precision medicine in the treatment of cancer patients.

CRISPR Cas9a has emerged as a functional genomics tool to study at genome level the factors that control cancer resistance to drugs. Recently, we used genome wide CRISPR-Cas9a screen to identify genes responsible for glioblastoma susceptibility to etoposide. We extended our inquiry to understand genes that control glioblastoma response to temozolomide by using genome scale CRISPR. This study shows that the unbiased genome-wide loss of function approach can be applied to discover genes that influence tumor resistance to chemotherapeutics and contribute to chemoresistance in glioblastoma.
Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance in sensory neurons
Authors: Smith Patrick R. , Loerch Sarah , Kunder Nikesh , Stanowick Alexander D. , Lou Tzu-Fang , Campbell Zachary T.
Published: August 2021

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies specifically in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splittingin vitro. Collectively, the data suggest that eEF2 phosphorylation defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.
ELICE16INDURES®: a plant immune-priming activator targeting jasmonate metabolism via TIFY and RHOMBOID proteins inHordeum vulgare
Authors: Hegedűs Géza , Kiniczky Márta , Nagy Ágnes , Pekker Péter , Lang Balázs , Gracza Lajos , Pallos József Péter , Thomas-Nyári Zsófia , Decsi Kincső , Kutasy Barbara , Székvári Kinga , Juhász Ákos , Virág Eszter
Published: August 2021

Priming activity of plant-based allelochemicals is advanced research nowadays meaning a high potential in sustainable agriculture. The ELICE16INDURES®(RIMPH LTD, Hungary) plant conditioner of CO2botanical extracts is rich in plant-active ingredients such as phenolic compounds, alkaloids, and flavonoids formulated in small multilamellar liposomes. This product was investigatedin autumn barley (Hordeum vulgare). Field experiments of ELICE16INDURES showed augmented NDVI values interconnected with higher photosynthetic activity and yield increase. Background of the better vitality of plants was investigated by whole genomic gene expression profiling and showed an enhanced response to wounding, jasmonic acid, oxidative detoxification, and chloroplast activity. Among top50 differentially expressed genes the TIFY domain protein TIFY11B and RHOMBOID-like protein 2 related to JA signaling were up-regulated in field-collected samples. Phytotron experiments of barley were set up to validate and evaluate the transcriptomic effect of ELICE16INDURES. Well-studied priming active agents such as salicylic acid and beta-aminobutyric acid were compared with ELICE16INDURES and confirmed as priming inducer material with positive regulation of TIFY11B, TIFY3B, TIFY9, TIF10A, and RHOMBOID like protein 2 by using NGS GEx and RT-qPCR methods.

One-sentence summary

ELICE16INDURES®is a plant conditioner agent with a high amount of allelochemicals encapsulated into small multilamellar liposomes and found as an immune priming activator tested inH. vulgarefield and phytotron cultures.
Data and Diversity Driven Development of a Shotgun Crystallisation Screen using the Protein Data Bank
Authors: Abrahams Gabriel , Newman Janet
Published: August 2021

Protein crystallisation has for decades been a critical and restrictive step in macro-molecular structure determination via X-ray diffraction. Crystallisation typically involves a multi-stage exploration of the available chemical space, beginning with an initial sampling (screening) followed by iterative refinement (optimisation). Effective screening is important for reducing the number of optimisation rounds required, reducing the cost and time required to determine a structure. Here, we propose an initial screen (Shotgun II) derived from analysis of the up-to-date Protein Data Bank (PDB) and compare it with the previously derived (2014) Shotgun I screen. In an update to that analysis, we clarify that the Shotgun approach entails finding the crystallisation conditions which cover the most diverse space of proteins by sequence found in the PDB - which can be mapped to the well known Maximum Coverage problem in computer science. With this realisation we are able to apply a more effective algorithm for selecting conditions, such that the Shotgun II screen outperforms the Shotgun I screen both in protein coverage and quantity of data input. Our data demonstrates that the Shotgun I screen, compared with alternatives, has been remarkably successful over the seven years it has been in use, indicating that Shotgun II is likely to be a highly effective screen.
Androgen synthesis inhibition but not gonadectomy reduces persistence during strategy set-shifting and reversal learning in male rats
Authors: Tomm Ryan J. , Seib Désirée R. , Kachkovski George V. , Schweitzer Helen R. , Tobiansky Daniel J. , Floresco Stan B. , Soma Kiran K.
Published: August 2021

Androgens regulate behavioural flexibility, which is essential to adapt to a changing environment and depends on the medial prefrontal cortex (mPFC). Testosterone (T) administration decreases behavioural flexibility. It is well known that T is produced in the gonads, but T is also produced in the mesocorticolimbic system, which modulates behavioural flexibility. It is unclear how T produced in the brain versus the gonads influences behavioural flexibility. Here, we assess the effects of the androgen synthesis inhibitor abiraterone acetate (ABI) and long-term gonadectomy (GDX) on behavioural flexibility in two paradigms. In Experiment 1, ABI independent of GDX reduced the number of trials to criterion and perseverative errors in a strategy set-shifting task. Similarly, in Experiment 2, ABI but not GDX reduced perseverative errors in a reversal learning task. In subjects from Experiment 1, we also examined tyrosine hydroxylase immunoreactivity (TH-ir), and ABI but not GDX increased TH-ir in the mPFC. Our findings suggest that neurally-produced androgens modulate behavioural flexibility via modification of dopamine signalling in the mesocorticolimbic system. These results suggest novel roles for neurosteroids and possible side effects of ABI treatment for prostate cancer.
MRG proteins are shared by multiple protein complexes with distinct functions
Authors: Devoucoux Maëva , Roques Céline , Lachance Catherine , Lashgari Anahita , Joly-Beauparlant Charles , Jacquet Karine , Alerasool Nader , Prudente Alexandre , Taipale Mikko , Droit Arnaud , Lambert Jean-Philippe , Hussein Samer M.I. , Côté Jacques
Published: August 2021

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain recognizing H3K36me3 in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing and DNA repair by homologous recombination. In order to get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the chromodomain, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase and PALB2/BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR-tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that can specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding chromodomain of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes which is essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.

Highlights

MRG15 and MRGX are stably associated with several different protein complexes important for genome expression and stability.

Several MRG-containing complexes are chromatin modifiers.

Specific point mutations in the MRG domain differentially affect associated complexes.

A major human complex homologous to the yeast TINTIN complex is identified.

The protein EP400NL competes with EP400 to functionally separate TINTIN from the NuA4/TIP60 complex.

TINTIN contains a bromodomain and a chromodomain to regulate transcription.
In vitro determination of inhibitory effects by humic substances complexing Zn and Se on SARS-CoV-2 virus replication
Authors: Hajdrik Polett , Pályi Bernadett , Kis Zoltán , Kovács Noémi , Veres Daniel S. , Szigeti Krisztián , Hegedűs Imre , Kovács Tibor , Bergmann Ralf , Máthé Domokos
Published: August 2021

Humic substances are well known human nutritional supplement materials and play important performance-enhancing roles as animal feed additives, too. For decades, ingredients of humic substances have also been proven to carry potent antiviral effects against different viruses. Here, the antiviral activity of a humic substance containing ascorbic acid, Se- and Zn2+ ions intended as a nutritional supplement material was investigated against SARS-CoV-2 virus B1.1.7 Variant of Concern (“Alpha Variant”) in a VeroE6 cell line. Results show that this combination has a significant in vitro antiviral effect at a very low concentration range of its intended active ingredients. Even picomolar concentration ranges of humic substances, vitamin C and Zn/Se ions in the given composition were enough to achieve fifty percent viral replication inhibition in the applied SARS-CoV-2 virus inhibition test.
Protection against SARS-CoV-2 Beta Variant in mRNA-1273 Boosted Nonhuman Primates
Authors: Corbett Kizzmekia S. , Gagne Matthew , Wagner Danielle A. , Connell Sarah O’ , Narpala Sandeep R. , Flebbe Dillon R. , Andrew Shayne F. , Davis Rachel L. , Flynn Barbara , Johnston Timothy S. , Stringham Christopher , Lai Lilin , Valentin Daniel , Van Ry Alex , Flinchbaugh Zackery , Werner Anne P. , Moliva Juan I. , Sriparna Manjari , O’Dell Sijy , Schmidt Stephen D. , Tucker Courtney , Choi Angela , Koch Matthew , Bock Kevin W. , Minai Mahnaz , Nagata Bianca M. , Alvarado Gabriela S. , Henry Amy R. , Laboune Farida , Schramm Chaim A. , Zhang Yi , Wang Lingshu , Choe Misook , Boyoglu-Barnum Seyhan , Shi Wei , Lamb Evan , Nurmukhambetova Saule T. , Provost Samantha J. , Donaldson Mitzi M. , Marquez Josue , Todd John-Paul M. , Cook Anthony , Dodson Alan , Pekosz Andrew , Boritz Eli , Ploquin Aurélie , Doria-Rose Nicole , Pessaint Laurent , Andersen Hanne , Foulds Kathryn E. , Misasi John , Wu Kai , Carfi Andrea , Nason Martha C. , Mascola John , Moore Ian N. , Edwards Darin K. , Lewis Mark G. , Suthar Mehul S. , Roederer Mario , McDermott Adrian , Douek Daniel C. , Sullivan Nancy J. , Graham Barney S. , Seder Robert A.
Published: August 2021

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 µg of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.β (heterologous), which encompasses the spike sequence of the B.1.351 (beta or β) variant. Reciprocal ID50pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the β variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost β-specific reciprocal ID50GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the β variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations.

One-sentence summary

mRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.
Towards the extended barcode concept: Generating DNA reference data through genome skimming of danish plants
Authors: Chua Physilia Y. S. , Leerhøi Frederik , Langkjær Emilia M. R. , Margaryan Ashot , Noer Christina L. , Richter Stine R. , Restrup Marlene E. , Bruun Hans Henrik , Hartvig Ida , Coissac Eric , Boessenkool Sanne , Alsos Inger G. , Bohmann Kristine
Published: August 2021

Background

Recently, there has been a push towards the extended barcode concept of utilising chloroplast genomes (cpGenome) and nuclear ribosomal DNA (nrDNA) sequences for molecular identification of plants instead of the standard barcode regions. These extended barcodes has a wide range of applications, including biodiversity monitoring and assessment, primer design, and evolutionary studies. However, these extended barcodes are not well represented in global reference databases. To fill this gap, we generated cpGenomes and nrDNA reference data from genome skims of 184 plant species collected in Denmark. We further explored the application of our generated reference data for molecular identifications of plants in an environmental DNA metagenomics study.

Results

We assembled partial cpGenomes for 82.1% of sequenced species and full or partial nrDNA sequences for 83.7% of species. We added all assemblies to GenBank, of which chloroplast reference data from 101 species and nuclear reference data from 6 species were not previously represented. On average, we recovered 45 genes per species. The rate of recovery of standard barcodes was higher for nuclear barcodes (>89%) than chloroplast barcodes (< 60%). Extracted DNA yield did not affect assembly outcome, whereas high GC content did so negatively. For thein silicosimulation of metagenomic reads, taxonomic assignments using the reference data generated had better species resolution (94.9%) as compared to GenBank (18.1%) without any identification errors.

Conclusions

Genome skimming generates reference data of both standard barcodes and other loci, contributing to the global DNA reference database for plants.
Cost-Benefit Analysis of a Children’s Spectacle Reimbursement Scheme: Evidence for Including Children’s Spectacles in Mongolia’s Social Health Insurance
Authors: Yong Ai Chee , Chuluunkhuu Chimgee , Chan Ving Fai , Stephan Tai , Congdon Nathan , O’Neill Ciaran
Published: August 2021

Background and aim

Globally, 12.8 million children have vision impairment due to uncorrected refractive error (URE). In Mongolia, one in five children needs but do not have access to spectacles. This cost-benefit analysis aims to estimate the net benefits of a children’s spectacles reimbursement scheme in Mongolia.

Methods

A willingness-to-pay (WTP) survey using the contingent valuation method was administered to rural and urban Mongolia respondents. The survey assessed WTP in additional annual taxes for any child with refractive error to be provided government-subsidised spectacles. Net benefits were then calculated based on mean WTP (i.e. benefit) and cost of spectacles.

Results

The survey recruited 50 respondents (mean age 40.2 ± 9.86 years; 78.0% women; 100% response rate) from rural and urban Mongolia. Mean WTP was US$24.00 ± 5.15 (95% CI US$22.55 to 25.35). The average cost of a pair of spectacles in Mongolia is US$15.00. Subtracting the average cost of spectacles from mean WTP yielded a mean positive net benefit of US$9.00.

Conclusion

A spectacle reimbursement scheme is a potentially cost-effective intervention to address childhood vision impairment due to URE in Mongolia. These preliminary findings support the inclusion of children’s spectacles into existing Social Health Insurance. A much larger random sample could be employed in future research to increase the precision and generalisability of findings.
Neutralization of VOCs including Delta one year post COVID-19 or vaccine
Authors: Havervall Sebastian , Marking Ulrika , Gordon Max , Ng Henry , Greilert-Norin Nina , Lindbo Sarah , Blom Kim , Nilsson Peter , Phillipson Mia , Klingström Jonas , Mangsbo Sara , Åberg Mikael , Hober Sophia , Thålin Charlotte
Published: August 2021

Background

SARS-CoV-2 variants, such as Alpha, Beta, Gamma and Delta, are raising concern about the efficiency of neutralizing antibodies (NAb) induced by wild-type infection or vaccines based on the wild-type spike.

Methods

We determined IgG and NAb against SARS-CoV-2 variants one year following mild wild-type infection (n=104) and two-dose regimens with BNT162b2 (BNT/BNT) (n=67), ChAdOx1 (ChAd/ChAd) (n=82), or heterologous ChAdOx1 followed by BNT162b2 (ChAd/BNT) (n=116).

Findings

Wild type spike IgG and NAb remained detectable in 80% (83/104) of unvaccinated participants one year post mild infection. The neutralizing capacity was similar against wild type (reference), Alpha (0.95 (0.92-0.98) and Delta 1.03 (0.95-1.11) but significantly reduced against Beta (0.54 (0.48-0.60)) and Gamma 0.51 (0.44-0.61). Similarly, BNT/BNT and ChAd/ChAd elicited sustained capacity against Alpha and Delta (1.01 (0.78-1.31) and 0.85 (0.64-1.14)) and (0.96 (0.84-1.09) and 0.82 (0.61-1.10) respectively), with reduced capacity against Beta (0.67 (0.50-0.88) and 0.53 (0.40-0.71)) and Gamma (0.12 (0.06-0.27) and 0.54 (0.37-0.80)). A similar trend was found following ChAd/BNT (0.74 (0.66-0.83) and 0.70 (0.50-0.97) against Alpha and Delta and 0.29 (0.20-0.42) and 0.13 (0.08-0.20) against Beta and Gamma).

Interpretation

Persistent neutralization of the wide-spread Alpha and Delta variants one year after wild-type infection may aid vaccine policy makers in low-resource settings when prioritizing vaccine supply. The reduced capacity of neutralizing Beta and Gamma strains, but not the Alpha and Delta strains following both infection and three different vaccine regimens argues for caution against Beta and Gamma-exclusive mutations in the efforts to optimize next generation SARS-CoV-2 vaccines.

Funding

A full list of funding bodies that contributed to this study can be found in the Acknowledgements section
B and T cell responses after a third dose of SARS-CoV-2 vaccine in Kidney Transplant Recipients
Authors: Schrezenmeier Eva , Rincon-Arevalo Hector , Stefanski Ana-Luisa , Potekhin Alexander , Staub-Hohenbleicher Henriette , Choi Mira , Bachmann Friederike , Pross Vanessa , Hammett Charlotte , Schrezenmeier Hubert , Ludwig Carolin , Jahrsdörfer Bernd , Lino Andreia C. , Eckardt Kai-Uwe , Kotsch Katja , Dörner Thomas , Budde Klemens , Sattler Arne , Halleck Fabian
Published: August 2021

Background

Accumulating evidence suggests that solid organ transplant recipients, as opposed to the general population, show strongly impaired responsiveness towards standard SARS-CoV-2 mRNA-based vaccination, demanding alternative strategies for protection of this vulnerable group.

Methods

In line with recent recommendations, a third dose of either heterologous ChAdOx1 (AstraZeneca) or homologous BNT162b2 (BioNTech) was administered to 25 kidney transplant recipients (KTR) without humoral response after 2 doses of BNT162b2, followed by analysis of serological responses and vaccine-specific B- and T-cell immunity.

Results

9/25 (36%) KTR under standard immunosuppressive treatment seroconverted until day 27 after the third vaccination, while one patient developed severe COVID-19 infection immediately after vaccination. Cellular analysis seven days after the third dose showed significantly elevated frequencies of viral spike protein receptor binding domain specific B cells in humoral responders as compared to non-responders. Likewise, portions of spike-reactive CD4+ T helper cells were significantly elevated in seroconverting patients. Furthermore, overall frequencies of IL-2+, IL-4+ and polyfunctional CD4+ T cells significantly increased after the third dose, whereas memory/effector differentiation remained unaffected.

Conclusions

Our data suggest that a fraction of transplant recipients benefits from triple vaccination, where seroconversion is associated with quantitative and qualitative changes of cellular immunity. At the same time, the study highlights that modified vaccination approaches for immunosuppressed patients still remain an urgent medical need.

Significance statement

Protection of solid organ transplant recipients against SARS-Cov-2 by vaccination remains an unmet need given the low immunogenicity of available vaccines in the presence of immunosuppression. Administration of a third dose to 25 kidney transplant recipients (KTR) resulted in seroconversion in 36% of patients, associated with significant quantitative and functional changes within the spike-antigen-specific B-cell- and CD4+ T-helper cell compartment. Our data support the need for individual humoral monitoring of immunosuppressed individuals after vaccination as well as continued efforts to adapt vaccination protocols for this at-risk group.
Hybrid immunity improves B cell frequency, antibody potency and breadth against SARS-CoV-2 and variants of concern
Authors: Andreano Emanuele , Paciello Ida , Piccini Giulia , Manganaro Noemi , Pileri Piero , Hyseni Inesa , Leonardi Margherita , Pantano Elisa , Abbiento Valentina , Benincasa Linda , Giglioli Ginevra , De Santi Concetta , Fabbiani Massimiliano , Rancan Ilaria , Tumbarello Mario , Montagnani Francesca , Sala Claudia , Montomoli Emanuele , Rappuoli Rino
Published: August 2021

To understand the nature of the antibody response to SARS-CoV-2 vaccination, we analyzed at single cell level the B cell responses of five naïve and five convalescent people immunized with the BNT162b2 mRNA vaccine. Convalescents had higher frequency of spike protein specific memory B cells and by cell sorting delivered 3,532 B cells, compared with 2,352 from naïve people. Of these, 944 from naïve and 2,299 from convalescents produced monoclonal antibodies against the spike protein and 411 of them neutralized the original Wuhan SARS-CoV-2 virus. More than 75% of the monoclonal antibodies from naïve people lost their neutralization activity against the B.1.351 (beta) and B.1.1.248 (gamma) variants while this happened only for 61% of those from convalescents. The overall loss of neutralization was lower for the B.1.1.7 (alpha) and B.1.617.2 (delta) variants, however it was always significantly higher in those of naïve people. In part this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in convalescents and generated potent and broadly neutralizing antibodies. Overall, vaccination of seropositive people increases the frequency of B cells encoding antibodies with high potency and that are not susceptible to escape by any of the four variants of concern. Our data suggest that people that are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control SARS-CoV-2 emerging variants.