bluuebunny/crossref_metadata_2025 huggingface dataset:
DDX41 is needed for pre-and post-natal hematopoietic stem cell differentiation in mice
Authors: Ma Jing , Mahmud Nadim , Bosland Maarten C. , Ross Susan R.
Published: August 2021

DDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms. DDX41 binds to DNA/RNA hybrids and interacts with spliceosome component (1, 2). How it affects hematopoiesis is still unclear. Using a knockout mouse model, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation. Lack of DDX41 particularly affected myeloid progenitor development, starting at embryonic day 13.5. DDX41-deficient fetal liver and adult bone marrow (BM) cells were unable to rescue mice from lethal irradiation after transplantation. DDX41 knockout stem cells were also defective in ex vivo colony forming assays. RNASeq analysis of lineage-negative, cKit+Sca1+ cells isolated from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered in DDX41 knockout cells. Furthermore, altered splicing of genes involved in key biological processes were observed. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through its regulation of gene expression programs and splicing.

Significance

DDX41 is a tumor suppressor in hematologic malignancies. However, whether DDX41 functions in hematopoiesis and myeloid cell differentiation is not known. Here we show that in mice, loss of DDX41 in hematopoietic stem cells (HSCs) leads to defects in hematopoietic development. The myeloid lineage was particularly affected as early as pre-natal stages. Transcriptional profiling of embryonic HSCs revealed that there were global changes in gene expression and splicing due to lack of DDX41. Collectively, the study uncovers a new function of DDX41 in HSC differentiation and could provide molecular targets for treatment of myeloid differentiation disorders.
Sequential in-cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster
Authors: Amândio Ana Rita , Beccari Leonardo , Lopez-Delisle Lucille , Mascrez Bénédicte , Zakany Jozsef , Gitto Sandra , Duboule Denis
Published: August 2021

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in-cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the mis-regulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, it seems that the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions and some doing both, whereas all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites amongst paralogous Hox clusters or between various vertebrates.
Over 1000 tools reveal trends in the single-cell RNA-seq analysis landscape
Authors: Zappia Luke , Theis Fabian J.
Published: August 2021

Recent years have seen a revolution in single-cell technologies, particularly single-cell RNA-sequencing (scRNA-seq). As the number, size and complexity of scRNA-seq datasets continue to increase, so does the number of computational methods and software tools for extracting meaning from them. Since 2016 the scRNA-tools database has catalogued software tools for analysing scRNA-seq data. With the number of tools in the database passing 1000, we take this opportunity to provide an update on the state of the project and the field. Analysis of five years of analysis tool tracking data clearly shows the evolution of the field, and that the focus of developers has moved from ordering cells on continuous trajectories to integrating multiple samples and making use of reference datasets. We also find evidence that open science practices reward developers with increased recognition and help accelerate the field.
Light-controlled elimination of PD-L1+ cells
Authors: Wong Judith Jing Wen , Selbo Pål Kristian
Published: August 2021

The programmed death ligand-1 (PD-L1), also known as CD274 or B7-H1, is mainly expressed on cancer cells and/or immunosuppressive cells in the tumor microenvironment (TME) and plays an essential role in tumor progression and immune escape. Immune checkpoint inhibitors (ICIs) of the PD-1/PD-L1 axis have shown impressive clinical success, however, the majority of the patients do not respond to immune checkpoint therapy (ICT). Thus, to overcome ICT resistance there is a high need for potent and novel strategies that simultaneously target both tumor cells and immunosuppressive cells in the TME. In this study, we show that the intracellular light-controlled drug delivery method photochemical internalization (PCI) induce specific and strongly enhanced cytotoxic effects of the PD-L1-targeting immunotoxin, anti-PD-L1-saporin (Anti-PDL1-SAP), in the PD-L1+ triple-negative breast cancer MDA-MB-231 cell line, while no enhanced efficacy was obtained in the PD-L1 negative control cell line MDA-MB-453. Using fluorescence microscopy, we reveal that the anti-PD-L1 antibody binds to PD-L1 on the surface of the MDA-MD-231 cells and overnight accumulates in late endosomes and lysosomes where it co-localizes with the PCI photosensitizer fimaporfin (TPCS2a). Moreover, light-controlled endosomal/lysosomal escape of the anti-PD-L1 antibody and fimaporfin into the cytosol was obtained. We also confirm that the breast MDA-MB-468 and the prostate PC-3 and DU-145 cancer cell lines have subpopulations with PD-L1 expression. In addition, we show that interferon-gamma strongly induce PD-L1 expression in the per se PD-L1 negative CT26.WT cells and enhance the PD-L1 expression in MC-38 cells, of which both are murine colon cancer cell lines. In conclusion, our work provides an in vitro proof-of-concept of PCI-enhanced targeting and eradication of PD-L1 positive immunosuppressive cells. This light-controlled combinatorial strategy has a potential to advance cancer immunotherapy and should be explored in preclinical studies.
Contrasting resistance and resilience to light variation of the coupled oxic and anoxic components of an experimental microbial ecosystem
Authors: Suleiman Marcel , Pennekamp Frank , Choffat Yves , Petchey Owen L.
Published: August 2021

Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used phototrophic oxic-anoxic micro-ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities is affected by light deprivation. Continuous measurements were performed to describe oxygen dynamics (mean/min/max/amplitude) and time-series of full-length 16S rRNA sequencing were used to quantify changes in alpha- and beta-diversity. In the top oxic layer, oxygen concentration decreased significantly under light deprivation, but showed resilience in mean, minimum and maximum after light conditions were restored. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha-diversity of the top oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient. In contrast, alpha-diversity of the anoxic bottom layer communities increased due to the stressor, but was resilient. Community composition changed significantly during light deprivation, showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha-diversity and the amplitude of oxygen within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function and structure, hence focusing on a single stability component could potentially be misleading.
Metabolomics and Transcriptomics unravel the mechanism of browning resistance in Agaricus bisporus
Authors: Cai Zhi-Xin , Chen Mei-Yuan , Lu Yuan-Ping , Guo Zhong-Jie , Zeng Zhi-Heng , Zheng Hui-Qing , Liao Jian-Hua
Published: August 2021

Agaricus bisporus is widely used on the world market. The easy browning of mushroom surface is one of the most intuitive factors affecting consumer purchase. A certain cognition on browning mechanism have been made after years of research. At present, people slow down the browning of mushrooms mainly by improving preservation methods. In addition, breeding is also a reliable way. In the production practice, we have identified some browning resistant varieties,and we selected a browning-resistant variety to compare with ordinary variety to reveal the resistance mechanism. Using transcriptomics and metabolomics, the differences in gene expression and metabolite levels were revealed, respectively. The results showed that differentially expressed genes (DEGs) like AbPPO4, AbPPO3 and AbPPO2 were differently expressed and these DEGs involved in many pathways that related to browning. The expression of AbPPO expression play an important role in the browning of A. bisporus and multiple PPO family members are involved in the regulation of browning. However, the resistance to browning cannot be judged only by the expression level of AbPPOs. Formetabolomics, most of the different metabolites were organic acids. These organic acids had a higher level in anti-browning (BT) than easy-browning varieties (BS), although the profile was very heterogeneous. On the contrary, the content of trehalose in BS was significantly higher than that in BT. Higher organic acids decreased pH and further inhibited PPO activity. In addition, the BS had a higher content of trehalose, which might play roles in maintain the activity of PPO. The difference of browning resistance between BS and BT is mainly due to the differential regulation mechanism of PPO.
Judgement Bias During Pregnancy in Domestic Pigs
Authors: Bushby Emily V. , Cotter Sheena C. , Wilkinson Anna , Friel Mary , Collins Lisa M.
Published: August 2021

In humans and rats, changes in mood and affect are known to occur during pregnancy, however it is unknown how gestation may influence mood in other non-human mammals. This study assessed changes in pigs’ judgment bias as a measure of affective state throughout gestation. Pigs were trained to complete a spatial judgement bias task with reference to positive and negative locations. We tested gilts before mating, and during early and late pregnancy, by assessing their responses to ambiguous probe locations. Pigs responded increasingly negatively to ambiguous probes as pregnancy progressed and there were consistent inter-individual differences in baseline optimism. This suggests that the pigs’ affective state may be altered during gestation, although as a non-pregnant control group was not tested, an effect of learning cannot be ruled out. These results suggest that judgement bias is altered during pregnancy in domestic pigs, consequently raising novel welfare considerations for captive multiparous species.
The temporal dynamics of the Stroop effect from childhood to young and older adulthood
Authors: Ménétré E. , Laganaro M.
Published: August 2021

It is well admitted that children and older adults tend to show longer response latencies at the Stroop task than young adults. The present study aims at clarifying the rational of such changes from childhood to adulthood and in ageing by comparing the impacted cognitive processes across age groups. More precisely, the aim was to clarify if all processes take more time to be executed, hence implying that longer latencies rely mainly on processing speed or if an additional process lengthens the resolution of the conflict in children and/or older adults. To this aim we recorded brain electrical activity using EEG in school-age children, young and older adults while they performed a classic verbal Stroop task. To decompose the signal in the underlying brain networks, we used microstates analyses and compared congruent, incongruent and neutral trials across the three age-groups. Behaviorally, children and older adults presented longer latencies and larger Stroop effects relative to young adults. The microstates results showed that children tend to present different brain configurations compared to both adult groups, even though some brain configurations remained identical among the three groups. In particular, additional brain networks were involved in children to perform the Stroop task, which party account for the longer latencies in this group. By contrast, in aging the results favor the general slowing hypothesis rather than a decline in a specific process since all involved brain networks were similar in the two adult groups but slowed down in the older one.
A bacterium-like particle vaccine displaying Zika virus prM-E induces systemic immune responses in mice
Authors: Jin Hongli , Bai Yujie , Wang Jianzhong , Jiao Cuicui , Liu Di , Zhang Mengyao , Li Entao , Huang Pei , Gong Zhiyuan , Song Yumeng , Xu Shengnan , Feng Na , Zhao Yongkun , Wang Tiecheng , Li Nan , Gao Yuwei , Yang Songtao , Xia Xianzhu , Wang Hualei
Published: August 2021

The emergence of Zika virus (ZIKV) infection, which is an unexpectedly associated with congenital defects, has prompted the development of safe and effective vaccines. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has emerged as a versatile and highly effective platform for delivering target proteins for vaccines. In this article, we developed a bacterium-like particle vaccine ZI-Δ-PA-GEM based on the GEM-PA system. The fusion protein ZI-Δ-PA, which contains the prM-E-Δ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3, was expressed. The fusion protein was successfully displayed on the GEM surface, forming ZI-Δ-PA-GEM. Moreover, when BALB/c mice were immunized intramuscularly with ZI-Δ-PA-GEM combined with 201 VG and poly(I:C) adjuvants, durable ZIKV-specific IgG and protective neutralizing antibody responses were induced. Potent B cell/DC activation was also be stimulated early after immunization. Remarkably, splenocyte proliferation, the secretion of multiple cytokines, T/B cell activation and central memory T cell responses were elicited. These data indicate that ZI-Δ-PA-GEM is a promising bacterium-like particle vaccine candidate for ZIKV.

Author summary

Because Zika virus (ZIKV) infection is considered as an example of “disease X”, the development of a safe and effective ZIKV vaccine is essential. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has been used in many vaccine studies due to its advantages. In this study, prM-E-△ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3 was fusion expressed, termed ZI-△-PA. Then the fusion protein ZI-△-PA could be displayed on the surface of GEM, forming ZI-△-PA-GEM. The author evaluated the immunogenicity of ZI-△-PA-GEM with the 201 VG and poly(I:C) adjuvants. The study demonstrates that ZI-△-PA-GEM induced mice to produce neutralizing antibody and specific cellular immune responses. The author believe that the bacterium-like particle vaccine ZI-△-PA-GEM has the potential to be used as the ZIKV vaccine.
Quantifying cumulative phenotypic and genomic evidence for procedural generation of metabolic network reconstructions
Authors: Moutinho Thomas J. , Neubert Benjamin C. , Jenior Matthew L. , Papin Jason A.
Published: August 2021

Genome-scale metabolic network reconstructions (GENREs) are valuable tools for understanding microbial community metabolism. The process of automatically generating GENREs includes identifying metabolic reactions supported by sufficient genomic evidence to generate a draft metabolic network. The draft GENRE is then gapfilled with additional reactions in order to recapitulate specific growth phenotypes as indicated with associated experimental data. Previous methods have implemented absolute mapping thresholds for the reactions automatically included in draft GENREs; however, there is growing evidence that integrating annotation evidence in a continuous form can improve model accuracy. There is a need for flexibility in the structure of GENREs to better account for uncertainty in biological data, unknown regulatory mechanisms, and context specificity associated with data inputs. To address this issue, we present a novel method that provides a framework for quantifying combined genomic, biochemical, and phenotypic evidence for each biochemical reaction during automated GENRE construction. Our method, Constraint-based Analysis Yielding reaction Usage across metabolic Networks (CANYUNs), generates accurate GENREs with a quantitative metric for the cumulative evidence for each reaction included in the network. The structure of a CANYUN GENRE allows for the simultaneous integration of three data inputs while maintaining all supporting evidence for biochemical reactions that may be active in an organism. CANYUNs is designed to maximize the utility of experimental and annotation datasets and to ultimately assist in the curation of the reference datasets used for the automatic reconstruction of metabolic networks. We validated CANYUNs by generating an E. coli K-12 model and compared it to the manually curated reconstruction iML1515. Finally, we demonstrated the use of CANYUNs to build a model by generating an E. coli Nissle CANYUN GENRE using novel phenotypic data that we collected. This method may address key challenges for the procedural construction of metabolic networks by leveraging uncertainty and redundancy in biological data.
A diabetic milieu increases cellular susceptibility to SARS-CoV-2 infections in engineered human kidney organoids and diabetic patients
Authors: Garreta Elena , Prado Patricia , Stanifer Megan , Monteil Vanessa , del Pozo Carmen Hurtado , Ullate-Agote Asier , Vilas-Zornoza Amaia , Romero Juan Pablo , Jonsson Gustav , Oria Roger , Leopoldi Alexandra , Hagelkruys Astrid , Moya-Rull Daniel , González Federico , Marco Andrés , Tarantino Carolina , Domingo-Pedrol Pere , HasanAli Omar , Ventura-Aguiar Pedro , María Campistol Josep , Prosper Felipe , Mirazimi Ali , Boulant Steeve , Penninger Josef M. , Montserrat Nuria
Published: August 2021

SARS-CoV-2 infections lead to a high risk of hospitalization and mortality in diabetic patients. Why diabetic individuals are more prone to develop severe COVID-19 remains unclear. Here, we established a novel human kidney organoid model that mimics early hallmarks of diabetic nephropathy. High oscillatory glucose exposure resulted in metabolic changes, expansion of extracellular membrane components, gene expression changes determined by scRNAseq, and marked upregulation of angiotensin-converting enzyme 2 (ACE2). Upon SARS-CoV-2 infection, hyperglycemic conditions lead to markedly higher viral loads in kidney organoids compared to normoglycemia. Genetic deletion of ACE2, but not of the candidate receptor BSG/CD147, in kidney organoids demonstrated the essential role of ACE2 in SARS-CoV-2 infections and completely prevented SARS-CoV-2 infection in the diabetogenic microenvironment. These data introduce a novel organoid model for diabetic kidney disease and show that diabetic-induced ACE2 licenses the diabetic kidney to enhanced SARS-CoV-2 replication.
Across functional boundaries: making non-neutralizing antibodies to neutralize HIV-1 and mediate Fc-mediated effector killing of infected cells
Authors: Richard Jonathan , Nguyen Dung N. , Tolbert William D. , Gasser Romain , Ding Shilei , Vézina Dani , Gong Shang Yu , Prévost Jérémie , Gendron-Lepage Gabrielle , Medjahed Halima , Gottumukkala Suneetha , Finzi Andrés , Pazgier Marzena
Published: August 2021

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or Cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, ‘closed’ Env trimer present on HIV-1 virions or expressed on HIV-1 infected cells. Antibodies targeting this region are therefore non-neutralizing and their potential as mediators of antibody depended cellular cytoxicity (ADCC) of HIV-1 infected cells diminished by a lack of available binding targets. Here we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or Cluster A specificity to the extracellular domain 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening ‘closed’ Env trimers on HIV-1 particles/infected cells to expose the Cluster A region and activate ADCC and neutralization against these non-neutralizing targets.

Importance

Highly conserved epitopes within the co-receptor binding site (CoRBS) and constant region 1 and 2 (C1C2 or Cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4, therefore they are not accessible on virions and infected cells where the expression of CD4 is downregulated. Here we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or Cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native “closed” Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1 infected cells through antibody depended cellular cytoxicity (ADCC) and inactivate HIV-1 virus through neutralization.
Two parallel sRNA amplification cycles contribute to RNAi inheritance in C. elegans
Authors: Ouyang John Paul Tsu , Zhang Wenyan , Seydoux Geraldine
Published: August 2021

RNA-mediated interference (RNAi) is a conserved mechanism that uses small RNAs (sRNAs) to tune gene expression. In C. elegans, exposure to dsRNA induces the production of gene-specific sRNAs that are propagated to progeny not exposed to the dsRNA trigger. We present evidence that RNAi inheritance is mediated by two parallel sRNA amplification loops. The first loop, dependent on the nuclear Argonaute HRDE-1, targets nascent transcripts, and reduces but does not eliminate productive transcription at the locus. The second loop, dependent on the conserved helicase ZNFX-1, targets mature transcripts and concentrates them in perinuclear condensates (nuage). Each amplification loop generates a distinct class of sRNAs, with the ZNFX-1 loop responsible for the bulk of sRNA production on the region targeted by the trigger. By independently targeting nascent and mature transcripts, the HRDE-1 and ZNFX-1 loops ensure maximum silencing in progeny not exposed to the trigger.
Vesalius: high-resolution in silico anatomization of Spatial Transcriptomic data using Image Analysis
Authors: Martin Patrick C.N. , Kim Hyobin , Lövkvist Cecilia , Hong Byung-Woo , Won Kyoung Jae
Published: August 2021

Characterization of tissue architecture promises to deliver insights into development, cell communication and disease. In silico spatial domain retrieval methods have been developed for spatial transcriptomics (ST) data assuming transcriptional similarity of neighboring barcodes. However, domain retrieval approaches with this assumption cannot work in complex tissues composed of multiple cell types. This task becomes especially challenging in cellular resolution ST methods. We developed Vesalius to decipher tissue anatomy from ST data by applying image processing technology. Vesalius uniquely detected territories composed of multiple cell types and successfully recovered tissue structures in high-resolution ST data including in mouse brain, embryo, liver, and colon. Utilizing this tissue architecture, Vesalius identified tissue morphology specific gene expression and regional specific gene expression changes for astrocytes, interneuron, oligodendrocytes, and entorhinal cells in the mouse brain.
Multiplein vivoroles for the C-terminal domain of the RNA chaperone Hfq
Authors: Kavita Kumari , Zhang Aixia , Tai Chin-Hsien , Majdalani Nadim , Storz Gisela , Gottesman Susan
Published: August 2021

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), theEscherichia colimutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression ofmutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.
Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins
Authors: Korona Dagmara , Dirnberger Benedict , Giachello Carlo N G , Queiroz Rayner M L , Minde David-Paul , Deery Michael J , Johnson Glynnis , Müller Karin H , Firth Lucy C , Earley Fergus G , Russell Steven , Lilley Kathryn S
Published: August 2021

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of receptor subunit composition is needed for effective design of insecticides. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Dα5, Dα6, Dα7 subunits. Finally, we report the localization of fluorescent tagged endogenous Dα6 during nervous system development. Taken together this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis
Authors: Zhang Wanlu , Khan Azqa , Vitale Jlenia , Neuner Annett , Rink Kerstin , Lüchtenborg Christian , Brügger Britta , Söllner Thomas H. , Schiebel Elmar
Published: August 2021

The integral membrane protein Apq12 is an important nuclear envelope (NE)/ER modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting upon Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE during NPC biogenesis.
The Zip4 protein directly couples meiotic crossover formation to synaptonemal complex assembly
Authors: Pyatnitskaya Alexandra , Andreani Jessica , Guérois Raphaël , De Muyt Arnaud , Borde Valérie
Published: August 2021

Meiotic recombination is triggered by programmed double-strand breaks (DSBs), a subset of these being repaired as crossovers, promoted by eight evolutionarily conserved proteins, named ZMM. Crossover formation is functionally linked to synaptonemal complex (SC) assembly between homologous chromosomes, but the underlying mechanism is unknown. Here we show that Ecm11, a SC central element protein, localizes on both DSB sites and sites that attach chromatin loops to the chromosome axis, which are the starting points of SC formation, in a way that strictly requires the ZMM protein Zip4. Furthermore, Zip4 directly interacts with Ecm11 and point mutants that specifically abolish this interaction lose Ecm11 binding to chromosomes and exhibit defective SC assembly. This can be partially rescued by artificially tethering interaction-defective Ecm11 to Zip4. Mechanistically, this direct connection ensuring SC assembly from CO sites could be a way for the meiotic cell to shut down further DSB formation once enough recombination sites have been selected for crossovers, thereby preventing excess crossovers. Finally, the mammalian ortholog of Zip4, TEX11, also interacts with the SC central element TEX12, suggesting a general mechanism.
Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages
Authors: Kriplani Nisha , Clohisey Sara , Fonseca Sonia , Fletcher Sarah , Lee Hui-Min , Ashworth Jordan , Kurian Dominic , Lycett Samantha J. , Tait-Burkard Christine , Kenneth Baillie J. , Woolhouse Mark E. J. , Carding Simon R. , Stewart James P. , Digard Paul
Published: August 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-κB signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.
A cooperative network at the nuclear envelope counteracts LINC-mediated forces during oogenesis in C. elegans
Authors: Liu Chenshu , Lung Zoe , Wang John S. , Wu Fan , Dernburg Abby F.
Published: August 2021

Oogenesis involves meiosis and oocyte maturation. Both processes rely on mechanical forces (Lee et al., 2015; Nagamatsu et al., 2019; Rog and Dernburg, 2015; Sato et al., 2009; Tsatskis et al., 2020; Wynne et al., 2012), which can be transduced from the cytoskeleton to the nuclear envelope (NE) through linker of nucleoskeleton and cytoskeleton (LINC) complexes (Burke, 2018; Chang et al., 2015; Fan et al., 2020; Link et al., 2014). Gametes must protect their genomes from damage in this mechanically stressful environment. In C. elegans, oocyte nuclei lacking the single lamin protein LMN-1 are vulnerable to nuclear collapse. Here we deploy the auxin-inducible degradation system to investigate the balance of forces that drive this collapse and protect oocyte nuclei. We find that nuclear collapse is not a consequence of apoptosis. It is promoted by dynein and a LINC complex comprised of SUN-1 and ZYG-12, which assumes polarized distribution at the NE in response to dynein-mediated forces. We also show that the lamin meshwork works in parallel with other inner nuclear membrane (INM) proteins to counteract mechanical stress at the NE during oogenesis. We speculate that a similar network may protect oocyte integrity during the long arrest period in mammals.
Comparative Analysis and Rational Design of dCas9-VP64 Variants for CRISPR Activation
Authors: Omachi Kohei , Miner Jeffrey H.
Published: August 2021

CRISPR/Cas9-mediated transcriptional activation (CRISPRa) is a powerful tool for investigating complex biological phenomena. Although CRISPRa approaches based on VP64 have been widely studied in both cultured cells and in animal models and exhibit great versatility for various cell types and developmental stages in vivo, different dCas9-VP64 versions have not been rigorously compared. Here, we compared different dCas9-VP64 constructs in identical contexts, including the cell lines used and the transfection conditions, for their ability to activate endogenous and exogenous genes. Moreover, we investigated the optimal approach for VP64 addition to VP64- and p300-based constructs. We found that MS2-MCP-scaffolded VP64 enhanced dCas9-VP64 and dCas9-p300 activity better than did direct VP64 fusion to the N-terminus of dCas9. dCas9-VP64+MCP-VP64 and dCas9-p300+MCP-VP64 were superior to VP64-dCas9-VP64 for all target genes tested. Furthermore, multiplexing gRNA expression with dCas9-VP64+MCP-VP64 or dCas9-p300+MCP-VP64 significantly enhanced endogenous gene activation to a level comparable to CRISPRa-SAM with a single gRNA. Our findings demonstrate improvement of the dCas9-VP64 CRISPRa system and contribute to development of a versatile, efficient CRISPRa platform.
Laboratory analysis of glucose, fructose, and sucrose contents in Japanese common beverages for the exact assessment of beverage-derived sugar intake
Authors: Ando Yoshitaka , Ohta Yoshiji , Munetsuna Eiji , Yamada Hiroya , Nouchi Yuki , Kageyama Itsuki , Mizuno Genki , Yamazaki Mirai , Fujii Ryosuke , Ishikawa Hiroaki , Suzuki Koji , Ohashi Koji
Published: August 2021

Background

The adverse health effects of sugar-sweetened beverage consumption have been studied worldwide. There are several reports on actual sugar contents in sugar-sweetened beverages. However, there is no recent report on actual sugar contents in Japanese sugar-sweetened beverages. Therefore, we attempted to analyze glucose, fructose, and sucrose contents in Japanese common beverages.

Methods

Glucose, fructose, and sucrose contents in 49 beverages including 8 energy drinks, 11 sodas, 4 fruit juices, 7 probiotic drinks, 4 sports drinks, 5 coffee drinks, 6 green tea drinks, and 4 tea drinks were determined using the enzymatic methods.

Results

Tow zero calorie drinks, 2 sugarless coffee drinks, and 6 green tea drinks contained no sugar. Three coffee drinks contained only sucrose. The orders of median glucose, fructose, and sucrose contents in categorized beverages containing sugars were as follows: for glucose, fruit juice > energy drink ≥ soda >> probiotic drink > black tea drink > sports drink; for fructose, probiotic drink ≥ energy drink > fruit juice > soda >> sports drink > black tea drink; and for sucrose, black tea drink > energy drink ≥ probiotic drink > fruit juice > soda > coffee drink >> sports drink. The rate of total fructose content in total sugar content in 38 sugar-containing beverages was approximately 40-60%. The total sugar content analyzed was not always equivalent to carbohydrate content indicated on the nutrition label.

Conclusions

These results indicate that actual sugar content in Japanese common beverages is necessary for the exact assessment of beverage-derived sugar intake.
Antimicrobial interactions between the phytoextracts of Callistemon citrinus and Eriobotrya japonica against Streptococcus mutans
Authors: Kavuo J. S , Namboko Jerald Kutosi , Bukusuba Samuel , Onen Healy
Published: August 2021

Streptococcus mutans is a gram-positive bacterium in the oral cavity that is most implicated in the dental caries progression. The condition is very expensive to manage and the most commonly used products such as fluoride tooth pastes and alcohol-based mouth washes are associated with many side effects. The current study therefore focused on providing a scientific evidence to guide the use of a combination of Eriobotrya japonica (EJ) and Callistemon citrinus (CC) as actives in development of an effective and cheaper herbal formulation for management of dental caries. The objective of this study was to determine antimicrobial interactions of Eriobotrya japonica and Callistemon citrinus phytoextracts proportions against Streptococcus mutans bacteria. The leaves of both plants (EJ and CC) were shade-dried and pulverized into a coarse powder which were then cold macerated using ethanol (60 %) for 24 h. Phytochemical screening was conducted for the two dry extracts obtained after fan drying before they were mixed in to five different proportions (1:0, 3:1, 1:1, 1:3 and 0:1). Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) assays against Streptococcus mutans were done for all the proportions above with ciprofloxacin and 2.5 % Dimethyl sulfoxide (DMSO) as the positive and negative controls respectively. Antimicrobial interactions between the two extracts were also evaluated using Fractional Inhibitory and Bacterial Concentration Indices (FICI/FBCI). Results showed that EJ and CC had percentage yields of 20.05 % and 15.45 % respectively. All the extracts showed similar phytochemical profiles. They also demonstrated an inhibitory effect on Streptococcus mutans with MIC and MBC values ranging from 521 to 3333 μg/ml and 1042 to 3667 μg/ml respectively. However, CC: EJ (1:0) had the lowest MIC and MBC comparable to that of the standard drug at P <0.05. The FICI/FBCI were between 1.5 and 3.917. Therefore, CC: EJ (1:0) proportion markedly demonstrated better antimicrobial activity against the test organism and there are no beneficial antimicrobial interactions between the two plant extracts to inform their combination as actives for dental caries product formulation.
Evidence for the Presence of Hyphae and Fruiting Body Calcium Oxalate Crystallites in Schizophyllum commune
Authors: Xiao Xiangyue , Huang Tianji , Zhang Jingyi , Su Qiuyi , Tao Lin , Chen Xiaojie , Zhang Yuchen , Jiang Yufan , Ding Yan , Yu Xiaoming , Liu Wei , Ji Hong , Song Chi
Published: August 2021

Biomineralization is a phenomenon in which organisms form crystals. Studies have shown that many fungi have the ability to biomineralize, it can exhibit calcium oxalate crystals on their hyphae and fruiting body. Schizophyllum commune is a common saprophytic fungus distributed all over the world, but there is little research on its biomineralization. In this paper, S. commune fruiting body from three different provinces of China were collected, and isolation for hyphal cultured to obtain several samples. Utilizing light microscope, FE-SEM, and EDAX, the existence of crystals on the fruiting body and mycelium of each strain was found, and their morphological characteristics and ion content were analyzed. It was ultimately established that biomineralization occurs on S. commune.
Non-Invasive Confocal Fluorescence Imaging of Mice Beyond 1700 nm Using Superconducting Nanowire Single-Photon Detectors
Authors: Wang Feifei , Ren Fuqiang , Ma Zhuoran , Qu Liangqiong , Gourgues Ronan , Xu Chun , Baghdasaryan Ani , Li Jiachen , Zadeh Iman Esmaeil , Los Johannes WN , Fognini Andreas , Qin-Dregely Jessie , Dai Hongjie
Published: August 2021

Light scattering by biological tissues sets a limit to the penetration depth of high-resolution optical microscopy imaging of live mammals in vivo. An effective approach to reduce light scattering and increase imaging depth is by extending the excitation and emission wavelengths to the > 1000 nm second near-infrared (NIR-II), also called the short-wavelength infrared (SWIR) window. Here, we developed biocompatible core-shell lead sulfide/cadmium sulfide (PbS/CdS) quantum dots emitting at ~1880 nm and superconducting nanowire single photon detectors (SNSPD) for single-photon detection up to 2000 nm, enabling one-photon fluorescence imaging window in the 1700-2000 nm (NIR-IIc) range. Confocal fluorescence imaging in NIR-IIc reached an imaging depth of ~ 800 μm through intact mouse head, and enabled non-invasive imaging of inguinal lymph nodes (LNs) without any surgery. In vivo molecular imaging of high endothelial venules (HEVs) with diameter down to ~ 6.6 μm in the lymph nodes was achieved, opening the possibility of non-invasive imaging of immune trafficking in lymph nodes at the single-cell/vessel level longitudinally.
Assembled chromosomes of the blood fluke Schistosoma mansoni provide insight into the evolution of its ZW sex-determination system
Authors: Buddenborg Sarah K , Tracey Alan , Berger Duncan J , Lu Zhigang , Doyle Stephen R , Fu Beiyuan , Yang Fengtang , Reid Adam J , Rodgers Faye H , Rinaldi Gabriel , Sankaranarayanan Geetha , Böhme Ulrike , Holroyd Nancy , Berriman Matthew
Published: August 2021

Background

Schistosoma mansoni is a flatworm that causes a neglected tropical disease affecting millions worldwide. Most flatworms are hermaphrodites but schistosomes have genotypically determined male (ZZ) and female (ZW) sexes. Sex is essential for pathology and transmission, however, the molecular determinants of sex remain unknown and is limited by poorly resolved sex chromosomes in previous genome assemblies.

Results

We assembled the 391.4 Mb S. mansoni genome into individual, single-scaffold chromosomes, including Z and W. Manual curation resulted in a vastly improved gene annotation, resolved gene and repeat arrays, trans-splicing, and almost all UTRs. The sex chromosomes each comprise pseudoautosomal regions and single sex-specific regions. The Z-specific region contains 932 genes, but on W all but 29 of these genes have been lost and the presence of five pseudogenes indicates that degeneration of W is ongoing. Synteny analysis reveals an ancient chromosomal fusion corresponding to the oldest part of Z, where only a single gene—encoding the large subunit of pre-mRNA splicing factor U2AF—has retained an intact copy on W. The sex-specific copies of U2AF have divergent N-termini and show sex-biased gene expression.

Conclusion

Our assembly with fully resolved chromosomes provides evidence of an evolutionary path taken to create the Z and W sex chromosomes of schistosomes. Sex-linked divergence of the single U2AF gene, which has been present in the sex-specific regions longer than any other extant gene with distinct male and female specific copies and expression, may have been a pivotal step in the evolution of gonorchorism and genotypic sex determination of schistosomes.
Artists as cultural intermediaries? Remediating practices of production and consumption
Authors: Hadley Steven
Published: August 2021

Purpose

The purpose of this paper is to discuss findings from an Arts and Humanities Research Council (AHRC)-funded research project into the heritage culture of British folk tales. The project investigated how such archival source material might be made relevant to contemporary audience via processes of artistic remediation. The research considered artists as “cultural intermediaries”, i.e. as actors occupying the conceptual space between production and consumption in an artistic process.

Design/methodology/approach

Interview data is drawn from a range of 1‐2‐1 and group interviews with the artists. These interviews took place throughout the duration of the project.

Findings

When artists are engaged in a process of remediation which has a distinct arts marketing/audience development focus, they begin to intermediate between themselves and the audience/consumer. Artist perceptions of their role as “professionals of qualification” is determined by the subjective disposition required by the market context in operation at the time (in the case of this project, as commissioned artists working to a brief). Artists’ ability (and indeed willingness) to engage in this process is to a great extent proscribed by their “sense-of-self-as-artist” and an engagement with Romantic ideas of artistic autonomy.

Originality/value

A consideration of the relationship between cultural intermediation and both cultural policy and arts marketing. The artist-as-intermediary role, undertaking creative processes to mediate how goods are perceived by others, enables value-adding processes to be undertaken at the point of remediation, rather than at the stage of intermediation.