bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multi-Tag: A modular platform of bioorthogonal probes for multi-modal (glyco)protein analysis
Authors: Driessen Marc D. , Junghans Hauke L. , Hartmann Laura , Gerling-Driessen Ulla I. M. 
Published: October 2022

ABSTRACTBioorthogonal chemistry is a well-established concept for tagging and analyzing targets of interest even in living cells, tissue or organisms. In particular glycans, which are, as a posttranslational modification, not amenable to genetic engineering, became analytically accessible through the establishment of metabolic oligosaccharide engineering and subsequent bioorthogonal tagging of chemical probes. Since many essential cellular processes involve glycoproteins, it is not surprising that especially aberrant glycosylation has been associated with the pathology of many diseases. Investigation of aberrant glycosylation in a disease background is complicated by the heterogeneity of glycans and dynamic changes in their composition. In order to create a meaningful information depth, it can be beneficial to analyze the same sample with different analytical methods. This becomes even more relevant for samples with limited access. Most of the currently existing bioorthogonal probes are designed for use in only one type of experiment. These design restrictions are mainly based on the limited synthetic accessibility of more complex bioorthogonal probes. Multi-step syntheses are often time consuming and cost-inefficient. Here, we introduce a fast and easily manageable strategy for the synthesis of complex bioorthogonal probes that allow an application in multiple coordinated experiments. Using established principles and conditions of solid-phase peptide synthesis, we combine different functional building blocks to generate multi-functional bioorthogonal probes (namedMulti-Tags). We show the easy synthesis of several multi-modal probes and demonstrate their applicability and versatility in exemplary assays.
GiRAFR improves gRNA detection and annotation in single cell CRISPR screens
Authors: Yu Qian , Van Minsel Paulien , Galle Eva , Thienpont Bernard 
Published: October 2022

AbstractNovel single cell RNA-seq analysis combined with CRISPR screens enables the high-throughput characterization of transcriptional changes caused by genetic perturbations. Dedicated software to annotate CRISPR guide RNA (gRNA) libraries and associate them with single cell transcriptomes are however lacking. Here, we generated a CRISPR droplet sequencing dataset. We demonstrate that the current default tool fails to detect mutant gRNAs. We therefore developed GiRAFR, a pysam-based software tool to characterize intact and mutant gRNAs. We show that mutant gRNAs are dysfunctional, and failure to detect and annotate them leads to an inflated estimate of the number of untransformed cells as well as an underestimated multiplet frequency. These findings are mirrored in publicly available datasets, where we find that up to 34 % of cells are transduced with a mutant gRNA. Applying GiRAFR hence stands to improve the annotation and quality of single cell CRISPR screens.
Pregranulosa cells engage a distinct transcriptional programme prior to cell-cycle dependent primordial follicle activation
Authors: Taylor Güneş , Frost Emily R , Crow Brendan N , Radley Arthur , Boeing Stefan , Galichet Christophe , Bucinskaite Barbora , Baker Mark A , Sutherland Jessie M , Lovell-Badge Robin 
Published: October 2022

AbstractPrimordial follicles are quiescent ovarian structures comprised of a single oocyte surrounded by a layer of somatic supporting pregranulosa cells. Primordial follicle activation is the first step towards oocyte maturation and, ultimately, ovulation. As the number of quiescent primordial follicles is finite, their rate of activation is a critical parameter of the duration of the female reproductive lifespan. Activation status is established by the presence of cuboidal and proliferative granulosa cells in primary follicles, rather than squamous and quiescent pregranulosa cells in primordial follicles. Here, using a continuous Entropy Sort Feature Weighting approach on single-cell RNA sequencing data, we identify a distinct transcriptomic signature of activating pregranulosa cells in neonatal wildtype mice. This signature contains several genes previously linked with mature granulosa cells as well several novel candidates:Slc18a2, Tnni3,Fam13aandMyo1e. We confirm expression ofSlc18a2and TNNI3 in the granulosa cells of activating follicles in embryonic, neonatal and adult mouse ovaries. Perturbation of cell cycle inhibitor p27kip1inCdkn1b-/-mice results in complete activation of all primordial follicles during this neonatal period. Contrary to previous reports on this established mouse model, we find substantial transcriptomic changes in embryonicCdkn1b-/-ovaries. Upon loss of cell-cycle inhibition we find increased expression of our signature of pregranulosa cell activation, particularly that of cardiac troponin I (Tnni3). We conclude that pregranulosa cells engage a distinct transcriptional programme prior to cell-cycle dependent primordial follicle activation.
A quarter-million-year-old polymorphism drives reproductive mode variation in the pea aphid
Authors: Rimbault M. , Legeai F. , Peccoud J. , Mieuzet L. , Call E. , Nouhaud P. , Defendini H. , Mahéo F. , Marande W. , Théron N. , Tagu D. , Le Trionnaire G. , Simon J.-C. , Jaquiéry J. 
Published: October 2022

AbstractAlthough asexual linages evolved from sexual lineages in many different taxa, the genetics of sex loss remains poorly understood. We addressed this issue in the pea aphidAcyrthosiphon pisum,whose natural populations encompass lineages performing cyclical parthenogenesis (CP) and producing one sexual generation per year, as well as obligate parthenogenetic (OP) lineages that can no longer produce sexual females but can still produce males. A SNP-based, whole-genome scan of CP and OP populations sequenced in pools (103 individuals from six populations) showed that a single X-linked region controls the variation in reproductive mode. This 840-kb region is highly divergent between CP and OP populations (FST= 34.9%), with >2000 SNPs or short Indels showing a high degree of association with the phenotypic trait. Comparison ofde novogenome assemblies built from long reads did not reveal large structural rearrangements between CP and OP lineages within the candidate region. This reproductive polymorphism still appears relatively ancient, as we estimated its age at ~0.25 million years from the divergence betweencpandopalleles. The low genetic differentiation between CP and OP populations at the rest of the genome (FST= 2.4%) suggests gene flow between them. Males from OP lineages thus likely transmit their op allele to new genomic backgrounds. This “contagious asexuality”, combined with environment-induced selection (each reproductive mode being favored under different climates) probably contributes to the long-term persistence of thecpandopalleles.SignificanceAsexual taxa occur in all major clades of Eukaryotes and derive from related sexual species. Yet, the genetic basis for these transitions is poorly known because crosses cannot generally be performed to genetically map the ability to propagate asexually. As a result, only one gene responsible for sex loss has been identified in one animal species. Here, using pooled genome sequencing, we identified an 840kb region (carrying 32 genes) that controls the transition to permanent asexuality in the pea aphid. We also revealed that sexual and asexual alleles diverged 0.25 million years ago and that asexual lineages likely persist through contagious asexuality, providing new insights into the mechanisms of coexistence of sexual and asexual lineages.
Cytogenetic screening of chromosomal abnormalities and genetic analysis of FSH receptor Ala307Thr and Ser680Asn genes in amenorrheic patients
Authors: Kanaan Bushra A. , Al-Ouqaili Mushtak T.S. , Murshed Rafal M. 
Published: October 2022

AbstractBackgroundAmenorrhea is a rare reproductive medical condition defined by the absence of menstruation during puberty or later life. This study aims to establish the frequency and pattern of chromosomal abnormalities (CA) in both primary amenorrhea (PA) and secondary amenorrhea (SA), and further to detect the genetic changes in exon 10 at nucleotide positions 919 and 2039 of the genotypes Thr307Ala, and Asn680Ser, respectively.MethodThis cross-sectional study was conducted on a sample of seventy amenorrhoeic women according to the Helsinki declaration rules of medical ethics, as divided into 40 (57.14%) with PA and 30 (42.86%) with SA, and 30 healthy women with normal menstruation as the control. The chromosomal karyotyping was performed according to the ISCN, 2020. PCR products were submitted to RFLP and Sanger sequencing for women with normal karyotype and high FSH serum levels.ResultThe classical Turner Syndrome was the most common CA in PA, followed by isochromosome X [46, Xi(X)(q10)], mosaicism of Turner and isochromosome X [45, X /46, Xi(X)(q10)], sex reversal (46, XY) and (46, XX,-3,+der3,-19,del 19 p). Abnormal SA cases were characterized by mosaicism Turner syndrome (45,X/46,XX) and (46,XX,-3,+der3,X,+derX). The homozygous genotypes AA and GG of Ala307Thr (rs6165) in the FSHR gene are most common in PA, while the homozygous genotype AA is more common in SA. GG and AG genotypes of Ser680Asn (rs6166) are more frequent in Iraqi patients with PA and SA compared to the healthy control women. Both PCR-RFLP and Sanger sequencing indicated a marked matching between genotypes.ConclusionsThe study emphasizes the need for cytogenetic analysis to determine the genetic basis of PA and SA. Further, genotyping for women with normal karyotype and high FSH serum concentrations via PCR-RFLP should be considered for the precise diagnosis and development of appropriate management of and counselling for these patients.
A Compensatory RNase E Variation Increases Iron Piracy and Virulence in Multidrug-ResistantPseudomonas aeruginosaduring Macrophage Infection
Authors: Vaillancourt Mylene , Galdino Anna Clara Milesi , Limsuwannarot Sam P. , Celedonio Diana , Dimitrova Elizabeth , Broerman Matthew , Bresee Catherine , Doi Yohei , Lee Janet S. , Parks William C. , Jorth Peter 
Published: October 2022

AbstractDuring chronic cystic fibrosis (CF) infections, evolvedPseudomonas aeruginosaantibiotic resistance is linked to increased pulmonary exacerbations, decreased lung function, and hospitalizations. However, the virulence mechanisms underlying worse outcomes caused by antibiotic resistant infections are poorly understood. Here, we investigated evolved aztreonam resistantP. aeruginosavirulence mechanisms. Using a macrophage infection model combined with genomic and transcriptomic analyses, we show that a compensatory mutation in thernegene, encoding RNase E, increased siderophore gene expression, causing macrophage ferroptosis and lysis. Macrophage killing could be eliminated by treatment with the iron mimetic gallium. RNase E variants were abundant in clinical isolates, and CF sputum gene expression data show that clinical isolates phenocopied RNase E variant functions during macrophage infection. Together these data show howP. aeruginosaRNase E variants can cause host damage via increased siderophore production and host cell ferroptosis but may also be targets for gallium precision therapy.
A 3D cell-free bone model shows collagen mineralization is driven and controlled by the matrix
Authors: van der Meijden Robin H.M. , Daviran Deniz , Rutten Luco , Walboomers X. Frank , Macías-Sánchez Elena , Sommerdijk Nico , Akiva Anat 
Published: October 2022

AbstractOsteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization and different degrees of mineralization where the intrafibrillar and extrafibrillar mineral is intergrown into one continuous network of oriented crystals.While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization, and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix.Through the remineralization of demineralized bone matrix, we demonstrate the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement. We then explore this cell-free in vitro system as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Combined Raman and electron microscopy indicates that glycosaminoglycans play a more prominent role than generally assumed in the matrix-mineral interactions. Our experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.
Surface protein profiling of milk and serum extracellular vesicles unveil body fluid and cell-type signatures and insights on vesicle biogenesis
Authors: Giovanazzi Alberta , van Herwijnen Martijn J.C. , van der Meulen Gerbrich N. , Wauben Marca H.M. 
Published: October 2022

ABSTRACTThe promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluid-specific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, which were used to model the potential biogenesis route of different EV subsets and their cellular origin.
Extracellular ATP drives pancreatic cancer cell invasion via purinergic receptor-integrin interactions
Authors: Bort Elena Tomas , Joseph Megan D. , Wang Qiaoying , Carter Edward P. , Roth Nicolas J. , Gibson Jessica , Samadi Ariana , Kocher Hemant M. , Simoncelli Sabrina , McCormick Peter J. , Grose Richard P. 
Published: October 2022

AbstractPancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet clinical need. Given the elevated ATP levels seen in PDAC, the purinergic axis represents an attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, it plays essential roles in fibrosis, inflammation response and immune function. We have analysed the PDAC purinome using publicly available databases to discern which members may impact patient survival. We identifiedP2RY2to be the purinergic gene with the strongest association to hypoxia, the highest cancer cell specific expression and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y2to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification or pharmacological strategies we identify the mechanism of this ATP-driven invasion to require direct protein-protein interactions between P2Y2and αV integrins. Using DNA-PAINT super-resolution fluorescence microscopy, we found that P2Y2regulates the amount and distribution of integrin αV in the plasma membrane. This work highlights a novel GPCR-integrin interaction in cancer invasion and its potential for therapeutic targeting.
An ER phospholipid hydrolase drives ER-associated mitochondrial constriction for fission and fusion
Authors: Nguyen Tricia T. , Voeltz Gia K. 
Published: October 2022

AbstractMitochondria are dynamic organelles that undergo cycles of fission and fusion at a unified platform defined by endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCSs). These MCSs or nodes co-localize fission and fusion machinery. We set out to identify how ER-associated mitochondrial nodes can regulate both fission and fusion machinery assembly. We have used a promiscuous biotin ligase linked to the fusion machinery, Mfn1, and proteomics to identify an ER membrane protein, Aphyd, as a major regulator of node formation. In the absence of Aphyd, fission and fusion machineries fail to recruit to ER-associated mitochondrial nodes and fission and fusion rates are significantly reduced. Aphyd contains an acyltransferase motif and an α/β hydrolase domain and point mutations in critical residues of these regions fail to rescue the formation of ER-associated mitochondrial hot spots. These data suggest a mechanism whereby Aphyd functions by altering phospholipid composition at ER-mitochondria MCSs. Our data present the first example of an ER membrane protein that regulates the recruitment of both fission and fusion machineries to mitochondria.
Rubella virus tropism and single cell responses in human primary tissue and microglia-containing organoids
Authors: Popova Galina , Retallack Hanna , Kim Chang N. , Wang Albert , Shin David , DeRisi Joseph , Nowakowski Tomasz J. 
Published: October 2022

AbstractRubella virus is an important human pathogen that can cause neurologic deficits in a developing fetus when contracted during pregnancy. Despite successful vaccination programs in the Americas and many developed countries, rubella remains endemic in many regions worldwide and outbreaks occur wherever population immunity is insufficient. Intense interest since rubella virus was first isolated in 1962 has advanced our understanding of clinical outcomes after infection disrupts key processes of fetal neurodevelopment. Yet it is still largely unknown which cell types in the developing brain are targeted. We show that in human brain slices, rubella virus predominantly infects microglia. This infection occurs in a heterogeneous population but not in a highly microglia-enriched monoculture in the absence of other cell types. By using an organoid-microglia model, we further demonstrate that rubella virus infection leads to a profound interferon response in non-microglial cells, including neurons and neural progenitor cells, and this response is attenuated by the presence of microglia.
Use of CD122+ natural killer cell precursors for superior anti-leukemia responses
Authors: Guglietta Silvia , Cardenas Luis , Krieg Carsten 
Published: October 2022

AbstractAcute Myeloid Leukemia (AML) is an aggressive blood cancer in adults. Intensive induction therapy successfully induces a complete response in up to 80% of the adult patients but a fraction of them is refractory or relapses. The fraction of non-responsive or relapsing patients is especially high amongst medically unfit and older patients, who cannot undergo aggressive chemotherapy treatment. Therefore, offering patients failing on first line intensive chemotherapy and medically unfit and older patients an effective treatment option for long-term control or even cure of disease represents a significant yet unmet clinical need.As a component of the innate immunity, NK cells have recently shown great promise for the treatment of patients with different malignancies. Here, we show that injection of human interleukin-2 (IL-2) complexes (IL-2cx) induce the de novo generation and massive expansion of NK cell precursorsin vivo. Furthermore, IL-2cx-expanded NK cells exert effector functions with the capacity to control the growth of non-self MHC class I-deficient RMA-S lymphoma cellsin vivo, while remaining tolerant towards MHC class-expressing self RMA cells.In an experimental setup mimicking the clinical case of refractory patients after intensive AML induction therapy, IL-2cx treatment mediated strong anti-AML responses following haploidentical bone marrow transplantation without the need for adoptive transfer of donor-derived orin vitroautologous expanded NK cells. Thus, this study demonstrates that IL-2cx immunotherapy allows thein vivogeneration and expansion of functionally mature NK cells to achieve long-term response in AML paving the way to an effective treatment option.
Early-life experience reorganizes neuromodulatory regulation of stage-specific behavioral responses and individuality types during development
Authors: Nasser Reemy Ali , Harel Yuval , Stern Shay 
Published: October 2022

AbstractEarly-life experiences may promote stereotyped behavioral alterations that are dynamic across development time, but also behavioral responses that are variable among individuals, even when initially exposed to the same stimulus. Here, by utilizing longitudinal monitoring ofC. elegansindividuals throughout development we show that behavioral effects of early-life starvation are exposed during early and late developmental stages and buffered during intermediate stages of development. We further found that both dopamine and serotonin shape the discontinuous behavioral responses by opposite and temporally segregated functions across development time. While dopamine buffers behavioral responses during intermediate developmental stages, serotonin promotes behavioral sensitivity to stress during early and late stages. Interestingly, unsupervised analysis of individual biases across development uncovered multiple individuality types that coexist within stressed and unstressed populations and further identified experience-dependent effects on their composition. These results provide insight into the complex temporal regulation of behavioral plasticity across developmental timescales, structuring shared and unique individual responses to early-life experiences.
Characterization of Three Variants of SARS-CoV-2in vivoShows Host-Dependent Pathogenicity in Hamsters
Authors: Toomer Gabriela , Burns Whitney , Melendez Monica , Garcia Liliana , Henry Gerelyn , Biancofiori Anthony , George Albert , Duffy Ciera , Chu Justin , Sides Morgan , Muñoz Melissa , Garcia Kelly , Nikolai-Yogerst Anya , Peng Xinjian , Westfall Landon , Baker Robert 
Published: October 2022

ABSTRACTAnimal models are used in preclinical trials to test vaccines, antivirals, monoclonal antibodies, and immunomodulatory drug therapies against SARS-CoV-2. However, these drugs often do not produce equivalent results in human clinical trials. Here, we show how different animal models infected with some of the most clinically relevant SARS-CoV-2 variants, WA1/2020, B.1.617.2/Delta, B.1.1.529/Omicron and BA5.2/Omicron, have independent outcomes. We show that in mice, B.1.617.2 is more pathogenic, followed by WA1, while B.1.1.529 showed an absence of clinical signs. Only B.1.1.529 was able to infect C57BL/6J mice, which lack the human ACE2 receptor. B.1.1.529-infected ACE2 mice had different T cell profiles compared to infected K18-hACE2 mice, while viral shedding profiles and viral titers in lungs were similar between the ACE2 and the C57BL/6J mice. These data suggest B.1.1.529 virus adaptation to a new host and shows that asymptomatic carriers can accumulate and shed virus. Next, we show how B.1.617.2, WA1 and BA5.2/Omicron have similar viral replication kinetics, pathogenicity, and viral shedding profiles in hamsters, demonstrating that the increased pathogenicity of B.1.617.2 observed in mice is host-dependent. Overall, these findings suggest that small animal models are useful to parallel human clinical data, but the experimental design places an important role in interpreting the data.IMPORTANCEThere is a need to investigate SARS-CoV-2 variants phenotypes in different animal models due to the lack of reproducible outcomes when translating experiments to the human population. Our findings highlight the correlation of clinically relevant SARS-CoV-2 variants in animal models with human infections. Experimental design and understanding of correct animal models are essential to interpreting data to develop antivirals, vaccines, and other therapeutic compounds against COVID-19.