A single cell characterisation of human embryogenesis identifies pluripotency transitions and putative anterior hypoblast centre

AbstractFollowing implantation, the human embryo undergoes major morphogenetic transformations that establish the future body plan. While the molecular events underpinning this process are established in mice, they remain unknown in humans. Here we characterise key events of human embryo morphogenesis, in the period between implantation and gastrulation, using single-cell analyses and functional studies. First, the embryonic epiblast cells transition through different pluripotent states and act as a source of FGF signals that ensure proliferation of both embryonic and extra-embryonic tissues. In a subset of embryos, we identify a group of asymmetrically positioned extra-embryonic hypoblast cells expressing inhibitors of BMP, NODAL and WNT signalling pathways. We suggest that this group of cells can act as the anterior singalling centre to pattern the epiblast. These results provide insights into pluripotency state transitions, the role of FGF signalling and the specification of anterior-posterior axis during human embryo development.

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A Review of Key Biological and Molecular Events Underpinning Transformation of Melanocytes to Primary and Metastatic Melanoma

Cancers ◽

10.3390/cancers11122041 ◽

2019 ◽

Vol 11 (12) ◽

pp. 2041 ◽

Cited By ~ 3

Author(s):

Louise A. Jackett ◽

Richard A. Scolyer

Keyword(s):

Treatment Strategies ◽

Genomic Analysis ◽

Molecular Therapy ◽

Health Concern ◽

Targeted Molecular Therapy ◽

Public Health Concern ◽

Molecular Events ◽

Key Events ◽

Potential Tool

Melanoma is a major public health concern that is responsible for significant morbidity and mortality, particularly in countries such as New Zealand and Australia where it is the commonest cause of cancer death in young adults. Until recently, there were no effective drug therapies for patients with advanced melanoma however significant advances in our understanding of the biological and molecular basis of melanoma in recent decades have led to the development of revolutionary treatments, including targeted molecular therapy and immunotherapy. This review summarizes our current understanding of the key events in the pathway of melanomagenesis and discusses the role of genomic analysis as a potential tool for improved diagnostic evaluation, prognostication and treatment strategies. Ultimately, it is hoped that a continued deeper understanding of the mechanisms of melanomagenesis will lead to the development of even more effective treatments that continue to provide better outcomes for patients with melanoma.

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Novel Role of Sphingolipid Synthesis Genes in Regulating Giardial Encystation

Infection and Immunity ◽

10.1128/iai.00116-08 ◽

2008 ◽

Vol 76 (7) ◽

pp. 2939-2949 ◽

Cited By ~ 23

Author(s):

Yunuen Hernandez ◽

Max Shpak ◽

Trevor T. Duarte ◽

Tavis L. Mendez ◽

Rosa A. Maldonado ◽

...

Keyword(s):

Gene Duplication ◽

Giardia Lamblia ◽

Phylogenetic Analyses ◽

Cyst Formation ◽

The Other ◽

Serine Palmitoyltransferase ◽

Cyst Production ◽

Molecular Events ◽

Key Events

ABSTRACT Although encystation (cyst formation) is important for the survival of Giardia lamblia outside its human host, the molecular events that prompt encystation have not been fully elucidated. Here, we demonstrate that sphingolipids (SLs), which are important for the growth and differentiation of many eukaryotes, play key roles in giardial encystation. Transcriptional analyses showed that only three genes in the SL biosynthesis pathways are expressed and transcribed differentially in nonencysting and encysting Giardia trophozoites. While the putative homologues of giardial serine palmitoyltransferase (gSPT) subunit genes (gspt-1 and -2) are differentially expressed in nonencysting and encysting trophozoites, the giardial ceramide glucosyltransferase 1 gene (gglct-1) is transcribed only in encysting cells. l-Cycloserine, an inhibitor of gSPT, inhibited the endocytosis and endoplasmic reticulum/perinuclear targeting of bodipy-ceramide in trophozoites, and this could be reversed by 3-ketosphinganine. On the other hand, d-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthesis, blocked karyokinesis and reduced cyst production in culture. PPMP also altered the expression of cyst wall protein transcripts in encysting cells. Phylogenetic analyses revealed that the gspt genes are paralogs derived from an ancestral spt sequence that underwent gene duplication early in eukaryotic history. This ancestral sequence, in turn, was probably derived from prokaryotic aminoacyl transferases. In contrast, gglct-1 is found in both prokaryotes and eukaryotes without any evidence of gene duplication. These studies indicate that SL synthesis genes are involved in key events in giardial biology and could serve as potential targets for developing new therapies against giardiasis.

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Ionic homeostasis and subcellular element compartmentation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010013434x ◽

1990 ◽

Vol 48 (2) ◽

pp. 150-151

Author(s):

Ann LeFurgey ◽

Peter Ingram ◽

J.J. Blum ◽

M.C. Carney ◽

L.A. Hawkey ◽

...

Keyword(s):

Leishmania Major ◽

Ionic Homeostasis ◽

X Ray ◽

Functional Studies ◽

Cell Compartments ◽

X Ray Imaging ◽

Resolution Electron ◽

Multiple Cell ◽

Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

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Inhibition of Aurora Kinase B activity disrupts development and differentiation of salivary glands

Cell Death Discovery ◽

10.1038/s41420-020-00393-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Abeer K. Shaalan ◽

Tathyane H. N. Teshima ◽

Abigail S. Tucker ◽

Gordon B. Proctor

Keyword(s):

Cell Cycle ◽

Embryonic Development ◽

Salivary Glands ◽

Aurora Kinase ◽

Serine Threonine Kinase ◽

Aurora Kinase B ◽

Development And Differentiation ◽

Differentiation Marker ◽

Key Events

AbstractLittle is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-βgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.

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Cross-species integration of single-cell RNA-seq resolved alveolar-epithelial transitional states in idiopathic pulmonary fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00594.2020 ◽

2021 ◽

Author(s):

Kevin Y. Huang ◽

Enrico Petretto

Keyword(s):

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Single Cell ◽

Transitional Cell ◽

Notch Pathway ◽

Alveolar Epithelium ◽

State Transitions ◽

Specific Cell ◽

Differentiation Process ◽

Cell State

Single-cell transcriptomics analyses of the fibrotic lung uncovered two cell states critical to lung injury recovery in the alveolar epithelium- a reparative transitional cell state in the mouse and a disease-specific cell state (KRT5-/KRT17+) in human idiopathic pulmonary fibrosis (IPF). The murine transitional cell state lies between the differentiation from type 2 (AT2) to type 1 pneumocyte (AT1), and the human KRT5-/KRT17+ cell state may arise from the dysregulation of this differentiation process. We review major findings of single-cell transcriptomics analyses of the fibrotic lung and re-analyzed data from 7 single-cell RNA sequencing studies of human and murine models of IPF, focusing on the alveolar epithelium. Our comparative and cross-species single-cell transcriptomics analyses allowed us to further delineate the differentiation trajectories from AT2 to AT1 and AT2 to the KRT5-/KRT17+ cell state. We observed AT1 cells in human IPF retain the transcriptional signature of the murine transitional cell state. Using pseudotime analysis, we recapitulated the differentiation trajectories from AT2 to AT1 and from AT2 to KRT5-/KRT17+ cell state in multiple human IPF studies. We further delineated transcriptional programs underlying cell state transitions and determined the molecular phenotypes at terminal differentiation. We hypothesize that in addition to the reactivation of developmental programs (SOX4, SOX9), senescence (TP63, SOX4) and the Notch pathway (HES1) are predicted to steer intermediate progenitors to the KRT5-/KRT17+ cell state. Our analyses suggest that activation of SMAD3 later in the differentiation process may explain the fibrotic molecular phenotype typical of KRT5-/KRT17+ cells.

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The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

Communications Biology ◽

10.1038/s42003-021-01963-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Anurag Kumar Sinha ◽

Kristoffer Skovbo Winther

Keyword(s):

Active Sites ◽

Molecular Switch ◽

Cell Physiology ◽

Synthetase Activity ◽

Wild Type ◽

Functional Studies ◽

Loop Region ◽

A Site ◽

Trna Binding

AbstractBacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114–130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

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Optimizing expression quantitative trait locus mapping workflows for single-cell studies

Genome Biology ◽

10.1186/s13059-021-02407-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Anna S. E. Cuomo ◽

Giordano Alvari ◽

Christina B. Azodi ◽

Davis J. McCarthy ◽

Marc Jan Bonder ◽

...

Keyword(s):

Gene Expression ◽

Quantitative Trait Locus ◽

Single Cell ◽

Quantitative Trait ◽

Cell Types ◽

Expression Quantitative Trait Locus ◽

Eqtl Mapping ◽

Trait Locus ◽

The Impact

Abstract Background Single-cell RNA sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states and promises to improve our understanding of genetic regulation across tissues in both health and disease. Results While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimize sc-eQTL mapping. Here, we evaluate the role of different normalization and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches. Conclusion We provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

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Interactions between the WEE-1.3 kinase and the PAM-1 aminopeptidase in oocyte maturation and the early C. elegans embryo

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab063 ◽

2021 ◽

Author(s):

Dorothy Benton ◽

Eva C Jaeger ◽

Arielle Kilner ◽

Ashley Kimble ◽

Josh Lowry ◽

...

Keyword(s):

Missense Mutation ◽

Oocyte Maturation ◽

Protective Role ◽

C Elegans ◽

Direct Target ◽

The One ◽

Actomyosin Cytoskeleton ◽

Embryonic Viability ◽

Anterior Posterior

Abstract Puromycin-sensitive aminopeptidases are found across phyla and are known to regulate the cell-cycle and play a protective role in neurodegenerative disease. PAM-1 is a puromycin-sensitive aminopeptidase important for meiotic exit and polarity establishment in the one-cell Caenorhabditis elegans embryo. Despite conservation of this aminopeptidase, little is known about its targets during development. In order to identify novel interactors, we conducted a suppressor screen and isolated four suppressing mutations in three genes that partially rescued the maternal-effect lethality of pam-1 mutants. Suppressed strains show improved embryonic viability and polarization of the anterior-posterior axis. We identified a missense mutation in wee-1.3 in one of these suppressed strains. WEE-1.3 is an inhibitory kinase that regulates maturation promoting factor. While the missense mutation suppressed polarity phenotypes in pam-1, it does so without restoring centrosome-cortical contact or altering the cortical actomyosin cytoskeleton. To see if PAM-1 and WEE-1.3 interact in other processes, we examined oocyte maturation. While depletion of wee-1.3 causes sterility due to precocious oocyte maturation, this effect was lessened in pam-1 worms, suggesting that PAM-1 and WEE-1.3 interact in this process. Levels of WEE-1.3 were comparable between wild-type and pam-1 strains, suggesting that WEE-1.3 is not a direct target of the aminopeptidase. Thus, we have established an interaction between PAM-1 and WEE-1.3 in multiple developmental processes and have identified suppressors that are likely to further our understanding of the role of puromycin-sensitive aminopeptidases during development.

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Proteomic Analysis of Synovial Fibroblasts and Articular Chondrocytes Co-Cultures Reveals Valuable VIP-Modulated Inflammatory and Degradative Proteins in Osteoarthritis

International Journal of Molecular Sciences ◽

10.3390/ijms22126441 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6441

Author(s):

Selene Pérez-García ◽

Valentina Calamia ◽

Tamara Hermida-Gómez ◽

Irene Gutiérrez-Cañas ◽

Mar Carrión ◽

...

Keyword(s):

Isotope Labeling ◽

Articular Chondrocytes ◽

Synovial Fibroblasts ◽

Immune Mediators ◽

Cartilage Extracellular Matrix ◽

Fibronectin Fragments ◽

Ecm Degradation ◽

Key Events ◽

First Time

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.

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Germline variant in REXO2 is a novel candidate gene in familial pheochromocytoma

Genetics Research ◽

10.1017/s0016672320000038 ◽

2020 ◽

Vol 102 ◽

Cited By ~ 1

Author(s):

Yael Laitman ◽

Shay Tzur ◽

Ruben Attali ◽

Amit Tirosh ◽

Eitan Friedman

Keyword(s):

Allelic Imbalance ◽

Chromosomal Region ◽

Cancer Susceptibility ◽

Functional Studies ◽

Cancer Susceptibility Genes ◽

Whole Exome ◽

Familial Pheochromocytoma ◽

The Family ◽

Recombination Processes

Abstract Pheochromocytoma (PCC) is a rare, mostly benign tumour of the adrenal medulla. Hereditary PCC accounts for ~35% of cases and has been associated with germline mutations in several cancer susceptibility genes (e.g., KIF1B, SDHB, VHL, SDHD, RET). We performed whole-exome sequencing in a family with four PCC-affected patients in two consecutive generations and identified a potential novel candidate pathogenic variant in the REXO2 gene that affects splicing (c.531-1G>T (NM 015523.3)), which co-segregated with the phenotype in the family. REXO2 encodes for RNA exonuclease 2 protein and localizes to 11q23, a chromosomal region displaying allelic imbalance in PCC. REXO2 protein has been associated with DNA repair, replication and recombination processes and thus its inactivation may contribute to tumorigenesis. While the study suggests that this novel REXO2 gene variant underlies PCC in this family, additional functional studies are required in order to establish the putative role of the REXO2 gene in PCC predisposition.

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