Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We ...show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.
► Cryo-EM images are blurred by sample motion during electron beam exposure ► The sample motion degrades signal especially at high resolution ► Aligning frames of movies recorded with a direct electron detector reduces blurring ► This procedure enhances the resolution obtained in 3D reconstructions of the sample
In cryo-EM, low-dose images are often affected by blurring caused by sample motion during beam exposure. Melody et al. show that aligning frames of movies recorded with a direct electron detector during beam exposure greatly reduces blurring, increases the efficiency of cryo-EM imaging, and enhances resolution.
We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical microregions, whose development, ...differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the cranial mesenchyme, composed of mixed neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. At E9.5 cells from the cranial mesenchyme, overlying olfactory placode/epidermal ectoderm, and underlying neuroepithelium, as well as the emerging mandibular and maxillary arches were sampled. At E10.5, as the facial prominences form, cells from the medial and lateral prominences, the olfactory pit, multiple discrete regions of underlying neuroepithelium, the mandibular and maxillary arches, including both their mesenchymal and ectodermal components, as well as Rathke׳s pouch, were similarly sampled and profiled using both microarray and RNA-seq technologies. Further, we performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm. Taken together, and analyzable by a variety of biological network approaches, these data provide a complementing and cross validating resource capable of fueling discovery of novel compartment specific markers and signatures whose combinatorial interactions of transcription factors and growth factors/receptors are responsible for providing the master genetic blueprint for craniofacial development.
•A gene expression atlas resource of craniofacial development is presented.•Laser capture microdissection isolation of craniofacial compartments.•Gene expression profiling by both microarray and RNA-seq.•Defining expression patterns of all growth factors and transcription factors.
We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells ...give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction.
The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly ...neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo-electron microscopy reconstruction and structural model of a cleaved, soluble Env trimer (termed BG505 SOSIP. 664 gp140) in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 angstrom resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1, and HR2 domains, as well as shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.
Influenza virus ribonucleoprotein complexes (RNPs) are central to the viral life cycle and in adaptation to new host species. RNPs are composed of the viral genome, viral polymerase, and many copies ...of the viral nucleoprotein. In vitro cell expression of all RNP protein components with four of the eight influenza virus gene segments enabled structural determination of native influenza virus RNPs by means of cryogenic electron microscopy (cryo-EM). The cryo-EM structure reveals the architecture and organization of the native RNP, defining the attributes of its largely helical structure and how polymerase interacts with nucleoprotein and the viral genome. Observations of branched-RNP structures in negative-stain electron microscopy and their putative identification as replication intermediates suggest a mechanism for viral replication by a second polymerase on the RNP template.
The Gsx2 homeodomain transcription factor promotes neural progenitor identity in the lateral ganglionic eminence (LGE), despite upregulating the neurogenic factor Ascl1. How this balance in ...maturation is maintained is unclear. Here, we show that Gsx2 and Ascl1 are co-expressed in subapical progenitors that have unique transcriptional signatures in LGE ventricular zone (VZ) cells. Moreover, whereas Ascl1 misexpression promotes neurogenesis in dorsal telencephalic progenitors, the co-expression of Gsx2 with Ascl1 inhibits neurogenesis. Using luciferase assays, we found that Gsx2 reduces the ability of Ascl1 to activate gene expression in a dose-dependent and DNA binding-independent manner. Furthermore, Gsx2 physically interacts with the basic helix-loop-helix (bHLH) domain of Ascl1, and DNA-binding assays demonstrated that this interaction interferes with the ability of Ascl1 to bind DNA. Finally, we modified a proximity ligation assay for tissue sections and found that Ascl1-Gsx2 interactions are enriched within LGE VZ progenitors, whereas Ascl1-Tcf3 (E-protein) interactions predominate in the subventricular zone. Thus, Gsx2 contributes to the balance between progenitor maintenance and neurogenesis by physically interacting with Ascl1, interfering with its DNA binding and limiting neurogenesis within LGE progenitors.
The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in ...situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.
The identity and degree of heterogeneity of glial progenitors and their contributions to brain tumor malignancy remain elusive. By applying lineage-targeted single-cell transcriptomics, we uncover an ...unanticipated diversity of glial progenitor pools with unique molecular identities in developing brain. Our analysis identifies distinct transitional intermediate states and their divergent developmental trajectories in astroglial and oligodendroglial lineages. Moreover, intersectional analysis uncovers analogous intermediate progenitors during brain tumorigenesis, wherein oligodendrocyte-progenitor intermediates are abundant, hyper-proliferative, and progressively reprogrammed toward a stem-like state susceptible to further malignant transformation. Similar actively cycling intermediate progenitors are prominent components in human gliomas with distinct driver mutations. We further unveil lineage-driving networks underlying glial fate specification and identify Zfp36l1 as necessary for oligodendrocyte-astrocyte lineage transition and glioma growth. Together, our results resolve the dynamic repertoire of common and divergent glial progenitors during development and tumorigenesis and highlight Zfp36l1 as a molecular nexus for balancing glial cell-fate decision and controlling gliomagenesis.
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•scRNA-seq reveals distinct transitional states of glial progenitors in the cortex•Intersectional analysis reveals malignant analogs of glial progenitor intermediates•Reprogramming OPC intermediates into a stem-like state for malignant gliomagenesis•Zfp36l1 is a critical regulator of glial fate specification and gliomagenesis
By applying lineage-targeted, single-cell transcriptomics analysis, Weng and colleagues uncover distinct intermediate glial progenitors in the neonatal brain and their malignant counterparts in murine and human gliomas. Lineage-driving network analysis further identifies Zfp36l1 as a pivotal regulator for glial fate specification and glioma growth.
Summary
Background
The prevalence and time trends of food allergy change during childhood depending on the age of the child and the type of food.
Objective
To study prevalence and longitudinal trends ...in food allergy from birth to 18 years in an unselected birth cohort in the Isle of Wight.
Method
Information on food allergy was collected at ages 1, 2, 4, 10 and 18 years from the Isle of Wight Birth Cohort (n = 1456). Skin prick testing (SPT) was performed at the age of 1 and 2 years in symptomatic children. At 4, 10 and 18 years of age, participants were tested to a panel of food and aeroallergens. Food allergy was diagnosed based on the criteria: symptoms suggestive of a typical IgE‐mediated reaction and reaction <4 hours following exposure to a known food allergen. McNemar's test was used to determine significance of changes in prevalence over time.
Results
The prevalence of food allergy remained relatively constant in early childhood (5.3%, 4.4% and 5.0% at 1, 2 and 4 years, respectively), with significant decline at 10 years (2.3%, P < .001 vs 4 years) followed by significant rise at 18 years (4%, P = .02 vs 10 years). Cow's milk (1.6%‐3.5%) and egg (1.1%‐1.4%) were the most common allergens in the first 10 years with peanut (1%) and tree nuts (0.5%) becoming more prevalent beyond 10 years. Fruit and wheat allergy were less common at 10 years, and shellfish and kiwi emerged during adolescence. The prevalence of food allergy plus positive SPT was 1.3%, 0.8%, 0.8%, 0.9% and 2.2% at 1, 2, 4, 10 and 18 years, respectively.
Conclusion
Food allergy is highly prevalent in infancy with partial resolution during late childhood. However, a number of children acquire new food allergy during adolescence resulting in a relatively higher prevalence at 18 years.
ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. ...Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes.
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•New tools enabled EM visualization of dynamic transporter conformations in lipids•Distinct conformational spectra of two homologous ABC transporters were revealed•P-gp prevails in inward-facing conformations under ATP hydrolysis conditions•MsbA with ATP displays both outward and a continuum of inward-facing conformations
Moeller et al. used single particle electron microscopy to delineate the entire conformational spectrum of two homologous ATP-binding cassette exporters and the influence of nucleotide and substrate binding. The data provide new insights into mechanistic and energetic regulation of conformational changes for the transport function.
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