Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been ...characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Post‐transcriptional silencing of plant genes using anti‐sense or co‐suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated ...the potential for constructs encoding self‐complementary ‘hairpin’ RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti‐sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron‐containing constructs (ihpRNA) generally gave 90–100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co‐suppression or anti‐sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high‐throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large‐scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
We use in situ Hi-C to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, ...achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ∼10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs “facing” one another. The inactive X chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats.
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•Contact domains (∼185 kb) segregate into six subcompartments with distinct histone marks•Loop anchors occur at domain boundaries and bind CTCF in a convergent orientation•Loops correlate with gene activation and are conserved across cell types and species•The inactive X chromosome contains large loops anchored at CTCF-binding repeats
Using 3D genome sequencing, we find ∼10,000 DNA loops across the human genome. Loop anchors typically occur at domain boundaries and bind CTCF in a convergent orientation, with the asymmetric motifs “facing” one another. On the inactive X chromosome, large loops are anchored at CTCF-binding repeats. Loops are conserved across cell types and species.
Monocyte chemoattractant protein 1 (MCP-1) is known to be an important chemokine for macrophage recruitment. Thus, targeting MCP-1 may prevent the perturbations associated with macrophage-induced ...inflammation in adipose tissue. However, inconsistencies in the available animal literature have questioned the role of this chemokine in this process. The purpose of this study was to examine the role of MCP-1 on obesity-related pathologies.
Wild-type and MCP-1-deficient mice on an friend virus B NIH (FVB/N) background were assigned to either low-fat diet or high-fat diet (HFD) treatment for a period of 16 weeks. Body weight and body composition were measured weekly and monthly, respectively. Fasting blood glucose and insulin, and glucose tolerance were measured at 16 weeks. Macrophages, T-cell markers, inflammatory mediators and markers of fibrosis were examined in the adipose tissue at the time of killing the mice.
As expected, HFD increased adiposity (body weight, fat mass, fat percent and adipocyte size), metabolic dysfunction (impaired glucose metabolism and insulin resistance) macrophage number (CD11b(+)F480(+) cells, and gene expression of EMR1 and CD11c), T-cell markers (gene expression of CD4 and CD8), inflammatory mediators (pNFκB and pJNK, and mRNA expression of MCP-1, CCL5, C-X-C motif chemokine-14, tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)) and fibrosis (expression of IL-10, IL-13, TGF-β and matrix metalloproteinase-2 (MMP2); P<0.05). However, contrary to our hypothesis, MCP-1 deficiency exacerbated many of these responses resulting in a further increase in adiposity (body weight, fat mass, fat percent and adipocyte size), metabolic dysregulation, macrophage markers (EMR1), inflammatory cell infiltration and fibrosis (formation of type I and III collagens, mRNA expression of IL-10 and MMP2; P<0.05).
These data suggest that MCP-1 may be a necessary component of the inflammatory response required for adipose tissue protection, remodeling and healthy expansion in the FVB/N strain in response to HFD feedings.
The human genome folds to create thousands of intervals, called “contact domains,” that exhibit enhanced contact frequency within themselves. “Loop domains” form because of tethering between two ...loci—almost always bound by CTCF and cohesin—lying on the same chromosome. “Compartment domains” form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.
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•We track the 4D Nucleome during cohesin loss and recovery, with 10 kb/20 min resolution•After cohesin loss, loop domains disappear; effects on transcription are modest•During cohesin recovery, loop domains form in minutes, consistent with fast extrusion•Superenhancers form loops, interchromosomal links, and higher-order hubs
Mapping the nucleome in 4D during cohesin loss and recovery reveals that cohesin degradation eliminates loop domains but has only modest transcriptional consequences.
The time–energy information of ultrashort X-ray free-electron laser pulses generated by the Linac Coherent Light Source is measured with attosecond resolution via angular streaking of neon 1s ...photoelectrons. The X-ray pulses promote electrons from the neon core level into an ionization continuum, where they are dressed with the electric field of a circularly polarized infrared laser. This induces characteristic modulations of the resulting photoelectron energy and angular distribution. From these modulations we recover the single-shot attosecond intensity structure and chirp of arbitrary X-ray pulses based on self-amplified spontaneous emission, which have eluded direct measurement so far. We characterize individual attosecond pulses, including their instantaneous frequency, and identify double pulses with well-defined delays and spectral properties, thus paving the way for X-ray pump/X-ray probe attosecond free-electron laser science.
G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. ...Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the β
-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-G
complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-G
complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.
Membranes are known to have modulatory effects on G protein-coupled receptors (GPCRs) via specific lipid interactions. However, the mechanisms of such modulations in physiological conditions and how ...they influence GPCR functions remain unclear. Here we report coarse-grained molecular dynamics simulations on the Adenosine A2a receptor in different conformational states embedded in an in vivo-mimetic membrane model. Nine lipid interaction sites were revealed. The strength of lipid interactions with these sites showed a degree of dependence on the conformational states of the receptor, suggesting that these lipids may regulate the conformational dynamics of the receptor. In particular, we revealed a dual role of PIP2 on A2aR activation that involves both stabilization of the characteristic outward tilt of TM6 and enhancement of A2aR-mini-Gs association. Our results demonstrated that the bound lipids allosterically regulate the functional properties of GPCRs. These protein-lipid interactions provide a springboard for design of allosteric modulators of GPCRs.
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•MD simulations show state-dependent binding of lipids to the Adenosine A2a receptor•Nine lipid interaction sites were revealed, for GM3, cholesterol and PIP2•Binding of PIP2 enhanced the association of mini-Gs with the A2a receptor•These results indicate lipids may allosterically regulate GPCRs
Song et al. reveal that the interactions of lipids with the Adenosine A2a receptor depend on the state of the receptor. PIP2 binding leads to enhanced interaction of mini-Gs with the A2aR. These results suggest a mechanism of allosteric regulation of GPCRs by lipid interactions.
A search for neutrinoless double-β decay (0νββ) in Xe136 is performed with the full EXO-200 dataset using a deep neural network to discriminate between 0νββ and background events. Relative to ...previous analyses, the signal detection efficiency has been raised from 80.8% to 96.4±3.0%, and the energy resolution of the detector at the Q value of Xe136 0νββ has been improved from σ/E=1.23% to 1.15±0.02% with the upgraded detector. Accounting for the new data, the median 90% confidence level 0νββ half-life sensitivity for this analysis is 5.0×1025 yr with a total Xe136 exposure of 234.1 kg yr. No statistically significant evidence for 0νββ is observed, leading to a lower limit on the 0νββ half-life of 3.5×1025 yr at the 90% confidence level.
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